Purf Brief 1

UNIT 1.1Overview of Protein Purification andCharacterization

AIMS AND OBJECTIVESProtein purification has an over 200-year

history: the first attempts at isolating sub-stances from plants having similar propertiesto “egg albumen,” or egg white, were reportedin 1789 by Fourcroy. Many proteins from plantswere purified in the nineteenth century, thoughmost would not be considered pure by modernstandards. A century later, ovalbumin was thefirst crystalline protein obtained (by Hofmeis-ter in 1889). The year 1989 may not go downin history as a milestone in protein chemistry,but since then there has been a resurgence ofinterest in proteins after more than a decade ofgene excitement.

The aims of protein purification, up until the1940s, were simply academic. To then, even thebasic facts of protein structure were not fullyappreciated, and pure proteins were needed justto study structure and test the rival theories ofthe pre-DNA days. During the Second WorldWar, an acute need for blood proteins led todevelopment of the Cohn fractionation proce-dure for purification of albumin and other pro-teins from serum (Cohn et al., 1946). This wasthe inception of large-scale protein purifica-tions for commercial purposes; Cohn fractiona-tion continues to be used to this day.

As more proteins, and particularly enzymes,were purified and crystallized, they started tobe used increasingly in diagnostic assays andenzymatic analyses, as well as in the large-scale food, tanning, and detergent industries.Many enzymes used in industry are not in factvery pure, but as long as they do the job, that issufficient. “Process” enzymes such as α-amy-lase, proteases, and lipases are pro-duced in tonquantities, mainly as secretion products in bac-terial cultures, and may undergo only limitedpurification processes to mini-mize costs. Atthe other extreme, enzyme products for re-search and analysis require a high degree ofpurification to ensure that contaminating activi-ties do not interfere with the intended use.Anyone familiar with molecular biology en-zymes will appreciate how minute levels ofcontamination of DNase or RNase can com-pletely destroy carefully planned experiments.

The 1960s and 1970s could be described asthe peak years for protein and enzyme research,and most of the methods used in protein puri-

fication were established by then, at least intheir principles. More recent developmentshave been mainly in instrumentation designedto optimize the application of each methodol-ogy. Developments in instrumentation havebeen stimulated by the rapid progress in mo-lecular biology, because gene isolation has of-ten been preceded by isolation of the geneproduct. Because such products can now becharacterized sufficiently (i.e., partially se-quenced) using minute amounts of protein, theneed for large-scale or even moderate-scaleprocedures has decreased. Hence there has beenan explosive development of modern equip-ment designed specifically for dealing withamounts of protein in the milligram to micro-gram range. On the other hand, structural stud-ies using X-ray crystallography and nuclearmagnetic resonance (NMR) require hundredsof milligrams of pure protein, so larger-scaleequipment and procedures are still needed inthe research laboratory.

The nature of the proteins studied has alsochanged substantially. Whereas enzymes wereonce the most favored subjects, they have nowbeen superceded by nonenzymatic proteinssuch as growth factors, hormone receptors, vi-ral antigens, and membrane transporters. Manyof these occur in minute amounts in the naturalsource, and their purification can be a majortask. Heroic efforts in the past have used kilo-gram quantities of rather unpleasant startingmaterials, such as human organs, and ended upwith a few micrograms of pure product. It isnow more usual, however, to take the geneticapproach: clone the gene before the protein hasbeen isolated or even properly identified, thenexpress it in a suitable host cell culture ororganism. The expression level may be ordersof magnitude higher than in the original source,which will make purification a relatively simpletask. It can be useful to know beforehand somephysical properties of the protein, to facilitatethe development of a suitable purification pro-tocol from the recombinant source. On the otherhand, there are now several ways of preparingfusion proteins, which can be purified by affin-ity techniques without any knowledge of theproperties of the target protein. Moreover, thereare ways of modifying the expressed productto simplify purification further.

Contributed by R.K. ScopesCurrent Protocols in Protein Science (1995) 1.1.1-1.1.6Copyright © 2000 by John Wiley & Sons, Inc. CPPS

1.1.1

Strategies ofProteinPurification andCharacterization

Thus the approach to protein purificationmust first take into account the reason it is beingdone, as the methods will vary greatly withdifferent requirements. At one extreme is theone-of-a-kind purification, in a well-financedand equipped laboratory, that is carried out toobtain a small amount of product for sequenc-ing so that gene isolation can proceed. In thiscase, expense of equipment and reagents maybe no problem, and a very low overall recoveryof product can be acceptable, provided it is pureenough. At the other extreme are the require-ments of commercial production of a protein inlarge amounts on a continuing basis, wherehigh recovery and economy of processing arethe chief parameters to be considered. There aremany intermediate situations as well.

Many publications in the area of proteinresearch are entitled “Purification and charac-terization of…,” and describe a purificationprocedure in sufficient detail that it can bereproduced in another laboratory. The charac-terization section may include structural, func-tional, and genetic information, and carryingout such studies is likely to require at leastmilligram quantities of pure protein. Ideally thepurification should involve a small number ofsteps, with good recovery at each step. If therecovery is poor (<50% at any step), however,there should be some indication of what hap-pened to the missing activity. Has it been dis-carded in the other fractions for the sake ofpurity, or does it represent a true loss of activity?If the latter, then the end-product may be lessthan fully active despite apparent homogeneityindicated by standard analysis. The choice be-tween recovery and purification at each step canbe problematical; taking a narrow cut of achromatographic peak may provide a very purefraction, at the expense of losing a good deal ofless pure active component on either side. Inmaking such decisions, the objective of theexercise must be kept in mind: if yield is notimportant, then the choice of poor yield for thesake of purity may be logical.

By far the most important requirement of apublication is reproducibility of the methodreported. It is not sufficient to have carried outthe process only once if it is expected that otherinvestigators will want to repeat it. There arealways factors that influence the process thatmay be overlooked at first, and which if variedslightly can have a major effect on the purifi-cation procedure. The reported process shouldalways be repeated exactly as described beforesubmitting the manuscript for publication.There is one exception, namely, the case where

purification was conducted simply to obtainenough protein for sequencing and gene isola-tion; if that was achieved, there should be noneed to provide instructions for repetition.

SOURCES OF MATERIAL FORPROTEIN PURIFICATION

For many people embarking on a proteinpurification project, there is no choice of mate-rial. They are studying a particular biologicaltissue or organism, and the objective is to purifya protein from that source. However, there maybe approaches that can make the project sim-pler. If, for instance, the source is difficult toobtain in large amounts, it may be best to carryout at least preliminary trials on a source speciesmore readily obtained. The most obvious andrelevant example is when the species beingstudied is Homo sapiens, and tissue samples arenot readily available for practical or ethicalreasons, or both. In this case, it is usual to goto where mammalian tissue is readily available(i.e., an abattoir) and work with bovine, ovine,or porcine sources. Alternatively, if quantity oftissue is not a problem, the humble laboratoryrat may suffice. Once a protocol for purifyingthe protein from substitute sources has beenworked out, it will be much easier to developone using human material—the identical pro-cedure may work satisfactorily. Proteins differto a fairly small extent between species thathave diverged within about 100 million years,a time frame that groups together most highermammals. Thus the behavior of proteins de-rived from different animals with respect to thevarious fractionation procedures is likely to besimilar, and a protocol worked out for pig tis-sues is likely to need only minor adjustmentsfor application to human tissues.

A second example is where the interest ismainly on the function of a protein, especiallyan enzyme, for which functions and actionshave generally been strongly conservedthrough evolution. In that case, a preliminaryscreening of potential sources, or, better still,the literature, should come up with a raw ma-terial that is best suited to the investigator’spurposes. Considerations should include thefollowing: (1) What functions are required ofthe end product? For instance, an enzyme hav-ing a low Km may be needed, so selecting thesource with the highest activity may not suffice.(2) How convenient is it to grow or obtain theraw material, and are there problems concern-ing pathogenicity or extractability? (3) Doesthe quantity of the protein vary with growthconditions or age, and does it deteriorate in situ

Current Protocols in Protein Science

1.1.2

Overview ofProtein

Purification andCharacterization

if left too long? Obviously one requires a sourcethat reliably produces the highest amount of thedesired protein per unit volume to maximizethe chances of developing a good purificationprocedure. (4) What storage conditions are re-quired for the raw material? It is important toconsider that fresh raw material may not beimmediately available whenever a purificationis attempted.

The above considerations are relevant to thetraditional situation for commencing a proteinpurification project. It is becoming increasinglycommon, however, for proteins to be purifiedas recombinant products using techniques inwhich the gene is expressed in a host organismor in cultured cells. This of course requires thegene encoding the protein of interest to beavailable. Until the mid-1980s, such materialwas usually obtained by hybridization of anoligonucleotide synthesized according toamino acid sequence information. This re-quired the protein to have been purified first, sothe initial task of protein purification stillneeded to be done at least once. More recently,genetic techniques have permitted the isolationof many genes encoding known proteins, eventhough the proteins may never have been stud-ied directly. Moreover, with the expansion ofthe Human Genome Project and related DNAsequencing efforts, many genes for both knownand unknown proteins will become availableand will be able to be expressed in recombinantform without ever being purified from the hostspecies. As a result some completely new con-siderations for protein purification come intoplay, including the possibility of modifying thegene structure not only to increase expressionlevel and alter the protein product itself toenhance a desired function, but equally impor-tantly to aid in purification. Recombinant pro-teins may be expressed in bacteria, yeasts, in-sect cells, and animal tissue cultures. Furtherdetails may be found in UNIT 1.2.

DETECTION AND ASSAY OFPROTEINS

During a protein purification procedurethere are two measurements that need to bemade, preferably for each fraction. Measure-ments both of the total protein and of theamount (usually bioactivity) of the desired pro-tein must be made. Details of the most com-monly used assay methods are given in Chapter3. It is not possible to isolate a protein withouta method of determining whether it is present;an assay, either quantitative or at least

semiquantitative, indicating which fractioncontains the most of the desired protein isessential.

Assays may range from the quick-and-easytype (e.g., instantaneous spectrophotometricmeasurement of enzyme activity) to long andtedious bioassays that may take days to producean answer. The latter situation is very difficult,because by the time one knows where the pro-tein is, it may be “was,” owing to degradationor inactivation. Moreover, this may not becomeclear until the next step has been completed andits products assayed. Any assay that is quick istherefore advantageous, even if it means a sac-rifice of accuracy for speed.

Measurement of total protein is useful, as itindicates the degree of purification at each step.However, unless the next step critically de-pends on how much protein is present, totalprotein measurement is not extremely impor-tant: a small sample can be put aside and meas-ured later, when the purification is complete. Itis, however, very important to know how muchprotein is present in the final, presumed puresample, as this will indicate the specific activity(if the protein has an activity), which can becompared with other preparations. The generalobject is to obtain as high a specific activity aspossible (taking into account recovery consid-erations), which means retaining as much of thedesired protein as possible while ending upwith as little total protein as possible.

METHODS FOR SEPARATIONAND PURIFICATION OF PROTEINS

The methods available for protein purifica-tion range from simple precipitation pro-cedures used since the nineteenth century tosophisticated chromatographic and affinitytechniques that are constantly undergoing de-velopment and improvement. Methods can beclassified in several alternative ways—perhapsone of the best is based on the properties of theproteins that are being exploited. Thus themethods can be divided into four distinct butinterrelated groups depending on protein char-acteristics: surface features, size and shape, netcharge, and bioproperties.

Methods Based on Surface Features ofProteins

Surface features include charge distributionand accessibility, surface distribution of hydro-phobic amino acid side chains, and, to a lesserextent, net charge at a given pH (see discussionof net charge). Methods exploiting surface fea-


1.1.3


tures mainly depend on solubility properties.Differences in solubility result in precipitationby various manipulations of the solvent inwhich the proteins are solubilized. Methods forobtaining an extract containing the desired pro-tein in soluble form are given in Chapter 4. Thesolvent, nearly always water containing a lowconcentration of buffer salts, can be treated toalter properties such as ionic strength, dielectricconstant, pH, temperature, and detergent con-tent, any of which may selectively precipitatesome of the proteins present. Conversely, pro-teins may be selectively solubilized from aninsoluble state by manipulation of the solventcomposition. The surface distribution of hydro-phobic residues is an important determinant ofsolubility properties; it is also exploited in hy-drophobic chromatography, both in the re-versed-phase mode (UNIT 11.6) and in aqueous-phase hydrophobic-interaction chromatogra-phy (UNIT 8.4).

Also included in this group is the highlyspecific technique of immunoaffinity chroma-tography, in which an antibody directed againstan epitope on the protein surface is used to pullout the desired protein from a mixture.

Methods Based on Whole Structure:Size and Shape

Although the size and shape of proteins canhave some influence on solubility properties,the chief method of exploiting these propertiesis gel-filtration chromatography (UNIT 8.3). Inaddition, preparative gel electrophoresis makesuse of differences in molecular size. Proteinsrange in size from the smallest classified asproteins rather than polypeptides, around 5000Da, up to macromolecular complexes of manymillion daltons. Many proteins in the bioactivestate are oligomers of more than one polypep-tide (see UNIT 1.2), and these can be dissociated,though normally with loss of overall structure.Thus many proteins have two “sizes”: that ofthe native state, and that (or those) of thepolypeptides in the denatured and dissociatedstate. Gel-filtration procedures normally dealonly with native proteins, whereas electro-phoretic procedures commonly involve separa-tion of dissociated and denatured polypeptides.

Methods Based on Net ChargeThe two techniques that exploit the overall

charge of proteins are ion-exchange chroma-tography (by far the most important) and elec-trophoresis (Chapter 10). Ion exchangers bindcharged molecules, and there are essentiallyonly two types of ion exchangers, anion and

cation. The net charge of a protein depends onthe pH—positive at very low pH, negative athigh pH, and zero at some specific point inbetween, termed the isoelectric point (pI). Itshould be stressed that at the pI a protein has agreat many charges; it just happens that at thispH the total negatives exactly equal the totalpositives. The most charged state (disregardingthe charge sign) is in the pH range 6.0 to 9.0.This is the most stable pH range for mostproteins, as it encompasses common physi-ological pH values. Ion exchangers consist ofimmobilized charged groups and attract oppo-sitely charged proteins. They provide the modeof separation that has the highest resolution fornative proteins. High-performance reversed-phase chromatography has equivalent or evenbetter resolution, but it generally involves atleast partial denaturation during adsorption andso is not recommended for sensitive proteinssuch as enzymes. Protein purification usingion-exchange chromatography has mainly em-ployed positively charged anion exchangers,for the simple reason that the majority of pro-teins at neutral pH are negatively charged (i.e.,have a low isoelectric point). Details of meth-odology are found in UNIT 8.2.

Methods Based on Bioproperties(Affinity)

A powerful method for separating the de-sired protein from others is to use a biospecificmethod in which the particular biological prop-erty of the protein is exploited. The affinityapproach is limited to proteins that have aspecific binding property, except that proteinsare theoretically able to be purified by immu-noaffinity chromatography (UNIT 9.1), which isthe most specific of all affinity techniques.Most proteins of interest do have a specificligand: enzymes have substrates and cofactors,and hormone-binding proteins and receptormolecules are designed to bind specifically andtightly to particular hormones and other factors.Immobilization of the ligand to which the pro-tein binds (or of antibody to the protein) enablesselective adsorption of the desired protein inthe technique known as affinity chromatogra-phy (Chapter 9). There are also nonchroma-tographic modes of exploiting biospecific in-teractions.

CHARACTERIZATION OF THEPROTEIN PRODUCT

Once a pure protein is obtained, it may beemployed for a specific purpose, such as enzy-matic analysis (e.g., glucose oxidase and lac-


1.1.4

Overview ofProtein


tate dehydrogenase), or as a therapeutic agent(e.g., insulin and growth hormone). However,it is normal, when a protein has been isolatedfor the first time, to characterize it in terms ofstructure and function. Several features are gen-erally expected in characterization of a newprotein. These include molecular weight, or atleast the size of the subunit(s), determined bySDS-polyacrylamide gel electrophoresis(Chapter 10) and/or gel filtration (UNIT 8.3).Spectral properties such as the UV spectrum(Trp and Tyr content), circular dichroism (CD)spectrum (secondary structure), and specialcharacteristics of proteins with prostheticgroups (e.g., quantitation and spectra) may bepresented. The quantity and nature of carbohy-drates on glycoproteins should be determined(Chapter 12). Also, if the gene has not alreadybeen reported, some amino-terminal sequenceanalysis should be given, if at all possible, alongwith the results of a database search for similarsequences (UNIT 2.1). Functional proteins shouldbe demonstrated to have the appropriate func-tion, and for enzymes detailed kinetic charac-terization is appropriate. Ultimately the fullthree-dimensional structure of the protein maybe determined, which will require crystals: anysuccessful crystallization attempts should bereported.

THE PROTEIN PURIFICATIONLABORATORY

The requirements for a protein purificationlaboratory cannot be exactly formulated be-cause they depend greatly on the types andamounts of proteins being isolated. To cover alleventualities, it would be necessary to have oneset of equipment to deal with submicrogramquantities and another set to deal with multi-gram quantities—a range of around 108! Onelaboratory dedicated to protein purificationmay not need small-scale equipment if, forexample, it works with plasma proteins that arealways available in large quantities. Anothermay have all the latest in high-performanceequipment but not be able (nor need) to handlequantities of protein in excess of a few milli-grams.

If it is assumed that neither extreme in quan-tity is to be attempted, and that the laboratoryis handling a variety of protein types andsources, then certain basic pieces of equipmentare needed. Obtaining the starting material andmaking an extract of it require homogenizationequipment and centrifuges to remove insolubleresidues. Preliminary fractionation, when start-ing with a crude extract of tissue or cells,

requires equipment and materials that will notbecome clogged by particulates. Adsorbentsand similar materials used at the first stepshould be relatively inexpensive so that whenperformance falls off after a few uses, owing tointransigent impurity buildup, they can be dis-carded. It is also relevant that a larger amountis handled at the initial step than later steps;therefore, reagent expense can be an importantconsideration. After the first one or two steps,the sample should be sufficiently clean andclear to enable use of high-performance equip-ment.

High-performance liquid chromatography,or HPLC, is a term with a variety of meanings.To some it refers exclusively to reversed-phasechromatography; to others it includes all sortsof chromatography provided that the equip-ment is fully automated and high-performanceadsorbents are used. A high-performance sys-tem designed specifically for proteins—intro-duced by Pharmacia Biotech (see SUPPLIERS AP-

PENDIX) called Fast Protein Liquid Chroma-tography, or FPLC—uses standard proteinchromatographies such as ion exchange, hydro-phobic interaction, and gel filtration. Scaleupis possible with larger equipment based on theFPLC design, so that laboratory developmentcan be quickly translated to large-scale produc-tion. FPLC is designed to separate proteins intheir native active configuration, whereas re-versed-phase HPLC often causes at least tran-sient denaturation during adsorption and elu-tion. Reversed-phase HPLC has a high resolv-ing power, but it is best suited to peptides andproteins smaller than ∼30 kDa. Chromatogra-phy run with older-style low-pressure adsor-bents is sometimes referred to as “low-perform-ance” or “open-column” chromatography; nei-ther of those descriptions is necessarilyaccurate. Simple fraction collector and moni-toring equipment is needed. This equipmentwill be used for larger-scale operations (tens ofmilligrams of protein and upward), probably atan earlier stage in the protocol than with HPLC.

Various columns, both prepacked with pro-prietary adsorbents and empty for self-packing,will be needed, with the sizes and types depend-ing on the scale of operations. Several anion-exchange columns (different sizes), one or twocation-exchange columns, and gel-filtrationmedia are essential, along with a range of alter-native adsorbents such as hydrophobic interac-tion materials, dyes, hydroxyapatite, and chro-matofocusing and specialist affinity media.

Fully equipped protein purification labora-tories should also have preparative electropho-


1.1.5


resis and isoelectric focusing apparatuses forrare occasions when other techniques fail togive sufficient separation.

In addition to equipment used in the actualfractionation processes, a variety of other itemsare needed. In particular it should be possibleto change buffers quickly and to concentrateprotein solutions with ease. These operationsrequire such things as dialysis membranes (AP-

PENDIX 3B), ultrafiltration cells, and gel-exclu-sion columns of various sizes (UNIT 8.3).

Finally, equipment for assaying and analyz-ing the preparations is needed. Most suchequipment is fairly standard in biochemicallaboratories and includes spectrophotometers,scintillation counters, analytical gel and capil-lary electrophoresis apparatuses, immunoblot-ting materials, and immunochemical reagents.A listing of standard equipment is found inAPPENDIX 2D.

Literature CitedCohn, E.J., Strong, L.E., Hughes, W.L., Mulford,

D.J., Ashworth, J.N., Melin, M., and Taylor, H.L.1946. Preparation and properties of serum andplasma proteins. IV. A system for the separationinto fractions of the proteins and lipoproteincomponents of biological tissues and fluids. J.Am. Chem. Soc. 68:459-475.

Key ReferencesDeutscher, M.P. (ed.) 1990. Guide to protein purifi-

cation. Methods Enzymol. 182:1-894.

Extensive collection of purification methods withsome general protocols and examples.

Janson, J.-C. and Ryden, L.G. 1989. Protein Purifi-cation: Principles, High Resolution Methods,and Applications. VCH Publishers, New York.

A useful collection of methods and examples.

Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recov-ery Processes for Biological Materials. JohnWiley & Sons, New York.

A useful introduction to the problems of large-scalemethods.

Kenny, A. and Fowell, S. (eds.) 1992. Practicalprotein chromatography. Methods Mol. Biol.11:1-327.

Extensive descriptions of affinity chromatographictechniques with protocols and recipes.

Scopes, R.K. 1993. Protein Purification, Principlesand Practice, 3rd ed. Springer-Verlag, New Yorkand Heidelberg.

General principles of all the main techniques usedin purifying proteins. A useful laboratory handbook;does not include recipes or procedures for specificproteins.

Contributed by R.K. ScopesLa Trobe UniversityBundoora, Australia


1.1.6

Overview ofProtein


UNIT 1.2Strategies for Protein Purification

CLASSIFICATION OF PROTEINSAs with most heterogeneous collections of

things, proteins can be classified in severaldifferent ways, such as by function, by struc-ture, or by physicochemical characteristics.Each protein species consists of identical mole-cules with exactly the same size, amino acidsequence, and three-dimensional shape. In thisway a solution of a mixture of proteins differsfrom a solution of synthetic polymers orsheared DNA, both of which contain a com-plete spectrum of possible sizes centeredaround the average. The protein mixture hasonly discrete sizes of molecules correspondingto each type of protein present. Although wecould classify proteins by size, it would be oflimited use, as there is usually no obviousrelationship between size and function.

A more useful structural classification takesinto consideration shape and oligomeric struc-ture (Table 1.2.1). In part, structure reflectsbiological location and origin. Simple, fairlyrigid protein molecules occur in the extracellu-lar environment, more complex and readilydeactivated molecules are found intracellularly,and hydrophobic proteins are associated withmembranes.

Classification by function is more relevant(Table 1.2.2). Proteins can be simply stores ofamino acids, can be structural, or can havespecific binding functions. The most “func-

tional” proteins are enzymes, which have bothbinding and catalytic roles. In part, this reflectsthe degree to which the detailed structure is arequirement for the protein’s function, whichin turn relates to conservation of structurethrough evolution. But as with every attempt atclassification, there are going to be examplesthat do not fit the pattern well. Most proteinsof interest to the pharmaceutical industry be-long to the general class of binding proteins,for instance, hormones [e.g., insulin and bovinesomatostatin (BST)], viral antigens (e.g., hepa-titis B antigen), growth factors [e.g., interfer-ons, interleukins and colony-stimulating fac-tors (CSFs)], and antibodies.

STRATEGIES FOR PROTEINPURIFICATION

Soluble Extracellular ProteinsThe source of soluble extracellular proteins

is the extracellular medium, whether it be ananimal source such as blood or spinal fluid, ora culture medium in which bacterial, fungal,animal, or plant cultures have been grown.Generally these do not contain a large numberof different proteins (blood is an exception),and the desired protein may be a major compo-nent, especially if produced as the result ofrecombinant expression. Nonetheless, the pro-tein in the starting material may be quite dilute,

Table 1.2.1 Classification of Proteins by Structural Characteristics

Structuralcharacteristic Examples Comments

Monomeric Lysozyme, growth hormone Usually extracellular; often havedisulfide bonds

Oligomeric

Identical subunits Glyceraldehyde-3-phosphatedehydrogenase, catalase, alcoholdehydrogenase, hexokinase

Mostly intracellular enzymes;rarely have disulfide bonds

Mixed subunits Aspartate carbamoyltransferase,pertussis toxin

Allosteric enzymes; differentsubunits have separate functions

Membrane-bound

Peripheral Mitochondrial ATPase, alkalinephosphatase

Readily solubilized by detergents

Integral Porins, cytochromes P450, insulinreceptor

Require lipid for stability

Conjugated Glycoproteins, lipoproteins,nucleoproteins

Many extracellular proteinscontain carbohydrate


1.2.1


and a large volume may therefore need to beprocessed. The starting fluid may also containmany compounds other than proteins, whosebehavior must be taken into account. The firststage should aim mainly to reduce the volumeand get rid of as much nonprotein material aspossible; some protein-protein separation isalso useful, but not essential. No general rulescan be given, but a batch adsorption methodusing an inexpensive material such as hydroxy-apatite, ion-exchange resin, immobilized metalaffinity chromatography (IMAC) medium, oraffinity adsorbent is best, if feasible. Followingthe first step, the sample should be in a formthat is amenable to standard purification proc-esses such as precipitation and column chroma-tography (Chapter 8).

Intracellular (Cytoplasmic) ProteinsTo obtain soluble intracellular proteins

(which are mainly enzymes), cells must bebroken open or lysed to release their solublecontents. The ease with which cell disruptioncan be accomplished varies considerably; ani-mal cells are readily broken, as are many bac-teria, but plants and fungi have tough cell walls.Methods for obtaining cell extracts are given inChapter 4. The macromolecular soluble con-tents of cells are mainly proteins, with nucleicacids as a minor but significant component.Bacterial extracts may be viscous unless DNaseis added to break down the long DNA mole-cules. Although chromatographic procedurescan be applied to crude extracts, valuable high-performance materials should not be employedin the first step, as there are always compounds,including unstable proteins, that may bind tothem and be difficult to remove.

Membrane-Associated ProteinsThere are two approaches to isolating a

membrane-associated protein. In one method,the relevant membrane fraction can first beprepared and then used to isolate the protein.Alternatively, whole tissue can be subjected toan extraction that solubilizes the membranesand releases the cytoplasmic contents as well.The former is much better in that purificationis accomplished by isolating the membranes:the specific activity of the solubilized mem-brane fraction will be much higher than in thesecond method. However, the process of puri-fying the membrane fraction may lead to sub-stantial losses, and it may be difficult to scaleup. If total recovery of the protein is moreimportant than purity, a whole-tissue extract islikely to be more appropriate. Although thismeans that a greater degree of purification isneeded, the fact that membrane proteins have,by definition, properties somewhat differentfrom those of cytoplasmic proteins permitssome very effective purification steps (e.g.,hydrophobic chromatography or fractionalsolubility separation).

Peripheral membrane proteins are onlyloosely attached and may be released by gentleconditions such as high pH, EDTA, or low(nonionic) detergent concentrations. Once insolution, some peripheral proteins no longerrequire the presence of detergent to maintaintheir solubility. Integral membrane proteins aremuch more difficult—they require high con-centrations of detergent for solubilization (i.e.,complete solubilization of the membrane isneeded to release them) and generally are nei-ther soluble nor stable in the absence of deter-gent. It is sometimes necessary to maintain

Table 1.2.2 Classification of Proteins by Function

Function Examples

Amino acid storage Seed proteins (e.g., gluten), milk proteins(e.g., casein)

Structural Inert Collagen, keratin With activity Actin, myosin, tubulin

Binding Soluble Albumin, hemoglobin, hormones Insoluble Surface receptors (e.g., insulin receptor),

antigens (e.g., viral coat proteins) With activity Enzymes, membrane transporters (e.g.,

amino acid uptake systems, ion pumps)


1.2.2

Strategies forProtein

Purification

natural phospholipids in association with theproteins in order to maintain activity. Eventhough the final objective may not require ac-tivity (e.g., amino acid sequencing), there is aneed for some sort of assay during the purifi-cation process to determine where the proteinis. If a particular band on a gel is known to bethe desired protein, then no other assay isneeded and loss of bioactivity can be allowed.

Purification processes may be affected bythe presence of detergents. The problem ofassociation with detergent micelles makes pu-rifying integral membrane proteins difficult;the close association of the different proteinsoriginating from membranes often results invery poor separation in conventional fractiona-tion procedures.

Insoluble ProteinsNatural proteins that are insoluble in normal

solvents are generally structural proteins,which are sometimes cross-linked by posttrans-lational modification. The first stage of purifi-cation is obvious—it involves extracting andwashing away all proteins that are soluble,leaving the residue containing the desired ma-terial. Further purification in a native state,however, may be impossible; extracting awayother proteins using more vigorous solvents orattempting to solubilize the target protein maydestroy the natural structure. Cross-linked pro-teins such as elastin or aged collagen cannot bedissolved without breaking the cross-links, andthe individual proteins may even be cross-linked together.

Insoluble Recombinant Proteins(Inclusion Bodies)

A major new class of insoluble proteins arerecombinant proteins expressed (usually in Es-cherichia coli) as inclusion bodies. These aredense aggregates found inside cells that consistmainly of a desired recombinant product, butin a nonnative state. Inclusion bodies may formfor a variety of reasons, such as insolubility ofthe product at the concentrations being pro-duced, inability to fold correctly in the bacterialenvironment, or inability to form correct, orany, disulfide bonds in the reducing intracellu-lar environment. Their purification is simple,since the inclusion bodies can be separated bydifferential centrifugation from other cellularconstituents, giving almost pure product; theproblem is that the protein is not in a nativestate, and is insoluble. Some methods for ob-taining an active product from inclusion bodiesare described in UNIT 6.3.

Soluble Recombinant ProteinsRecombinant proteins that are not expressed

in inclusion bodies either will be soluble insidethe cell or, if using an excretion vector, will beextracellular (or, if E. coli is the host, possiblyperiplasmic). They can be purified by conven-tional means. In some systems, expression is sogood that the desired product is the major pro-tein present and its purification is relativelysimple. In systems where the expression levelis low, the purification process can be tedious,though easier, it is hoped, than isolation fromthe natural source. It should be rememberedthat a procedure developed for isolating a pro-tein from natural sources may not work suc-cessfully with the recombinant product, be-cause the nature of the other proteins presentinfluences many fractionation procedures.

Because of the difficulties often experiencedin purifying recombinant proteins, a variety ofvector systems (see UNIT 6.1 and Sassenfeld,1990, for some examples) have been developedin which the expressed prod-uct is a fusionprotein containing an N-terminal polypeptidethat simplifies purification. Such “tags” can besubsequently removed using a specific pro-tease. A further advantage is that the expressionlevel is dictated mainly by the transcription andtranslation signals for the fusion portion of theprotein, which are optimized. Tags used includeproteins and polypeptides for which there is aspecific antibody, binding proteins that willinteract with columns containing a specific li-gand, polyhistidine tags with affinity to immo-bilized metal columns, sequences that result inbiotinylation by the host and enable purifica-tion on an avidin column, and sequences thatconfer insolubility under specified conditions(UNIT 6.1 & UNIT 6.5).

Unstable proteins may be modified by themolecular biological technique of site-directedmutagenesis to remove the site of instability—for instance, an oxidizable cysteine. Such tech-niques are appropriate for commercial produc-tion of proteins, but may of course alter naturalfunctioning parameters. Increased thermo-stability can be one modification, although it isnot easy to predict mutations that will improvethat parameter. Thermostable proteins originat-ing from thermophilic bacteria do not needstructural modification and, if expressed inlarge amounts, can be purified satisfactorily inone step by simply heat-treating the extract at70°C for 30 min, which denatures virtually allthe host proteins (e.g., see Oka et al., 1989).

The host bacteria used for production ofrecombinant proteins are usually Escherichia


1.2.3


coli, or Bacillus subtilis; they may expressproteins at 1% to over 50% of the cellularprotein, depending on such variables as thesource, promoter structure, and vector type.Generally the proteins are expressed intracel-lularly, but leader sequences for excretion maybe included. In the latter case, the protein isgenerally excreted into the periplasmic space,which limits the amount that can be produced.Excretion from gram-positive species such asB. subtilis sends the product into the culturemedium, with little feedback limitation on totalexpression level.

Literature CitedOka, M., Yang, Y.S., Nagata, S., Esaki, N., Tanaka,

M., and Soda, K. 1989. Overproduction ofthermostable leucine dehydrogenase of Bacillusstearothermophilus and its one-step purificationfrom recombinant cells of Escherichia coli.Biotechnol. Appl. Biochem. 11:307-316.

Sassenfeld, H.M. 1990. Engineering proteins forpurification. Trends Biotechnol. 8:88-93.



Extensive collection of purification methods withsome general protocols and examples.



Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recovery Processes for Biological Materials.John Wiley & Sons, New York.






Contributed by R.K. ScopesLa Trobe UniversityBundoora, Australia


1.2.4

Strategies forProtein

Purification

UNIT 1.3Protein Purification Flow Charts

Protein purification flow charts are pre-sented to give a broad outline of the methodsused for different types of proteins. They cannotgive any detail, as the process appropriate foreach protein will have its own variations at eachstage. In most cases, the first stage is to obtaina solution containing the desired protein, afterwhich it can be dealt with by the many separa-tion techniques described in the followingchapters. In some cases the insolubility of thedesired protein can be exploited by removingsoluble fractions. Purification procedures arecommonly divided into three stages: (a) theprimary steps, which deal with crude mixturesof proteins and other molecules present in theraw material; (b) the secondary processing,which generates a product near to homogene-ity; and (c) the polishing steps, which removeminor contaminants, a process that is especiallyimportant for therapeutic proteins.

SOLUBLE RECOMBINANTPROTEINS

Proteins expressed in a recombinant mannermay be (1) soluble in the cytoplasm, (2) insol-uble as inclusion bodies (see section on Insol-uble Recombinant Proteins), (3) excreted fromthe cells into the culture medium, (4) excretedinto the periplasmic space (e.g., in gram-nega-tive bacteria), or (5) associated with organellesor membrane fractions. In addition they maybe expressed (6) as the normal, mature, natu-rally occurring protein, (7) containing a naturalleader peptide that would normally be proc-essed, (8) as a fusion protein with a peptide thatis not natural to the protein, or (9) lackingglycosylation or other post-translational modi-fication, or incorrectly modified. Possibilities(1) to (5) affect the method of extraction usedto obtain the starting material for purification.Cases (6) to (9) can affect the methods used forpurification.

The scheme for purifying soluble recombi-nant proteins is outlined in Figure 1.3.1. Thefirst stage is to obtain a clarified solution con-taining the desired protein, with as little in theway of unwanted proteins as possible. For sol-uble cytoplasmic proteins, case (1), it is notnormally possible to exclude any significantamount of unwanted soluble proteins, but incases (2) to (5) the compartmentalization awayfrom the cytoplasm allows such separation inthe initial stage.

It may be necessary to carry out a concen-

tration step before proceeding, especially if theprotein has been excreted into the culture me-dium. Normally ultrafiltration is used, althoughother techniques are possible, especially if theextract contains particulates that block ultrafil-tration membranes.

Recombinant expression in the cytoplasmof bacteria, followed by extraction via total celldisruption, results in large amounts of nucleicacids being solubilized with the protein. A num-ber of treatments to remove nucleic acids arepossible. Streptomycin is used to precipitateribosomal material, and cationic polymers suchas protamine (a basic protein) and polyethyl-enimine will form insoluble complexes (at lowionic strength) with nucleic acids. In addition,viscosity caused by DNA can be reduced byadding small amounts of DNase.

INSOLUBLE RECOMBINANTPROTEINS

It has been found that many proteins ex-pressed in bacteria (mainly in Escherichia coli)do not fold correctly, and as a result aggregationoccurs, leading to large insoluble inclusionbodies within the cytoplasm of the cells (seeChapter 6). Although this creates major diffi-culties in obtaining satisfactory amounts ofactive native product, it greatly simplifies theinitial stage of purification.

The purification scheme for recombinantinsoluble proteins is outlined in Figure 1.3.2.After cell disruption, inclusion bodies can beobtained in a fairly pure state by differentialcentrifugation. They must now be solubilized,however, and the active protein generated byencouraging correct folding. Solubilization isusually accomplished with guanidine hydro-chloride and/or urea, and thiols such as 2-mer-captoethanol or glutathione are included to dis-rupt any disulfides that have formed and pre-vent more from forming. Folding the proteincorrectly may require a variety of additions tothe solution, as well as slow removal of thedenaturant. The latter can be carried out bysimple dilution or by dialysis. Folding occursbest at low protein concentrations, so dilutionmay be adequate. If the native protein doescontain disulfides, then it is important to createredox conditions such that some (but not exces-sive) oxidation of thiols can occur. A combina-tion of oxidized and reduced glutathione iscommonly used. In addition, the action of theenzyme protein disulfide isomerase, which can


1.3.1


make and unmake disulfides by exchange reac-tions, has been found to be beneficial in manycases. If the native protein is of intracellularorigin, it probably will not contain disulfides;it will, however, contain cysteines, so a fullreducing potential should be maintained. Spe-cific methodology is discussed in UNIT 6.5.

Not all proteins can fold unassisted by othercellular components. Chaperonins are proteinswhose role is to refold denatured proteins suchas those that form during heat shock (Zeilstra-

Ryalls et al., 1991). The most studied, and justbecoming commercially available as of 1995,are the E. coli chaperonins GroEL and GroES,both of which are needed, together with ATP,to renature many proteins. Proline residuescan adopt two isomeric conformations in pro-teins, and the wrong conformation is switchedto the correct one by the enzyme prolylisomerase, aiding the process of protein fold-ing. At present these are not large-scale pros-pects, both because of the cost of the chap-

cells withprotein soluble in cytoplasm

cells withprotein excreted

to medium

cells with proteinin periplasmic

space

cells withprotein organelle-

associated

break up cells;centrifuge orultrafilter to

removeinsolubles

centrifuge or filterto remove

cells

gently treatwith lysozyme to

minimize celllysis

separate organelles;perform differential

centrifugation

treat to removenucleic acids

remove protoplastsand cell debris

extract tosolubilize proteins,remove insolubles

performconcentration

step

starting material:soluble fraction containing

the protein

perform purification steps

use salt fractionation,ion-exchange chromatography,hydrophobic chromatography,

affinity and pseudoaffinitychromatography, gel filtration,

or other less conventional steps(i.e., preparative electrophoresis)

for tagged fusion protein,use affinity column

chromatography andpolishing steps

purified protein

Figure 1.3.1 Purification scheme for soluble recombinant proteins, which may be excreted orlocated in the periplasm, in the membrane fraction, or most commonly the cytoplasm. The first stepis to obtain an extract containing the desired protein in soluble form. After this, conventionalpurification steps may be carried out, or affinity purification of tagged fused proteins can beperformed.


1.3.2

ProteinPurificationFlow Charts

eronins and because the agents operate best invitro at very low protein concentrations.

Once the proteins are folded, the purificationprocess consists of removing small amounts ofstill incorrectly folded protein plus any otherhost proteins that were trapped with the originalinclusion bodies. The former may be difficult,since incorrectly folded species have a size andcharge similar to those of the correct product.However, subtle differences arising from thefolded conformation can be exploited by chro-matographic techniques. In ideal cases immu-noaffinity techniques using antibodies specificfor either the incorrectly folded form or thecorrect one can be used to resolve the mixture.

SOLUBLE NONRECOMBINANTPROTEINS

There are so many sources of soluble pro-teins that it is not possible to give a completeoverview of methods used to obtain startingextracts from which a desired protein can beisolated. The sources can be classified as eithermicroorganisms, plants, or animals, as shownin Figure 1.3.3, but these in turn should besubdivided according to how the starting ex-tract is obtained. In particular there is a distinc-tion between extracellular and intracellularproteins. With the latter it is necessary to disruptthe cells and release the proteins, whereas withthe former, if the extracellular fluid can beobtained directly, there need be no contamina-tion with intracellular proteins. Extracellular

cells containing protein

disrupt cells

perform differential centrifugation

wash inclusion bodies

dissolve in denaturing agents

dilute with buffer or dialyzeto dilute denaturing agents

add appropriate reducing agentsand/or folding factors

concentrate

perform purification procedure:remove unwanted proteins and

incorrectly folded species(e.g., by ion-exchange chromatography,

immunoaffinity methods, gel filtration)

purified protein

Figure 1.3.2 Purification scheme for insoluble recombinant proteins that are produced as inclu-sion bodies in the cytoplasm of host cells. The cells must be broken open, and then the insolubleinclusion bodies are separated by differential centrifugation. Solubilization is achieved by the useof denaturing solvents, and renaturation of the dissolved protein occurs on removal of thedenaturant. Further polishing steps will be needed to remove small amounts of contaminatingproteins as well as incorrectly folded species. Additional information can be found in UNIT 6.3-6.5.


1.3.3


sources include microorganism culture me-dium, plant and animal tissue culture medium,venoms, milk, blood, and cerebrospinal fluids.Soluble proteins may also occur within organ-elles such as mitochondria; these may be bestobtained by first isolating the organelle, thendisrupting it to release the contents.

The starting extract normally contains be-

tween 5 and 20 mg protein per milliliter, thoughlesser concentrations can be dealt with, espe-cially if working on a small scale. It may benecessary to include a concentration step beforestarting the purification process in order toapproach that level. There are exceptions toevery rule, however, and very high proteinconcentrations can be handled, for example,

microorganisms plants animals

disrupt cells,e.g., using French press,bead mill, or sonication

with 5-10 vol buffer

disrupt cells,e.g., using French pressor by grinding with sand

using 1-2 vol buffer

homogenize with2-5 vol buffer ormince and stir

with buffer

treat to removenucleic acids

treat to removephenols

extracellularproteins starting extract

(5-20 mg/ml protein)

perform initial fractionation:use salt fractionation, precipitation

with organic solvents,affinity methods, and/ortwo-phase partitioning

perform secondary fractionation:use ion-exchange chromatography,

hydrophobic chromatography,affinity methods, other adsorbents,

and/or gel filtration

perform polishing step:use HPLC– reversed phase,

HPLC– ion exchange,or isoelectric focusing

purified protein

centrifuge

Figure 1.3.3 Purification scheme for soluble proteins present in their natural host cells. Cells mustbe disrupted to release the proteins, usually in the presence of 2 to 10 ml of a suitable buffer pergram weight. After removal of insoluble material, the process will generally require several steps,using various standard fractionation procedures in a suitable order. For production of highly pureprotein, a final polishing step may be required to remove final trace contaminants. Additionalinformation can be found in UNIT 6.2.


1.3.4


with two-phase partitioning (Walter and Jo-hansson, 1994). When isolating proteins on alarge scale, the volumes being manipulatedbecome of increasing concern, so maximizingprotein concentration can be an important aim.The starting extract should be clarified, usuallyby centrifugation; on a large scale, ultrafiltra-tion methods are becoming more widely used.Pretreatment of certain extracts to remove ex-

cessive amounts of nucleic acids, phenolics,and lipids may be necessary in order to obtainan extract that is amenable to standard frac-tionation procedures.

Fractionation procedures can somewhat ar-bitrarily be divided into three steps: initial frac-tionation, secondary fractionation, and polish-ing. Initial processing, which deals with a largeamount of extract that is not all protein, may

whole tissue

homogenizewithout detergent;

centrifuge

disrupt cells; gentlyisolate organelles

supernatant residue

homogenizewith detergent;

centrifuge

supernatant containingmembrane-associated

proteins

residue containinginsoluble proteins

perform primary fractionation:use hydrophobic chromatography,precipitation with organic solvents,

ion-exchange chromatography,and/or phase partitioning

perform secondary fractionation:use ion-exchange chromatography,

affinity methods, and/orgel filtration

perform polishing step:use HPLC or isoelectric focusing

purified protein

suspend and extractwith other solvents

(e.g., ethanol, urea, or SDS)

insolubleresidue

purifiedprotein

Figure 1.3.4 Purification scheme for membrane-associated and poorly soluble proteins (nonre-combinant). An initial purification can be achieved by isolation of organelles containing the desiredprotein. Membrane proteins are normally solubilized with a nonionic detergent, although looselyassociated proteins may be extracted without detergent at high pH, with EDTA, or with smallamounts of an organic solvent such as N-butanol. Normal fractionation procedures may need somemodification if the detergent is required throughout to maintain the integrity of the protein.


1.3.5


involve materials that become soiled and un-able to be used many times. Consequently,preferred methods do not require expensivereagents or adsorbents such as are used in high-performance liquid chromatography (HPLC).Classic salt fractionation and the less-used or-ganic solvent fractionation can achieve, if nota high degree of purification, a useful level ofconcentration and removal of much unwantednonproteinaceous material. Alternatively, ahighly selective affinity procedure may be usedas the first step, but only if the affinity materialis inexpensive to make and/or the extract is asimple, clear solution, as opposed to a turbidwhole-cell homogenate.

Secondary processing achieves the main pu-rification, and may involve two or more stepsin difficult situations. Ion-exchange and hydro-phobic-interaction chromatography, gel filtra-tion, and affinity techniques (see Chapter 8 andChapter 9) are the main procedures. Finally, itmay be necessary to remove traces of contami-nants by “polishing,” using high-resolutionprocedures such as reversed-phase HPLC (UNIT

11.6) and isoelectric focusing (IEF; UNIT 10.2 &

UNIT 10.4). Because every protein has uniquecharacteristics, it is impossible to make generalstatements about procedures to be followed.

MEMBRANE-ASSOCIATED ANDINSOLUBLE NONRECOMBINANTPROTEINS

Proteins that are not physiologically solublecan be purified after extracting and removingsoluble proteins, thereby achieving a substan-tial degree of purification at the extraction step(Fig. 1.3.4; also see UNITS 6.1 & 6.2). To carry outa purification it is nearly always necessary toobtain the desired protein in a soluble form,which will often require the addition of solubi-lizing agents such as detergents. Some proteinsremain insoluble even with detergent treatment,and so can be substantially purified by remov-ing the soluble fractions. Some membrane-as-sociated proteins become partly solubilizedduring breaking up of the tissue, and recoveryin the particulate fraction may be poor. In suchcases it may be best to solubilize the wholetissue by including detergent in the homogeniz-ing buffer. Extraction of insoluble residues us-ing detergents can be done differentially; someproteins are released at low detergent concen-tration, whereas others require complete solu-bilization of the membrane fraction. Suitabledetergents include nonionic (e.g., Triton) andweakly acidic types (e.g., cholic acid deriva-

tives). Strongly acidic detergents such as sulfateesters (e.g., sodium dodecyl sulfate) usuallyresult in denaturation.

Detergents can be removed either by adsorp-tion of the protein on a column, and subsequentelution without detergent, by use of specialdetergent-adsorbing beads, or even by extrac-tion with nonmiscible organic solvents inwhich the detergent partitions. On the otherhand, many membrane proteins require thepresence of detergent at all times in order toremain in solution and in a native conformation.These include most integral membrane pro-teins, for example, cytochrome: P450, trans-membrane receptors, and transporters. Themost sensitive proteins require a particularcombination of natural lipids (in addition to thedetergent) to maintain structural integrity. Pu-rification methods include most of those usedfor soluble proteins, but some techniques arenot recommended if detergent is needed at alltimes. For instance, ammonium sulfate precipi-tation will often cause a detergent-protein com-plex to come out of solution and float ratherthan sink on centrifugation—this can be useful,but the “floatate,” when redissolved, may havea high detergent content. Hydrophobic chroma-tography can be very useful, as membrane pro-teins are naturally hydrophobic.

Integral membrane proteins that are com-pletely insoluble in normal detergents may besolubilized by denaturation using compoundssuch as sodium dodecyl sulfate and guanidinehydrochloride. Some cross-linked proteinssuch as elastin are not soluble without disrup-tion of the covalent linkages.

Literature CitedWalter, H. and Johansson, G. (eds.) 1994. Aqueous

two-phase systems. Methods Enzymol. 228:1-725.

Zeilstra-Ryalls, J., Fayet, O., and Georgopoulos, C.1991. The universally-conserved GroE (Hsp60)chaperonins. Annu. Rev. Microbiol. 45:301-325.



Extensive collection of purification methods, withsome general protocols and examples.




1.3.6


Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Recov-ery Processes for Biological Materials. JohnWiley & Sons, New York.






Contributed by R. K. ScopesLa Trobe UniversityBundoora, Australia


1.3.7


UNIT 1.4Purification of Glutamate Dehydrogenasefrom Liver and Brain

Because of the wide variety of procedures that may be used to purify any given enzyme,it is not possible to provide a single protocol that includes them all. In this unit twoprotocols for the purification of glutamate dehydrogenase (GDH)—also referred to asL-glutamate-NAD(P)+ oxidoreductase (deaminating; EC 1.4.1.3:GDH)—have been se-lected as examples because they present two very different approaches to the purificationof the enzyme. In the first (see Basic Protocol 1), affinity chromatography using a columnconsisting of the allosteric inhibitor guanosine 5′-triphosphate (GTP) bound to Sepharoseis described. Support protocols detail the preparation of the GTP-Sepharose needed forthis procedure (see Support Protocol 1), assessment of incorporation of GTP into theGTP-Sepharose gel, if necessary (see Support Protocol 2), the activation of Sepharose 4Bwith CNBr, if necessary (see Support Protocol 3), and reuse and equilibration of thechromatography media (see Support Protocols 4 and 5).

An alternative purification procedure (see Alternate Protocol) uses a bifunctional ligandcomposed of two NAD+ molecules linked together by a spacer arm to precipitate theenzyme. Protocols for the synthesis of the bis-NAD compound (see Support Protocol 7),resolution of NAD+ derivatives (see Support Protocol 8), and instructions for a pilotaffinity study (see Support Protocol 9) complement this alternative approach.

Of value to both studies are methods for concentrating the protein product (see SupportProtocol 6), determining the protein concentration (see Basic Protocol 2), and assayingfor GDH activity (see Basic Protocol 3). Although some of these techniques can be foundelsewhere in this manual, instructions are included in this unit that are specific to thesystems mentioned here.

As discussed below, some tissues, including the brain but not the liver, contain anadditional isoenzyme of glutamate dehydrogenase (GLUD2) as a relatively minor com-ponent. The procedures described here are for purifying the major form (GLUD1).

Since this unit is concerned with the purification of mammalian glutamate dehydrogenase,a detailed review of its properties and functions is not appropriate. However, some of theessential properties are briefly summarized below. Further details can be found in thereferences cited. References to earlier work on the enzyme can be found in Tipton andCouée (1988) and are not repeated here. GDH catalyzes the reversible oxidative deami-nation of glutamate to 2-oxoglutarate (α-ketoglutarate). Either NAD+ or NADP+ can serveas cofactor, although the kinetic and regulatory behavior depend on which is used. Thenative enzyme is a hexamer of six identical subunits, each consisting of 505 amino acids,with approximate relative molecular mass of 56,700. However, at high protein concen-trations, the hexamers may aggregate further to form high-molecular-weight polymers.The crystal structure of the enzyme has been recently determined (Peterson and Smith,1999; Smith et al., 2001).

The enzyme activity is allosterically modulated by a range of metabolites, includingadenosine, guanosine, and inosine di- and trinucleotides, as well as L-leucine (see Couéeand Tipton 1989a). The exact behavior of these compounds depends on the conditionsunder which activity is measured and whether NAD+ or NADP+ is used as cofactor. Thekinetic behavior in the direction of glutamate oxidation is complex. Substrate inhibitionby high concentrations of 2-oxoglutarate occurs in the reaction leading to NADPHoxidation, but this is not apparent in the reaction pathway leading to NADH oxidation.For that reason, activity determinations are usually performed in the direction of 2-oxo-glutarate amination with NADH as the cofactor. Phospholipids, such as cardiolipin, also

Supplement 29

Contributed by Martha Motherway, Keith F. Tipton, Alun D. McCarthy, Ivan Couée, and Jane IrwinCurrent Protocols in Protein Science (2002) 1.4.1-1.4.34Copyright © 2002 by John Wiley & Sons, Inc.

1.4.1


affect enzyme activity (Couée and Tipton, 1989b) and the enzyme is also inhibited byantipsychotic drugs such as perphenazine (Couée and Tipton, 1990a,b; Yoon et al., 2001).

GDH is localized in the mitochondrial matrix, although small amounts of the activity havebeen detected associated with microtubules (Rajas and Rousset, 1993) and endoplasmicreticulum (Lee et al., 1999). A nuclear-associated activity has also been reported(McDaniel, 1995) and the enzyme has been found to be capable of functioning as anRNA-binding protein (Preiss et al., 1993).

In addition to the ubiquitously distributed GDH isoenzyme (GLUD1), which is encodedby a gene on chromosome 10, brain, retina, and testis tissue also contain a second formof the enzyme (GLUD2), encoded on the X chromosome (see Shashidharan et al., 1994).This isoenzyme differs in its kinetic and regulatory properties from the major form ofGDH (see e.g., Cho et al., 1995; Shashidharan et al., 1997). The possible functions ofthese two GDH isoenzymes in brain have been discussed by Plaitakis and Zaganas (2001).

NOTE: Unless otherwise stated, all required chemicals can be obtained from Sigma-Aldrich or BDH. They should be of the highest purity available.

BASICPROTOCOL 1

PURIFICATION OF OX BRAIN OR LIVER GLUTAMATEDEHYDROGENASE BY AFFINITY CHROMATOGRAPHY ONGTP-SEPHAROSE

The initial steps in this protocol involve the classical procedures of salt precipitation andion-exchange chromatography. These are then followed by affinity chromatography onGTP Sepharose. GTP is an allosteric inhibitor of glutamate dehydrogenase when NAD+

is used as the substrate (see McCarthy and Tipton, 1985; Couée and Tipton, 1989a). Theprocedure described here is that of McCarthy et al. (1980). The steps involved aresummarized in Figure 1.4.1.

Materials

Fresh ox (beef) brain (∼400 g) or liver (∼50 g) from slaughterhouse0.32 M sucrose (optional; if tissue samples are to be stored before use)Homogenization buffer (see recipe), 4°CAmmonium sulfate [(NH4)2SO4]20 mM and 200 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E), 4°CAffinity chromatography buffer (see recipe)400 mM KCl in affinity chromatography buffer (see recipe for buffer)Glycerol

ScissorsWaring blender or kitchen liquidizerRefrigerated centrifuge (e.g., Sorvall RC-5C)Dounce (hand-held) homogenizer with loosely-fitting pestle (clearance, ∼0.03 cm)250 ml or 500 ml polycarbonate centrifuge tubesDialysis tubing, 12,000 molecular weight cut-off (MWCO)Conductivity meterGradient mixer (e.g., UNIT 8.2)Chromatography columns (see Support Protocol 5 for equilibration; column

lengths may be varied somewhat according to availability): 40 × 3–cm packed to height of 30 cm with DEAE-cellulose (DE-52,

Whatman) and equilibrated with 20 mM sodium phosphate buffer, pH 7.4 (see APPENDIX 2E for buffer)

30 × 2.5–cm packed to height of 22 cm with Sephadex G-25 (Pharmacia Biotech) and equilibrated with affinity chromatography buffer (see recipe for buffer)

Supplement 29 Current Protocols in Protein Science

1.4.2

Purification ofGlutamate

Dehydrogenasefrom Liverand Brain

6 × 2–cm packed to height of 4 cm with ∼10 ml of hydrazide-Sepharose 4B and equilibrated with affinity chromatography buffer (see Support Protocol 1, steps 1 to 3 for resin; see recipe for buffer)

6 × 2–cm packed to height of 4 cm with GTP-Sepharose 4B and equilibrated with affinity chromatography buffer (see Support Protocol 1, steps 1 to 7 for resin; see recipe for buffer)

homogenize for 1 min in 3 volumes(w/v) of 4 mM sodium phosphate

buffer, pH 7.4, containing0.5mM EDTA and 0.1 mM PMSF

homogenize for 1 min in 500 mlof 4 mM sodium phosphatebuffer, pH 7.4, containing

0.5 mM EDTA and 0.1 mM PMSF

add solid (NH4)2SO4 (113 g/liter), stir for 20 min

add solid (NH4)2SO4 (188 g/liter), stir for 20 min

dialyse against 20 mM sodium phosphate buffer,pH 7.4 and apply to DEAE-cellulose column

gel-filter into affinity chromatography buffer;pass through hydrazide-Sepharose; and

apply to GTP Sepharose column

concentrate by ultrafiltration

purified glutamate dehydrogenase

centrifugediscard pellet

centrifugediscard supernatant

elute with 20 to 200 mM sodiumphosphate, pH 7.4, gradient

elute with 0 to 15 mM KCl gradientin affinity-chromatography buffer

dialyze against 20 mM sodiumphosphate buffer, pH 7.4

stage 1(homogenate)

stage 2(NH4)2SO4supernatant

stage 3(dialyzedpellet)

stage 4(DEAEeluate)

stage 5(GTP-Sepharoseeluate)

ox brain (400 g) ox liver (50 g)

Figure 1.4.1 Flow chart for the purification of ox liver and brain glutamate dehydrogenaseaccording to the Basic Protocol 1. Full details of the steps involved are given in the text.

Current Protocols in Protein Science Supplement 29

1.4.3


Additional reagents and equipment for dialysis (UNIT 4.4 and APPENDIX 3B),determining protein concentration (see Basic Protocol 2 and UNIT 3.4), assayingGDH activity (see Basic Protocol 3), and concentrating protein solutions byultrafiltration (see Support Protocol 6)

NOTE: Unless indicated otherwise, all steps are performed at 0° to 4°C in a cold room orrefrigerated cabinet.

Prepare and homogenize the tissue1. Obtain one ox brain (∼400 g) or liver (∼50 g) from a freshly slaughtered animal

transported to the laboratory on ice to be used as the starting material for thepurification.

The brain may be used fresh or stored at −70°C, using the procedure as follows: Place thefresh tissue in ice-cold 0.32 M sucrose solution and keep it in a −20°C freezer until it hasfrozen. Transfer to a −70°C freezer until required. Before use place the vessel containingthe frozen material in a water bath at 37°C and allow it to thaw, with occasional agitation.The larger amount of enzyme in ox liver permits the use of a smaller amount of startingmaterial (e.g., 50 g in the example purification shown here) to be used.

2a. For brain tissue: Divide the brain tissue into three equal portions, weigh, and suspendeach portion in 3 volumes (w/v) of cold homogenization buffer.

2b. For liver tissue: Weigh the liver tissue and suspend in 10 volumes (w/v) of coldhomogenization buffer.

3. Chop the tissue into smaller pieces using scissors, then homogenize in a Waringblender at full speed for 1 min. Retain a small aliquot of this homogenate fordetermining protein concentration by the biuret procedure (see Basic Protocol 2) andassaying GDH activity (see Basic Protocol 3).

A kitchen liquidizer can be used instead of a Waring blender. However, extra care is thenneeded with any biohazard material (see Critical Parameters) since kitchen liquidizers canrelease aerosols. These are generally less powerful than the Waring blender and it is thusnecessary to increase the homogenization time (e.g., 1.5 min using a Kenwood liquidizerat full speed).

The volumes given in the subsequent steps refer to the preparation of GDH from brain. Thecorresponding values for liver are given in Table 1.4.6 (see Critical Parameters andTroubleshooting).

Precipitate with ammonium sulfate4. Add solid (NH4)2SO4 slowly in four approximately equal amounts to the homogenates

while they are still in the blender, homogenizing for 1 min between each addition, toyield a final 20% saturated solution (113 g/liter). Continue homogenization foranother 30 sec at full speed after the final addition. For brain tissue, repeat thisprocedure with the other two tissue homogenates.

5. Combine the ammonium sulfate–treated homogenates in a beaker on ice and stir themixture for 20 to 30 min. Centrifuge 30 min at 14,000 × g, 4°C. Carefully decant thesupernatants and discard the pellets. Retain a small aliquot of this material fordetermining protein concentration by the biuret procedure (Basic Protocol 2) andassaying GDH activity (Basic Protocol 3).

6. Add 188 g/liter of solid (NH4)2SO4 slowly with continuous stirring. Stir the mixturefor an additional 20 to 30 min, then centrifuge again as described in step 5.

This results in 50% saturation with ammonium sulfate.


1.4.4



7. Decant and discard the supernatants. Resuspend the pellets in 20 mM sodiumphosphate buffer, pH 7.4, using a Dounce homogenizer, to yield a total volume of200 to 250 ml (160 ml for liver). Retain a small aliquot of this material for determiningprotein concentration by the biuret procedure (Basic Protocol 2) and assaying GDHactivity (Basic Protocol 3).

The ammonium sulfate pellet can be resuspended by careful stirring if a Dounce homoge-nizer is not available.

8. Dialyze (UNIT 4.4 and APPENDIX 3B) for 18 hr against 20 volumes (v/v) of cold 20 mMsodium phosphate buffer, pH 7.4, with at least two changes of buffer, using an MWCO12,000 dialysis membrane.

9. Centrifuge the material from the dialysis bag 30 min at 40,000 × g, 4°C, and retainthe supernatants. Resuspend the pellets in the 20 mM sodium phosphate buffer, pH7.4, and centrifuge again for 30 min at 40,000 × g, 4°C. Combine the supernatantswith those from the first centrifugation and discard the pellets. Retain a small aliquotof this material for determining protein concentration by the biuret procedure (BasicProtocol 2) and assaying GDH activity (Basic Protocol 3).

Extraction by resuspension and recentrifugation of the pellet after dialysis and centrifuga-tion increases the yield of enzyme, since otherwise some glutamate dehydrogenase activityis lost in the pellet.

As is common with salt precipitation in enzyme purification, this step does not give a highdegree of purification of glutamate dehydrogenase. However, it is an effective way ofreducing the volume of material for the subsequent steps, and it also removes somecontaminants.

Perform chromatography on DEAE-cellulose10. Pour the supernatant carefully onto the top surface of a 40 × 3–cm chromatography

column packed to 30 cm with DEAE-cellulose, equilibrated with 20 mM sodiumphosphate buffer, pH 7.4 (Support Protocol 5). After the protein solution has run intothe column, continue to pass buffer through it and determine the absorbance ofaliquots of the effluent at 280 nm (A280). Continue washing the column until the A280

of the effluent is <0.1.

11. Pass a 2-liter linear gradient from 20 to 200 mM sodium phosphate buffer, pH 7.4,through the column. Collect fractions (∼10 ml) and determine their A280 (see BasicProtocol 2), conductivity, and enzyme activities (see Basic Protocol 3). Pool thefractions containing glutamate dehydrogenase activity. Retain a small aliquot of thispooled material for determining protein concentration by the biuret procedure (BasicProtocol 2) and assaying GDH activity (Basic Protocol 3).

The combined active fractions require concentration before the next step; this can be doneeither by ultrafiltration or ammonium sulfate precipitation. The authors have used both ofthese procedures and they work equally well.

12a. Concentrate protein by ultrafiltration: Concentrate the combined fractions contain-ing GDH activity in an Amicon ultrafiltration cell using either an Ultracel AmiconYM 30 or Ultracel PLTK membrane, as described in Support Protocol 6, until thevolume remaining in the cell is reduced to ∼20 ml.

12b. Concentrate protein by ammonium sulfate precipitation: Add 312 g/liter solid(NH4)2SO4 slowly, over a period of 30 min with continuous stirring (this brings thesaturation to 50%). Continue stirring for a further 20 min, then centrifuge 20 min at14,000 × g, 4°C. Discard the supernatant and resuspend the pellet in ∼20 ml of 20mM sodium phosphate buffer, pH 7.4.


1.4.5


Perform chromatography on GTP-Sepharose13. Desalt the sample by passing the concentrated enzyme solution through a 30 × 2.5–cm

chromatography column packed to 22 cm with Sephadex G-25, equilibrated withaffinity chromatography buffer.

The next chromatographic steps should be performed as quickly as possible, since theenzyme is slightly unstable in the affinity chromatography buffer.

14. Apply the enzyme solution to a 6 × 2–cm chromatography column packed to 4 cmwith hydrazide-Sepharose 4B equilibrated with affinity chromatography buffer. Eluteby passing an additional 50 ml of affinity chromatography buffer through the column.

Glutamate dehydrogenase activity is not appreciably retarded by this material, andessentially complete recovery of the applied activity is obtained when the applied volumeplus an additional 50 ml of buffer has been passed through the column. This procedureremoves any components which bind nonspecifically to the Sepharose or to the spacer arm.

15. Apply the enzyme solution eluted from the above column to a 6 × 2–cm chromatog-raphy column packed to 4 cm with GTP-Sepharose 4B, equilibrated with affinitychromatography buffer. Use a flow rate of not more than ∼1 ml/min. Pass affinitychromatography buffer through the column until the A280 of the effluent has fallen to<0.01 (∼150 ml of buffer should be required).

Once the enzyme solution has passed into the column the flow rate may be increased to 1.5ml/min, and this flow rate should be used for step 16.

16. Elute the enzyme activity with a linear 400-ml gradient from 0 to 400 mM KCl inaffinity chromatography buffer. Collect fractions (∼10 ml) and monitor the A280 (seeBasic Protocol 2), conductivity, and enzyme activity (see Basic Protocol 3) in each.

17. Combine fractions containing glutamate dehydrogenase activity and concentrate byultrafiltration, followed by osmotic dehydration, as described in Support Protocol 6,to yield a protein concentration of at least 3 mg/ml.

18. Dialyze (UNIT 4.4 and APPENDIX 3B) overnight against 3 liters of 20 mM sodiumphosphate buffer, pH 7.4, using MWCO 12,000 dialysis membrane. Add glycerol tothe dialyzed enzyme solution to yield a final concentration of 30% (v/v) and store ina sealed container, which has been flushed with N2, at 4°C.

There may be some small variations between different preparations of the GTP-Sepharosein the sharpness of the peak of activity that emerges and in the salt concentration necessaryto elute the enzyme. This may reflect varying concentrations of bound ligand, but does notaffect the degree of purification obtained.

SUPPORTPROTOCOL 1

PREPARATION OF GTP-SEPHAROSE

Although GTP-agarose is available from Sigma, the preparation described in this protocolgenerally yields more satisfactory and reproducible results than commercial preparations.The steps involved are outlined in Figure 1.4.2.

GTP is coupled to Sepharose 4B by the method of Jackson et al. (1973) as modified byGodinot et al. (1974), where L-glutamic acid γ-methyl ester is used as the spacer insteadof ε-aminohexanoic acid methyl ester. After the spacer is coupled to the activatedSepharose and the ester group is treated with hydrazine (Godinot et al., 1974), a portionof the gel (∼10 ml) is retained and used as the hydrazide gel in the purification procedure.


1.4.6



Materials

Cyanogen bromide–activated Sepharose 4B (Pharmacia Biotech, or see SupportProtocol 3).

Sodium bicarbonate buffer: prepare 0.1 M NaHCO3 and adjust pH to 9.0 withNaOH

L-glutamic acid γ-methyl esterHydrazine hydrateGuanosine 5′-triphosphate (GTP)0.1 M disodium hydrogen phosphate adjusted to pH 5.0 with 1 M citric acidSodium periodateEthylene glycol

OHOH

CNBr

OO

C

Sepharose

NH

COOH

NHCHCH2CH2COCH3OH

H2NNH2

COOH

NHCHCH2CH2CNHNH2OH

O

L-glutamic acid γ -methyl ester

O

hydrazine derivative

COOH

NHCHCH2CH2CNHN

OH

O

GTP-Sepharose G

CH2OPOPOP

CH2OPOPOP

O

O

G

HO

HO

CH2OPOPOP

GTPG

NaIO4

O

O

O

Figure 1.4.2 The chemical processes involved in the synthesis of GTP-Sepharose according tothe method described in Support Protocol 1. The activation of Sepharose by cyanogen bromide(Support Protocol 3) is also shown in simplified form; alternative activated products are formed inthis step.


1.4.7


Nitrogen source50 mM sodium phosphate buffer, pH 6.8 (APPENDIX 2E) containing 5 mM EDTAButanol

Sintered-glass filter funnel (Pyrex, porosity G2)70°C water bath

Prepare the hydrazide gel1. Wash ∼4.5 g of cyanogen bromide–activated Sepharose 4B in a sintered-glass filter

funnel with ice-cold water, then wash with 100 ml ice-cold sodium bicarbonate buffer.Finally, suspend the resin in sodium bicarbonate buffer to a volume of 16 ml.

The cyanogen bromide–activated Sepharose 4B contains dextran and lactose as stabilizers.It is necessary to remove these by washing.

It is possible to activate Sepharose with cyanogen bromide in the laboratory as describedin Support Protocol 3. This is a less expensive alternative, but cyanogen bromide is toxicand should be handled with great care.

2. Dissolve 1.12 g of L-glutamic acid γ-methyl ester in 4 ml sodium bicarbonate buffer.Add this solution to the ∼16 ml of the gel suspension and stir this mixture for 16 to18 hr at 4°C. Finally, filter the mixture and wash it with cold water in a sintered-glassfilter funnel before suspending it in water to a volume of 30 ml.

The following steps should be performed in a fume hood.

3. Add 28 ml of hydrazine hydrate (99% to 100%) slowly to 30 ml of the suspended geland heat the reaction mixture to 70°C in a water bath for 15 min. Allow the mixtureto cool and then filter and wash with cold water on a sintered-glass filter.

This material, the hydrazide gel, may be overlaid with butanol to impede bacterial growth,and stored at 4°C. Retain a 10-ml portion of this hydrazide gel for use in the purificationprocedure. After use, the hydrazide gel can be recycled as described in Support Protocol3.

Prepare GTP-Sepharose4. Dissolve 14.2 mg of GTP in 5 ml of 0.1 M disodium hydrogen phosphate that has

been adjusted to pH 5.0 with 1 M citric acid. Dissolve 8.6 mg of sodium periodate in0.4 ml of the same buffer. Mix these two solutions and incubate in the dark for 30min at room temperature. Add 8 µl of ethylene glycol to remove the excess periodate,and incubate the mixture for a further 15 min. Bubble nitrogen through the solutionfor 5 to 10 min.

Bubbling with N2 is effected by connecting a Pasteur pipet to a nitrogen cylinder by plastictubing. The Pasteur pipet is placed in the tube containing the reacted mixture and thenitrogen is allowed to bubble through slowly. This procedure removes any formaldehydeproduced during the reaction, which would compete with the oxidized GTP for reactionwith the hydrazide gel.

5. Dilute the mixture from step 4 (oxidized GTP solution) to 10 ml with 0.1 M disodiumhydrogen phosphate that has been adjusted to pH 5.0 with 1 M citric acid. If necessary,remove the butanol layer from the hydrazide gel (from step 3) by aspiration and washthe gel with cold water, then suspend it in 20 ml of cold water. Mix 10 ml of this gelsuspension with the oxidized GTP solution and stir the mixture at room temperaturefor 2 hr.


1.4.8



6. Filter the gel on a sintered-glass funnel and wash with at least 100 ml of cold 50 mMsodium phosphate buffer, pH 6.8, containing 5 mM EDTA. Retain washings if step8 is to be performed. Store the resultant GTP-Sepharose at 4°C with an overlay ofbutanol to retard bacterial growth. Remove this butanol layer by aspiration and washthe gel with buffer prior to use of the gel.

After use, the GTP-Sepharose gel can be recycled as described in Support Protocol 4.

SUPPORTPROTOCOL 2

ASSESSMENT OF INCORPORATION OF GTP INTO THE HYDRAZIDE GEL

The following modification to Support Protocol 1 may be applied if there are any problemsusing the GTP-Sepharose medium prepared in Support Protocol 1 in the affinity purifi-cation steps (see Basic Protocol 1, steps 13 to 16). It is generally not necessary to resortto this procedure.

CAUTION: When working with radioactivity, take appropriate precautions to avoidcontamination of the experimenter and the surroundings. Carry out the experiment anddispose of wastes in an appropriately designated area, following the guidelines providedby the local radiation safety officer (also see APPENDIX 2B).

Additional Materials (also see Support Protocol 1)

[8-14C]GTP (Amersham Pharmacia Biotech)250:500:1 (v/v/w) Triton X-100/toluene/2,5-diphenyloxazole (PPO)

Carry out Support Protocol 1 with the following modifications at the indicated steps.

4a. Add 2.7 µCi of [8-14C]GTP to the solution of unlabeled GTP before periodateoxidation.

7b. Determine radioactivity in a sample of the gel washings by scintillation counting ina mixture by scintillation counting in 250:500:1 Triton X-100/toluene/PPO.

About 35% of the added GTP should be found to be tightly bound to the gel, correspondingto 0.6 �mol of GTP per ml of settled gel (see Table 1.4.1).

Table 1.4.1 Recovery of [8-14C]GTP After Coupling to Sepharose 4B

Aqueous phase Radioactivity(cpm)a,b

Total radioactivity(cpm)a,b

10 µM of initial 14C-GTP solution + 1 ml H2O 14760 3.77 × 106

1 ml gel washings (from 475 ml) 5167 2.44 × 106

10 µM of initial 14C-GTP solution + 1 ml gelwashings

20100 —

a9 ml of 250:500:1 (v/v/w) Triton X-100:toluene:PPO were mixed with 1 ml of the aqueous phase and theradioactivity determined in a liquid scintillation counter.bSince the cpm obtained with the 10 µl 14C-GTP solution + 1 ml gel washings equals the sum obtained for thetwo solutions separately, there is no significant difference in the quenching for the two solutions and therefore itis unnecessary to convert the results to dpm.


1.4.9


SUPPORTPROTOCOL 3

ACTIVATION OF SEPHAROSE 4B WITH CYANOGEN BROMIDE

This protocol is given as an alternative to the purchase of preprepared cyanogen-bromide-activated Sepharose.

Materials

Sepharose 4B (Pharmacia Biotech)Cyanogen bromideAcetonitrile5 M sodium hydroxideSodium bicarbonate buffer: prepare 0.1 M NaHCO3 and adjust pH to 9.0 with

NaOH

CAUTION: Because cyanogen bromide is very toxic, it should be handled with extremecare. See APPENDIX 2A for general precautions.

1. Wash 10 ml Sepharose 4B with water and suspend in 40 ml water.

2. Stir this mixture slowly in an ice-bath in a fume-hood. Add 2.4 g cyanogen bromidedissolved in 1.7 ml acetonitrile and monitor the pH of the resultant mixture constantly,maintaining it at ≥pH 11 by the addition of 5 M sodium hydroxide.

Use extreme caution as hydrocyanic acid may be formed in this step.

3. After hydrogen ion release has subsided (i.e., the pH ceases to fall), filter the gel ona sintered-glass funnel. Wash with at least 100 ml of ice-cold sodium bicarbonate andresuspend in 16 ml of that buffer.

SUPPORTPROTOCOL 4

REUSE OF CHROMATOGRAPHY MEDIA

Hydrazide-Sepharose (prepared as in Support Protocol 1, steps 1 to 3)

Wash the resins after use with 500 ml of 2 M KCl, followed by 1 liter of water. Store at4°C with an overlay of butanol, to retard bacterial growth. Remove the butanol layer byaspiration and equilibrate with affinity chromatography buffer (see recipe) immediatelybefore use.

GTP Sepharose (prepared as in Support Protocol 1, steps 1 to 7)

Follow the same procedure as described for the hydrazide-Sepharose, above. Over aperiod of several months, the properties of the GTP-Sepharose change and the enzymeactivity is eluted earlier in the gradient, probably as a result of instability of the boundGTP. The gel should therefore be discarded after 6 months.

DEAE-Cellulose

The manufacturers provide full instructions for recycling this material for reuse. Afteruse, remove tightly bound material from the ion-exchange material by passing 2 columnvolumes of 2 M NaCl through the column. Equilibrate the column with the buffer to beused and then pass 1 column volume of the same buffer containing 0.02% sodium azidethrough the column. Before use, wash the column with azide-free buffer.

If a small portion at the top of the column becomes discolored as a result of tightly boundmaterial, that portion may be removed and replaced by fresh DEAE-cellulose. Removalof the top few centimeters from the column may also be effective if the flow rate throughthe column becomes excessively slow. After removing the discolored top resin, transferthe remaining resin from the column to a beaker. Add the appropriate amount of fresh


1.4.10



DEAE-cellulose from which the fines have been removed and which has been equilibratedwith 20 mM sodium phosphate buffer, pH 7.4 (see Support Protocol 5), and repack thecolumn.

If the characteristics of the DEAE-cellulose change after protracted use, it may beregenerated by the following procedure.

1. Empty the contents of the column into a beaker. Stir with 15 volumes of 0.5 M HClfor 30 min.

Stirring can be continued for up to, but no longer than, 2 hr if convenient.

2. Filter the DEAE-cellulose on a sintered-glass funnel and wash it with water until thepH of the effluent reaches 4.

3. Stir the DEAE-cellulose with 15 volumes of 0.5 N NaOH for 30 min.

4. Allow to settle, remove the supernatant by decantation, and repeat step 3.

5. Filter on a sintered glass funnel and wash with water until the pH of the effluent isclose to neutrality.

The DEAE-cellulose is then ready for re-equilibration and reuse, although it may benecessary to remove the fines (see Support Protocol 5).

For longer-term storage, remove the DEAE-cellulose from the column and store it in asaturated solution of NaCl, in a tightly-sealed container.

Sephadex G-25

Sephadex G-25 is supplied in dry form and must be swollen before use. Full instructionsfor swelling and recycling this material are provided in the literature from the manufac-turers.

SUPPORTPROTOCOL 5

EQUILIBRATION OF CHROMATOGRAPHIC MEDIA

The chromatographic medium must never be allowed to dry. When using it in columnsthere should always be a layer of the appropriate buffer above the surface.

Equilibration of DEAE-Cellulose

DEAE-cellulose (Whatman DE-52) may contain small amounts of relatively small-sizedparticles (“fines”) which can slow down the rate of flow through a column if not removed.The following steps are appropriate for the DEAE-cellulose chromatography performedin Basic Protocol 1 and the Alternate Protocol. For the DEAE-cellulose chromatographyperformed in Support Protocol 7 (i.e., step 15 of that protocol), the procedure is the sameas that described below except that 1 M NH4HCO3 may be used for removing the fines.A total of 10 column volumes of 1 M NH4HCO3 should then be used to equilibrate thecolumn, which is then washed with 8 liters of water.

Materials

DEAE-cellulose (Whatman DE-52)200 mM and 20 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E)

Chromatography column; 40 × 3–cm (length × diameter)Conductivity meter (e.g., Linton, type WPA CMD80)

1. Suspend the DEAE-cellulose resin in 5 volumes of 200 mM sodium phosphate buffer,pH 7.4. Allow the mixture to settle and remove the buffer and fines by decantation.Repeat this process at least three times, after which the supernatant, upon settling,should be clear.


1.4.11


2. Pour a suspension of this material into a 40 × 3–cm chromatography column toproduce a final bed volume of 30 × 3 cm. Allow 20 mM sodium phosphate buffer,pH 7.4, to flow through the column until the effluent has the same pH and conductivityas the in-flowing buffer.

The column is now ready for use.

Equilibration of Dowex AG 1X2

Dowex AG 1X2 medium is used in Support Protocol 7. Suspend the Dowex AG 1X2 (200to 400 mesh, Cl− form) in 3 M HCl and pour into a column of 4 cm diameter to give asettled bed volume of 10 cm. Wash the column before use with 1 liter of 3 M HCl, followedby exhaustive washing with at least 20 liters of water, until the pH of the effluent reachesneutrality.

Equilibration of Sephadex G-25

Pass at least 2.5 column volumes of the appropriate buffer through the packed columnbefore use.

SUPPORTPROTOCOL 6

CONCENTRATION OF PROTEIN-CONTAINING SOLUTIONS

Some of the purification procedures used in Basic Protocol 1 and the Alternate Protocolresult in some degree of concentration. Precipitation with ammonium sulfate followed byresuspension of the pellet in a smaller volume, as well as ion-exchange chromatography,can also be effective concentration procedures. In contrast, however, dialysis and gelfiltration tend to dilute samples. The procedures described below are specifically forconcentration purposes. For additional information, see UNIT 4.4.

It is important to note that in both of the procedures described below, inattention mayresult in the concentration proceeding until all the fluid has been removed. It is essentialto monitor the progress of the procedure and stop when the desired volume has beenreached, because attempts to recover enzyme activity from material that has been takento dryness by either of these procedures will result in significant losses.

Ultrafiltration

The Amicon ultrafiltration cell uses pressure of nitrogen gas to force the solution througha membrane that retains molecules above a pre-determined size.

Prepare the apparatus according to the manufacturer’s instructions using the ultrafiltrationcell from Amicon either an Amicon Ultracel YM 30 or Ultracel PLTK membrane. Slowlystir the contents during the concentration process, which is carried out at 4°C. Continueuntil the required volume remains in the cell.

Earlier work used Amicon Diaflo XM-50 membranes for this procedure, but these are nolonger readily available. The Ultracel Amicon YM 30 or Ultracel PLTK membranes areappropriate substitutes.

Centriplus concentrators (Amicon) may be an alternative for concentrating the enzymefrom volumes of less than 60 to 70 ml. They rely on centrifugation to force solvent througha membrane (e.g., Centriplus 50, MWCO 50 kDa). The sample containing enzyme isretained by the membrane. The authors have found this procedure to be successful withseveral other dehydrogenases.


1.4.12



Osmotic Dehydration

Although ultrafiltration is a highly effective procedure for concentrating relatively largevolumes, it may be less effective and convenient with small volumes and high proteinconcentrations. Osmotic dialysis relies on the use of a high concentration of materialoutside a dialysis tube to draw water and small-molecule solutes from its contents byosmosis, leaving a more concentrated protein solution within the dialysis tubing.

Either sucrose or polyethylene glycol (PEG; Mr = 15,000 to 20,000) may be used. Howeverthe purity of PEG can be variable and it is important to use preparations that are as pureas possible to avoid contamination of the enzyme solution from low-Mr impurities thatmay be inhibitory.

Put the enzyme solution in a length of dialysis tubing (see APPENDIX 3B for preparation)that has been knotted at the lower end, and then tie a knot in the other end of the tubing.Surround the resulting tube with solid sucrose or PEG. This may be done by heaping thesucrose or PEG on a sheet of cooking foil, embedding the dialysis tubing in the sucrose,and then folding the foil to form a parcel around the sucrose or PEG-covered dialysistube. Alternatively, the dialysis tubing may be buried in the material within a plastic boxof appropriate dimensions. Leave at 4°C until the solution in the dialysis bag has reachedthe required volume; this will take several hours. Rinse the outside of the dialysis tubewith water or buffer to remove adhering sucrose or PEG. This also dampens the dialysismembrane, which becomes brittle during the concentration procedure.

Whereas with ordinary dialysis it is necessary to leave an empty space in the tubing toallow for the osmotic volume increase, this need not be done for osmotic dehydrationwhere a decrease in volume occurs.

In Basic Protocol 1 and the Alternate Protocol, this concentration procedure is followedby dialysis. In order to avoid too great a degree of dilution occurring at this stage, reknotthe dialysis tube to confine the enzyme solution in a smaller volume of the tubing.

BASICPROTOCOL 2

DETERMINATION OF PROTEIN CONCENTRATION

The first two methods presented under this heading are the Biuret and Bio-Rad proteinassays. A spectrophotometer and 1-cm light-path cuvettes (either glass or plastic) will berequired for each of these determinations. In the case of the third method presented below,determination of protein concentration by absorbance at 280 nm (A280), the cuvettes mustbe quartz or some other UV-transmitting material.

Other procedures for determining protein concentration may also be used, including theLowry-Folin method, which is described in adequate detail in Dawson et al. (1989). UNIT

3.4 also provides additional information on determination of protein concentration.

Biuret Protein Assay

Materials

Biuret reagent (see recipe).1.5% (w/v) sodium deoxycholate (1.5 g sodium deoxycholate in 100 ml distilled

H2O)50 mg/ml bovine serum albumin (BSA), or alternative protein calibration standard

1. Prepare a series of standards in triplicate according to the scheme described in Table1.4.2.


1.4.13


2. For enzyme protein determination use 20 to 100 µl of appropriately diluted sampleand prepare as described in Table 1.4.2.

3. Mix each sample and allow the reaction to proceed at room temperature for ∼30 min.

4. Measure absorbance at 540 nm using the samples containing no protein as blanks.

5. Prepare a standard curve by plotting absorbance at 540 nm against the proteinconcentration.

6. Use the standard curve to determine the protein concentration of the enzyme samplefrom its absorbance. Determine the absorbance of the sample against a blankcontaining the same medium. Dilute, if necessary, to ensure that the absorbance is<1.0.

Bio-Rad Protein Assay

Materials

Bio-Rad Dye Reagent Concentrate0.1 mg/ml bovine serum albumin (BSA), or alternative protein calibration standard

(this can be conveniently prepared by diluting some of the stock solutionprepared for the biuret assay; see above)

Method1. Prepare a series of standards in triplicate according to the scheme described in Table

1.4.3.

2. For enzyme protein determination use 20 to 100 µl of appropriately diluted sampleand prepare as described in Table 1.4.3.

Table 1.4.3 Preparation of Standard Curve for Bio-Rad Protein Assay

µg protein0.1 mg/ml BSA(µl)

H2O(µl)

Bio-Rad Dye ReagentConcentrate (µl)

0 0 800 200 2 20 780 200 5 50 750 20010 100 700 20015 150 650 20020 200 600 200

Table 1.4.2 Preparation of Standard Curve for Biuret Protein Assay

mg protein50 mg/ml BSA(µl)

1.5% (w/v) sodiumdeoxycholate (µl)

H2O(µl)

Biuret reagenta

(ml)

0 0 100 900 40.25 5 100 895 40.5 10 100 890 40.75 15 100 885 41.0 20 100 880 41.5 30 100 870 42.0 40 100 860 4aSee recipe in Reagents and Solutions.


1.4.14



3. After mixing, leave for a period of 5 to 60 min, then determine the absorbance at 595nm using the samples containing no protein as blanks.

4. Prepare a standard curve by plotting absorbance at 595 nm against the proteinconcentration.

5. Use the standard curve to determine the protein concentration of the enzyme samplefrom its absorbance. Determine the absorbance of the sample against a blankcontaining the same medium. Dilute, if necessary, to ensure that the absorbance is<1.0.

Note that the Bio-Rad assay involves determination of the absorbance of 1 ml of mixture.The optical arrangements of some spectrophotometers may not permit this in standard1-cm2 cross-section cuvettes. In such cases microcuvettes should be used. However, someolder spectrophotometers may not focus the light beam to pass through the narrower pathof a microcuvette. It is a good idea to check the instructions supplied with the spectro-photometer in regard to this. If all else fails, the volumes shown for this procedure can allbe increased proportionally.

Absorbance at 280 nm

Using quartz or other non-UV-absorbing cuvettes, determine the absorbance at 280 nmof the sample against a blank composed of the medium in which the sample is dissolved.A UV-Vis spectrophotometer is required. Use the rough approximation that a 1 mg/mlprotein solution has an absorbance of 1.0 at this wavelength.

BASICPROTOCOL 3

ASSAY OF GLUTAMATE DEHYDROGENASE ACTIVITY

Glutamate dehydrogenase catalyzes the reaction:

The assay described here determines the decrease of absorbance at 340 nm as NADH isoxidized to NAD+ concomitant with the reductive amination of 2-oxoglutarate to L-glu-tamate.

Materials

Enzyme preparation (see appropriate steps of Basic Protocol 1 or AlternateProtocol)

0.4% (v/v) Triton X-100 in 50 mM sodium phosphate buffer, pH 7.4100 mM potassium cyanide (KCN)50 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2E)2.5 M ammonium sulfate in 50 mM sodium phosphate buffer, pH 7.44 mM NADH in 50 mM sodium phosphate buffer, pH 7.4 (prepared fresh on day

of use)0.25 M 2-oxoglutarate (α-ketoglutarate), in 50 mM sodium phosphate buffer, pH

7.4

Spectrophotometer with cuvette holder set at 37°C1-cm path-length cuvettes (quartz or plastic with good light transmittance at 340

nm)

NOTE: All GDH enzyme assays are performed at 37°C. In order to ensure rapidtemperature equilibration, the assay buffer should be prewarmed to that temperature.

+L3 22-oxoglutarate NH NADH H -glutamate H O NAD++ + + + +�


1.4.15


Other hints on the performance and validation of enzyme assays can be found in Tipton(2002).

1. For crude enzyme preparations (see Basic Protocol 1, step 1): Incubate 50 µl of theenzyme solution with 2.68 ml of 0.4% Triton X-100 in 50 mM sodium phosphatebuffer, pH 7.4, and 30 µl of 100 mM KCN (which will give a final KCN concentrationof 1 mM in the complete assay) for 2 min at 37°C before proceeding to step 2.

For assaying the enzyme at later steps of the purification, begin with step 2.

Triton X-100 serves to disrupt particulate material, and KCN decreases any contaminatingNADH oxidase activity. It has been shown that neither Triton X-100 nor KCN affect theactivity of purified GDH at these concentrations.

These assays are performed in phosphate buffer. Do not attempt to perform them in Trisbuffer since the enzyme is unstable in Tris and either no activity or a rapid decrease inactivity may be observed.

2. Add reagents to the cuvettes in the order listed, in a final volume of 3 ml:

2.71 ml 50 mM sodium phosphate buffer, pH 7.460 µl 2.5 M ammonium sulfate (final concentration 50 mM)120 µl 4 mM NADH (final concentration 160 µM)50 µl enzyme preparation.

3. Cover the cuvette with Parafilm, mix the contents by inversion and incubate in thespectrophotometer for 3 min. Initiate the reaction by the addition of 60 µl of 0.25 M2-oxoglutarate (final concentration 5 mM), invert to mix and allow the reaction toproceed for 10 min in the spectrophotometer.

4. Determine the initial (linear) rate of decrease in absorbance as NADH is oxidized toNAD+, subtracting any blank rate occurring before the addition of the 2-oxoglutarate(see Critical Parameters).

The initial absorbance resulting from the NADH present will be ∼1.0, although impuritiesin the enzyme solution may add to this. The assay has been found to proceed in a linearfashion until the absorbance has decreased by 0.15 absorbance units. In all cases it willbe necessary to dilute the enzyme preparation in order to obtain a rate that is sufficientlyslow to measure with accuracy. See Critical Parameters for a detailed discussion of dilutionconsiderations.

5. Calculate the specific activity.

Since the molar absorbance coefficient of NADH in a 1-cm path-length cuvette is 6220M-1cm-1, the production of 1 �mol of glutamate, or the reductive amination of 1 �mol2-oxoglutarate in an assay volume of 3.0 ml will give an absorbance change of 2.073. Thus,for a 1-cm path-length cuvette, the specific activity of the enzyme (�mol product formed.permin per mg protein) will be related to the rate of decrease in absorbance per min (∆A/min),which will be given by:

It is most common to express enzyme activities in these terms. 1 unit (U) of enzyme activityis defined as the amount catalyzing the production of 1 �mol of product (or the disappear-ance of 1 �mol of substrate) in 1 min under stated conditions.

∆= ×/min assay volume (ml)specific activity

6.22 protein (mg)

A


1.4.16



ALTERNATEPROTOCOL

PURIFICATION OF RAT OR OX LIVER GDH INVOLVING AFFINITYPRECIPITATION WITH bis-NAD+

Affinity precipitation of enzymes was developed by Mosbach and co-workers (seeFlygare et al., 1983; Larsson et al., 1984). The bifunctional ligand bis-NAD+ (N2,N2′-adipodihydrazido-bis-(N6-carbonyl-methyl)NAD+) comprises two molecules ofNAD+ joined by a spacer arm (see Fig. 1.4.5). This separates the NAD+ groups by asufficient distance (∼1.7 nm) to allow them to bind to the coenzyme-binding sites of twodifferent molecules of glutamate dehydrogenase. Mammalian GDH is a polymer, soextensive cross-linking of the enzyme molecules occurs and hence they precipitate fromsolution, as illustrated in Figure 1.4.3. Since there are many oligomeric dehydrogenases

+

glutamatedehydrogenase

bis-NAD+

Figure 1.4.3 A diagrammatic view of the cross-linking of glutamate dehydrogenase hexamers bybis-NAD+, which results in the formation of an insoluble lattice. The “locking-on” in the presence ofglutarate is not shown. Further details of the process are given in the Alternate Protocol.


1.4.17


in the cell, this process might be expected to be somewhat nonspecific, causing theprecipitation of a number of enzymes containing binding sites for NAD+. However, theprocedure can be made specific for the precipitation of a selected dehydrogenase by theuse of a compound that is a competitive inhibitor of the enzyme with respect to its othersubstrate. Thus, in the case of GDH, glutarate, which is a competitive inhibitor towardsglutamate, is used. This strengthens the binding between the bis-NAD+ and glutamatedehydrogenase, allowing it to be used at concentrations below those that would benecessary to precipitate other dehydrogenases.

ox liver (46 g) rat liver (10g)

homogenize for 1 min in 10 volumes (w/v) of 4 mM sodiumphosphate buffer, pH 7.4, containing 0.5 mM EDTA and 0.1 mM PMSF

centrifuge and discard pelletdialyze against 20 mM sodium phosphate buffer, pH 7.4

apply to DEAE-cellulose column

affinity precipitate with the appropriate amountdetermined from pilot precipitation experiment

(Support Protocol 9) of bis-NAD+ in the presence of79 mM glutaric acid and keep on ice overnight

redissolve pellet by stirring for 6 hr in 1 ml of buffercontaining 0.6 mM NADH

purified glutamate dehydrogenase

Stage 1(homogenate)

Stage 2[dialyzed(NH4)2SO4 pellet]

Stage 3(DEAE eluate)

Stage 4(purified enzyme)

dialyze against 20 mM sodiumphosphate buffer, pH 7.4

centrifugediscard supernatant

elute with 20 to 200 mM sodiumphosphate, pH 7.4, gradientand concentrate to ~10 ml

add (NH4)2SO4 (113 g/liter), stir for 20 min

centrifuge and discard pelletadd (NH4)2SO4 (188 g/liter), stir for 20 min

Figure 1.4.4 Outline scheme for the purification of ox and rat liver glutamate dehydrogenaseaccording to the Alternate Protocol. Full details of the steps involved are given in the text.


1.4.18



This behavior, which was termed the “locking-on” effect by O’Carra (see O’Carra et al.,1996; O’Flaherty et al., 1999; Mulcahy and O’Flaherty, 2001), was originally developedfor increasing the strength of enzyme binding to an immobilized ligand. It depends onthe analog of the substrate that is oxidized by NAD+ binding to the enzyme⋅NAD+ binarycomplex and thus displacing the coenzyme-binding equilibrium by ternary complexformation:

This “locking-on” behavior occurs only if the oxidizable substrate, or a competitiveinhibitor that is an analog of the oxidizable substrate, binds to the enzyme⋅NAD+ binarycomplex and not to the free enzyme. The basic concept is that, although several enzymesbind NAD+, only the GDH⋅NAD+ complex binds glutarate. The majority of dehydro-genases have ordered sequential kinetic mechanisms in which the coenzyme binds beforethe second substrate. To date, the technique has been found to work well for thepurification of yeast alcohol dehydrogenase and lactate dehydrogenase (see Irwin andTipton, 1996), and the synthesis of the corresponding bis-ATP analog has extended theapplication of bis-coenzymes to kinases (Beattie et al., 1987).

The precipitated dehydrogenase can then be dissolved by incubation in the presence ofNADH, which effectively competes with bis-NAD+ for binding to the enzyme. Theprocedure described here is that of Graham et al. (1985), as modified by Irwin and Tipton(1996). The steps involved are summarized in Figure 1.4.4.

Additional Materials (also see Basic Protocol 1)

Bis-NAD+ (see Support Protocol 7)0.7 M glutaric acid, adjusted to pH 7.0 with NaOH

Additional reagents and equipment for pilot-scale precipitation (see SupportProtocol 9)

NOTE: Unless indicated otherwise all steps are performed at 0° to 4°C.

1. Homogenize the sample and perform ammonium sulfate precipitation and DEAE-cellulose chromatography steps (see Basic Protocol 1, steps 1 to 12), concentratingthe eluate from the DEAE-cellulose column to ∼10 ml at step 12a or b.

Although it is possible to perform affinity precipitation with preparations that have notbeen subjected to chromatography on DEAE-cellulose, the yield is much lower and theprecipitated material is not completely pure.

2. Dialyze the enzyme preparation overnight at 4°C against 200 ml of sodium phosphatebuffer, pH 7.4, with at least one change of buffer. Carry out a pilot-scale precipitationas described in Support Protocol 9 to determine the optimal amount of bis-NAD+ touse. Add that amount of bis-NAD+ to the remaining dialyzed enzyme preparation andadd 0.7 M glutaric acid (preadjusted to pH 7) to a final concentration of 79 mMglutarate.

Keep the mixture on ice overnight.

3. Centrifuge 15 min at 10,000 × g, 4°C. Redissolve the pellet by stirring for 6 hr in 1ml of 20 mM sodium phosphate buffer, pH 7.4, containing 0.6 mM NADH. Dialyzethe redissolved pellet overnight (using an MWCO 12,000 dialysis membrane) against200 ml of sodium phosphate buffer, pH 7.4. Add glycerol and store as described inBasic Protocol 1, step 18.

+NAD glutarate+ +GDH GDH NAD GDH NAD glutarate��


1.4.19


SUPPORTPROTOCOL 7

SYNTHESIS OF N2, N2′-ADIPODIHYDRAZIDO-bis-(N6-CARBONYLMETHYL)NAD+ (bis-NAD)

This reagent is used in the Alternate Protocol. The steps involved are outlined in Figure 1.4.5.

Materials

Iodoacetic acid (fresh)2 M and 1 M lithium hydroxide (LiOH)NAD+ (98%, free acid)2 M and 6 M HCl96% (v/v) ethanol0.24 M NaHCO3

1 M NaOHNitrogen sourceSodium dithionite2 M Tris (base)Acetaldehyde, redistilledYeast alcohol dehydrogenase (crystalline, Sigma-Aldrich or Roche Diagnostics)5 mM CaCl2, pH 2.7, and 50 mM CaCl2, pH 2.02 M Ca(OH)2

Adipic acid dihydrazide dichloride (Sigma-Aldrich)0.5 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC;

Sigma-Aldrich)0.25, 1, and 2 M ammonium bicarbonate (NH4HCO3)

Sintered-glass funnel (≥10 cm diameter)Rotary evaporatorVacuum desiccator75°C water bath4 × 10–cm Dowex AG 1X2 anion-exchange column (Support Protocol 5)Gradient mixer (e.g., UNIT 8.2)30 × 2.5–cm chromatography column packed with Whatman DE-52 resin and

equilibrated (see Support Protocol 5) with 1 M NH4HCO3, pH 8.0

Additional reagents and equipment for resolution of NAD+ derivatives (seeSupport Protocol 8)

Synthesize N(1)-carboxymethyl-NAD+

1. Dissolve 9 g fresh iodoacetic acid in ∼10 ml of water, neutralize the solution with 2M LiOH, and add 3 g of NAD+. Adjust the pH to 6.5 with 2 M HCl (the total volumeshould be ∼30 ml) and leave the solution in darkness for 7 days at room temperature(∼20°C), or alternatively, at 37°C for 2 days. Check the pH daily (or every 4 to 6 hrat the higher temperature) and readjust to 6.5 with 2 M HCl as required.

The progress of the reaction should also be followed by TLC and/or HPLC (see SupportProtocol 8).

The solid iodoacetic acid should be white; if it is discolored as a result of decompositionit should not be used. Solutions of iodoacetic acid should be prepared fresh.

2. When reaction is complete, adjust pH to 3.0 with 6 M HCl and add 2 volumes of 96%(v/v) ethanol to produce a milky, pink-tinged suspension. Add a further 10 volumesof cold 96% ethanol to complete the precipitation.

The water/ethanol ratio is important; using a wet vessel can give rise to the formation ofa brown, sticky substance which is water-soluble.


1.4.20



3. Filter the crude N(1)-carboxymethyl-NAD+ on a sintered funnel, wash with ethanoland diethyl ether, and dry in a rotary evaporator at 35°C under vacuum or bylyphilization (average yield 2.9 g). Store the product at −20°C under vacuum.

Carry out rearrangement of N(1)-carboxymethyl-NAD+ to N6-carboxymethyl-NAD+

4. Dissolve the crude N(1)-carboxymethyl-NAD+ in 0.24 M NaHCO3 (90 ml), whichgives a pale orange solution, and adjust the pH to 8.5 with 1 M NaOH.

5. Deoxygenate the solution by bubbling N2 gas through it for 2 min, add 1.5 g of sodiumdithionite, and leave the solution in the dark until maximum reduction is achieved.Monitor progress of reduction by taking samples, diluting them 1:50 or 1:100, andmeasuring the increase in absorbance at 340 nm (A340).

6. Terminate the reaction by stirring vigorously for 10 min to oxygenate the solutionand then bubble N2 gas through it for 2 min. Adjust the pH to 11.5 with 1 M NaOHand leave in a water bath at 75°C to allow the rearrangement from the N(1)- to theN6-substituted derivative to occur (Dimroth rearrangement; see Engel, 1975). Moni-tor the rearrangement by HPLC or TLC (see Support Protocol 8).

7. Cool the reaction mixture to room temperature and add 6 ml of 2 M Tris (base) and1.5 ml of redistilled acetaldehyde. Adjust the pH to 7.5 with 1 M HCl and add 8 to24 mg (2500 to 7500 U) of yeast alcohol dehydrogenase.

8. Monitor the reaction at 340 nm as in step 5. When a minimum A340 is reached, add 1volume of 96% ethanol and pour the milky flocculent precipitate into 10 volumes ofvigorously stirred 96% ethanol. Leave for 30 min (or overnight, if desired) and collectthe precipitate by filtration. Store the crude N6-carboxymethyl-NAD+ at −20°C in avacuum desiccator for up to a month (yield ∼2.6 g).

Purify the crude N6-carboxymethyl-NAD+

9. Dissolve the crude powder (2.6 g, from step 7) in 30 ml water, adjust the pH to 8.0with 1 M LiOH, and apply this solution to a 2.5 × 30–cm Dowex AG 1X2 column.

See Support Protocol 5 for details of packing and equilibrating the Dowex column.

10. After applying the coenzyme solution, wash the column with 0.5 liter of water,followed by 1 liter of 5 mM CaCl2, pH 2.7, until the pH of the effluent is 2.8. Applya 2-liter linear gradient from 5 mM CaCl2, pH 2.7, to 50 mM CaCl2, pH 2.0. Collect10 to 20 ml fractions and monitor the absorbance at 260 nm.

The composition of the fractions can be monitored by TLC (see Support Protocol 8).

11. Pool the fractions containing the N6 derivative, adjust the pH to 7.0 with 2 M Ca(OH)2,and concentrate to 5 to 10 ml by rotary evaporation at 40°C.

12. Precipitate with 96% ethanol as in step 2, above, and lyophilize.

This gives a pale yellow compound (yield 0.82 g, 25%); ε = 19,300 M−1cm−1 at 266 nm.

Synthesis of bis-NAD+

13. Dissolve 0.82 g of purified N6-carboxymethyl-NAD+ and 105 mg of adipic aciddihydrazide dihydrochloride in 20 ml water, to produce a brownish solution.

14. Adjust the pH to 4.6 with 1 M HCl and then add 0.5 M EDC at 0°C in 15- to 100-µlaliquots over a period of 35 min. Monitor the pH and readjust it to 4.6 before eachaddition.

15. Add water (2 liters) to dilute the solution 100-fold, adjust the pH to 8.0 with 2 MNH4HCO3, and apply the solution to a 30 × 2.5–cm DEAE-cellulose (DE-52) columnequilibrated with 1 M NH4HCO3, pH 8.0 (Support Protocol 5). Wash the column with


1.4.21


water until the A260 is <0.1 and apply a 2-liter linear gradient from 0 to 0.25 MNH4HCO3, pH 8.0.

16. Monitor the Rf values of the fractions by TLC (Support Protocol 8).

The yield from pure N6-carboxymethyl-NAD+ is ∼14%. ε= 42,800 M−1 cm−1 at 266 nm.

17. Concentrate the product-containing fractions to ∼3 to 5 ml by rotary evaporation at35°C and store at –20°C.

For longer-term storage (>7 to 10 days) the material may be lyophilized and stored in avacuum desiccator over silica gel at –20°C.

N N

NN

NH2

R

N+N

NN

NH2

R

CH2COO–ICH2COO–

NAD+

pH 6.5, 25°C, 5 to 7 days

NN

NN

NHCH2COO–

RH

Dimrothrearrangement

pH 11.5, 75°C,2 hr

N6-carboxymethyl-NADH

N(1) carboxymethyl-NAD+

Sodium dithionitepH 8.5, 2 to 4 hr

N N

NN

NH2

RH

CH2COO–

N(1) carboxymethyl-NADH

+

alcohol dehydrogenase,acetaldehyde, pH 7.5

N N

NN

NHCH2COO–

R

NN

NN

R bis-NAD+

HN CH2 C NH NH C (CH2)4 C NH NH C CH2 NH

0 0 0 0

NN

N N

R

EDC= CH3CH2-N C N (CH2)3NH(CH2)2Cl-

1-ethyl-3-(3)-dimethylaminopropyl) carbodiimide hydrochloride

N6-carboxymethyl-NAD+

H2NOC(CH2)4CONH2+ EDC

pH 4.6, 0°C, 35 min

+N

CONH2

R =

OH H

H H

OH OH

CH2O P O P OCH2

O

O

O– OH

OO

H HOH OH

H HO

Figure 1.4.5 Outline of the steps involved in the synthesis of bis-NAD+. Full details of the stepsinvolved are given in Support Protocol 7.


1.4.22



SUPPORTPROTOCOL 8

RESOLUTION OF NAD+ DERIVATIVES

Thin-layer Chromatography (TLC)

The techniques of TLC are not described in detail here since they are adequately describedin many textbooks (e.g., Wilson and Walker, 2000; Hahn-Deinstrop, 2000).

Two different TLC solvent systems have been used for monitoring the products of thesynthesis of bis-NAD+:

System A: 60:100:2 (w/v/v) (NH4)2SO4/0.1 M potassium phosphate, pH 6.8/1-propanol.

System B: 5:3 (v/v) isobutyric acid/1 M aqueous NH3, saturated with disodium EDTA.

Apply the samples as spots to TLC plates (aluminium backed, silica gel 60, fluorescentindicator F254, layer thickness 0.2 mm; Merck), dry, and develop by ascending chroma-tography in a TLC developing tank. After developing the plates and allowing them to dry,the spots can be visualized by their fluorescence under UV light (wavelength 254 nm).They appear purple on a green background. The Rf values of bis-NAD+ and the compoundsinvolved as intermediates in its synthesis are shown in Table 1.4.4.

High-Performance Liquid Chromatography (HPLC)

Inject the samples onto the HPLC column (4 mm × 30-cm C18 reversed-phase; e.g.,Waters µBondapak C18) and elute isocratically with 0.1 M KH2PO4, pH 6.0, containing10% methanol, at a flow rate of 0.7 to 1.0 ml/min. The elution profile is determined bycontinuously monitoring the absorbance of the eluate at 259 to 266 nm using a UV detectorflow cell.

Good separation between NAD+ and N(1)-carboxymethyl-NAD+ can be achieved, andN6-carboxymethyl-NAD+ and bis-NAD+ can be well separated, but this system does notresolve N(1)-carboxymethyl-NAD+ and N6-carboxymethyl-NAD+ satisfactorily. Reten-tion times for NAD+ derivatives are given in Table 1.4.4.

The retention times are decreased if buffer containing 20% methanol is used. A gradientof 0% to 20% methanol may be used to separate the N(1)- and N6-substituted NAD+

derivatives. The advantage of HPLC over TLC is that it is easier to quantify the levels ofany impurities present in the product.

Table 1.4.4 Some Properties of bis-NAD+ and its Precursors

CompoundRf

a (TLC) Retention timeb

(HPLC)Molar absorbance(M-1cm-1)Solvent Ac Solvent Bd

NAD+ 0.41 0.44 4.6 min 16,900 (at 259 nm)N(1)-carboxymethyl-NAD+ 0.31 0.27 3.7 min 17,100 (at 259 nm)N6-carboxymethyl NAD+ 0.22 0.22 3.6 min 19,300 (at 266 nm)Bis- NAD+ 0.09 0.05 6.9 min 42,800 (at 266 nm)aThe Rf values given for TLC of coenzyme derivatives are not exact. They may vary by up to ±0.05. This may result fromslight changes in the solvent composition over time, possibly as a result of evaporation.bThe HPLC retention times were obtained for samples chromatographed on a reversed-phase µBondapak C-18 column(4 mm × 30 cm) and eluted with 0.1 M KH2PO4 containing 10% methanol.cSolvent A: 60:100:2 (w/v/v) (NH4)2SO4;/0.1 M potassium phosphate, pH 6.8/1-propanol.d5:3 (v/v) isobutyric acid/1 M aqueous NH3, saturated with disodium EDTA.


1.4.23


SUPPORTPROTOCOL 9

PILOT-SCALE AFFINITY PRECIPITATION STUDY

In order to obtain optimal results from the purification procedure described in theAlternate Protocol, it is necessary to perform a small-scale (pilot) study to determine theoptimum concentrations of protein and bis-NAD+ to use (see Fig. 1.4.6). This will varysomewhat between different enzyme preparations, owing to differences in enzymeconcentration and the presence of other contaminants that may bind the bis-coenzyme.The optimum ratio of NAD+ equivalents/enzyme subunit may vary with the preparationused; for example, with a preparation of beef liver enzyme that had a specific activity of2.7 U/mg (see Basic Protocol 3), about half the activity was found to precipitate at a ratioof 2 NAD+ equivalents/GDH subunit. However, a preparation with the lower specificactivity of 0.8 U/mg required ∼8 NAD+ equivalents/GDH subunit to precipitate half theenzyme activity. The precipitation yield also depends on the protein concentration, asshown, for example, in Figure 1.4.7.

Materials

Sample of the enzyme preparation (Alternate Protocol)Bis-NAD+ (see Support Protocol 7)700 mM glutaric acid, pH 7.0

Additional reagents and equipment for assaying protein concentration (see BasicProtocol 2) and GDH activity (see Basic Protocol 3)

1. Take an aliquot of the enzyme-containing solution from the DEAE-cellulose purifi-cation step (see Alternate Protocol and Basic Protocol 1) and assay it for GDH activity(see Basic Protocol 3) and protein concentration (see Basic Protocol 2) to determineits specific activity.

0 5 10 15 200

50

100

[bis-NAD+] (mol/mol GDH subunit)

Pre

cipi

tatio

n (%

)

Figure 1.4.6 An example of a pilot-scale affinity precipitation of ox liver glutamate dehydrogenasepurified according to steps 1 to 3 of the Alternate Protocol. The experimental details are describedin Support Protocol 9. The precise behavior will vary between preparations. The affinity precipitationis expressed as a percentage of the activity remaining in the control sample.


1.4.24



2. Calculate the approximate concentration of enzyme, based on the specific activityand subunit relative molecular mass.

Assuming each subunit to have a relative molecular mass of 56,700 and the pure enzymeto have a specific activity of 40 �mol/min/mg protein, this corresponds to ∼2267 molproduct/min/mol enzyme subunit. Thus:

3. Measure effects of bis-NAD+ concentration as follows:

a. Add a 100-µl sample of the enzyme preparation to each of 10 microcentrifuge tubes.

b. To each tube, add 12.7 µl of 700 mM glutaric acid, pH 7.0, and varying amountsof bis-NAD+ within the range 0 to 10 coenzyme equivalents/enzyme subunit.

c. Also prepare sample containing no bis-NAD+ (to which water is added in place ofthe coenzyme derivative) as well as a control tube that contains no bis-NAD+ orglutarate, so that the percentage inhibition resulting from the presence of theglutarate can be calculated.The concentration of bis-NAD+ can be calculated from the absorbance at 266 nm and themolar absorbance coefficient given in Table 1.4.4. A 1 M solution of bis-NAD+ will givean absorbance, in a 1-cm light-path cuvette, of 42,800, and each mole of bis-NAD+ contains2 NAD+ equivalents. Thus:

( )µ

× 6

activity mol/minmol of subunit =

2267 10

=2662( )coenzyme equivalent concentration in mol/liter

42,800

A

0

50

100

Pre

cipi

tatio

n (%

)

0 0.05 0.1 0.2 0.3 0.4 1 5 10

[Protein] (mg/ml)

Figure 1.4.7 An example of the effects of protein concentration on the pilot-scale affinity precipi-tation of ox liver glutamate dehydrogenase by bis-NAD+. These results are from a sampleexperiment performed as described in Support Protocol 9 in which the enzyme-containing samplewas diluted to the indicated protein concentration before precipitation with a constant amount ofbis-NAD+.


1.4.25


4. Allow the tubes to stand at 4°C for at least 30 min (or overnight, if required), thenmicrocentrifuge 5 min at maximum speed (∼13,000 × g), 4°C.

5. Assay the GDH activity remaining in the supernatant (see Basic Protocol 3) of eachsample and express it as a percentage of the activity remaining in the control sample.

The maximum affinity precipitation has occurred in the tube with the minimal residualactivity and thus gives the appropriate concentration of bis-coenzyme to add to obtainmaximum affinity precipitation.

These experiments should be performed at least in duplicate.

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock solutions,see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Affinity chromatography buffer100 mM Tris acetate1 mM KH2PO4

0.1 mM EDTAAdjust pH to 7.15 at 4°CStore at 0° to 4°C up to 1 week; check pH before use and adjust if necessary

Biuret reagentDissolve 1.5 g copper sulfate (CuSO4⋅5H2O) and 6.0 g sodium potassium tartrate(C4H4KNaO6⋅4H2O) in 500 ml distilled H2O. Add 300 ml of 10% (w/v) sodiumhydroxide and dilute the mixture to 1 liter with distilled water.

Homogenization buffer4 mM sodium phosphate buffer, pH 7.4 (APPENDIX 2A)0.5 mM EDTAStore with above components at 0° to 4°C up to 1 week; check pH before use and

adjust if necessary0.1 mM phenymethanesulfonyl fluoride (PMSF; add immediately before use as

described below)

PMSF is unstable in aqueous solution and must be added to the buffer immediately beforeuse. This is done by adding a suitable volume of a 100 mM PMSF solution in acetone to asmall quantity (approximately one-tenth of the final volume) of boiling buffer, and immedi-ately mixing this solution with the remainder of the ice-cold buffer.

COMMENTARY

Background InformationThere are several factors that must be con-

sidered in the choice of a purification proce-dure. The major factors are how much enzymeis needed for the planned studies, how long theprocedure will take, and how much it will cost.To these should be added the question ofwhether the purified enzyme is different fromthe material that was in the tissues to start with.As discussed below, GDH purified by someolder procedures has been shown to have un-dergone limited proteolysis, which alters itskinetic and regulatory behavior. Clearly, suchpreparations can give misleading informationabout the behavior of the enzyme.

Several alternative procedures have beenused to purify GDH from different mammaliansources. The procedures described in this unitare preferable to the earlier procedures thatinvolved precipitation with ethanol or acetoneand/or detergent extraction followed by frac-tional precipitation with ammonium sulfateand/or sodium sulfate (see e.g., Fahien et al.,1969). Although that procedure resulted in acrystalline preparation of the enzyme, it hasbeen shown that limited proteolysis results in aloss of a tetrapeptide from the amino-terminalend of the protein (McCarthy et al., 1980). Thismodification, which results in a slightly fastermobility on SDS (0.1% w/v) PAGE in 13%


1.4.26



polyacrylamide gels (McCarthy et al., 1980),significantly affects the kinetic behavior of theenzyme (McCarthy and Tipton, 1984) and itsinteract ions with allosteric effectors(McCarthy and Tipton, 1985; Couée and Tip-ton, 1989a) and with antipsychotic drugs suchas perphenazine (Couée and Tipton, 1990a,b).

An obvious alternative to the hard work ofpurifying an enzyme is to buy it from a com-mercial source. Purified preparations of ox(beef) liver GDH have been available fromseveral commercial sources: Boehringer-Mannheim (now Roche Diagnostics,Mannheim), Calbiochem, PL Biochemicals(now Pharmacia P-L Biochemicals), Sigma,and United States Biochemical (USB), al-though some of these sources no longer supplypurified preparations of GDH. However, theauthors have found the enzyme from all thesesources to have suffered the same, or more,proteolytic modification as that suffered by theenzyme prepared by the earlier procedures dis-cussed above (see McCarthy et al., 1980).

Cloning and expression of GDH is an alter-native source of enzyme (Shashidharan et al.,1994; Plaitakis et al., 2000). An expressionsystem has the advantage that it permits site-di-rected mutagenesis studies (see Fang et al.,2002; Zaganas and Plaitakis, 2002). It is also,of course, useful in cases where there are rathersmall quantities of enzyme in the chosen sourcematerial. This is not the case with the majorisoform of GDH (GLUD1) in liver, but it is thecase for the so-called “nerve tissue–specific”form (GLUD2) which is present in much lowerabundance in the brain (see Zaganas and Plai-takis, 2002).

It is still necessary to purify such an ex-pressed enzyme if one needs to work with ahomogeneous preparation. In some cases, thiscan be facilitated by the insertion of a tag intothe gene to be expressed. Such tags range fromoligohistidine tails to another protein, such asglutathione-S-transferase. It is common not toremove a tag after purification, and indeed insome cases it is not easy to do so. The assump-tion that the presence of such a tag will notaffect the behavior of the expressed enzymemay sometimes be true, but it is certainly notalways so (see e.g., Halliwell et al., 2001; Hiseret al., 2001; Ledent et al., 1997). In the case ofGDH, as already discussed, modification at theamino-terminal end results in alterations in itsbehavior, and the C-terminal region appears tobe involved in its allosteric regulation (Petersonand Smith, 1999). This suggests that expressionof the protein with a tag at either end would be

likely to produce artefactual results. Indeed, itis necessary to use conventional procedures,involving a GTP affinity column, to purify theenzyme after cloning and expression (see Fanget al., 2002; Zaganas and Plaitakis, 2002).

To assess the purity of the protein, polyacry-lamide gel electrophoresis (PAGE) in the pres-ence and absence of sodium dodecyl sulfate(SDS) remains the accepted standard for as-sessing protein purity (UNITS 10.2-10.4). The hex-americ glutamate dehydrogenase from ox andsome other mammalian species forms higheraggregates at higher protein concentrations(see, e.g., McCarthy et al.,1981). Although thisprocess is reversible, with a dissociation con-stant of ∼1 mg/ml (Cohen et al., 1976), ithampers attempts to determine the behavior onnondenaturing PAGE. However PAGE in thepresence of SDS reveals that the enzyme pre-pared as described here contains only a fewminor impurities (see McCarthy et al., 1980).

Critical Parameters andTroubleshooting

A critical aspect of protein purification pro-cedures is to have everything that will beneeded prepared in advance. For example, it isa recipe for disaster to finish one step and thenfind that the column needed for the next has notyet been equilibrated. Many enzymes are rela-tively unstable and susceptible to degradationby contaminating peptidases. Generally, toavoid losses of enzyme activity, the procedureshould be completed as rapidly as possible.

The essence of any robust purification pro-cedure is that any minor problems with one stepare corrected by the others. Thus, for example,a somewhat lower degree purification on theDEAE-cellulose column may be compensatedfor in the affinity-chromatographic step. Someproblems that are common to many purificationprocedures are listed below.

Failure to use the highest purity reagents.Analytical grade chemicals should be used forsuch procedures as preparing buffers and am-monium sulfate precipitation. Less pure mate-rials may contain compounds that inhibit en-zyme activity.

Working at the wrong temperature. Most ofthe procedures involved should be executed at0° to 4°C. This is most easily accomplished ina cold room, although cooling the material inan ice bucket is satisfactory for some steps.Performing those steps at higher temperatureswill result in enzyme inactivation.

Overzealous stirring. This can cause froth-ing of protein solutions, which results in some


1.4.27


denaturation. Stirring speeds should be fastenough to ensure adequate mixing, but nofaster; if frothing does occur the stirring is toofast. Note that some frothing does occur duringsteps 1 and 2 of Basic Protocol 1, but this doesnot result in significant losses of GDH activity,probably because of the high protein concen-trations at those stages.

Failure to equilibrate chromatographic col-umns. Equilibration can be judged to have oc-curred when the pH and conductivity of theout-flowing buffer is the same as that of thein-flowing equilibration buffer. To use a col-umn before complete equilibration has oc-curred will usually result in poor resolution ofthe sample components.

Using a chromatographic column that is tooold. The degradation of GTP-Sepharose after∼6 months of storage is discussed in SupportProtocol 4. DEAE-cellulose is quite stable ifstored under the conditions described in Sup-port Protocol 4. However, with time, the accu-mulation of tightly-bound material may affectthe performance of columns, in which case therecycling procedure outlined in Support Proto-col 4 should be used, or, alternatively, freshDEAE cellulose can be used.

Failure to check that the pH of buffers iscorrect. The pH should be adjusted at the tem-perature at which it will be used. Some bufferscontain additional components, such as EDTA,and the pH should be checked and, if necessary,adjusted after all ingredients have been added.

Failure to desalt samples before chroma-tographic steps. The presence of high concen-trations of salt may prevent compounds of in-terest from binding to the column. In all caseswhere the enzyme is meant to bind to thecolumn, it is important to retain the materialthat passes unretarded through the column and toassay it for enzyme activity as soon as possible.If material that should have bound passes straightthrough the column, it may be possible to rescueit by further desalting and application to a freshlyequilibrated column. If in doubt about effectivedesalting, check the conductivity.

The flow rate through the column is veryslow, or there is apparently no flow at all. Donot load the enzyme-containing material onto acolumn that is running more slowly than speci-fied. Very slow flow rates may occur if the materialin the column has become compacted. This canarise if the column is run at too high a pressure.Always follow the manufacturers’ instructionsabout the maximum hydrostatic pressure that canbe applied, and do not exceed that value. Theaccumulation of denatured material at the top of

a column can also slow the flow rate. This maybe rectified by removing some of the chroma-tographic material from the top of the column,which is often identifiable by discoloration,adding an equivalent amount of fresh material,and repacking the column (see Support Proto-col 4). In the case of DE-52 DEAE-cellulose,failure to remove the “fines” before using theresin (see Support Protocol 5) can result ininadequate flow rates. Flow rates through col-umns that are flowing well are likely to slowconsiderably if the sample applied containsappreciable amounts of particulate material. Ifparticulate material is present, it should beremoved by centrifugation, or, where appropri-ate, filtration, before the sample is applied tothe column.

Air bubbles or cracks are present in col-umns. This can result in the applied material notcoming into adequate contact with the chroma-tographic material. Air bubbles can occur all tooeasily in gel-filtration media (e.g., Sephadex andSepharose, including GTP-Sepharose) unless themanufacturer’s instructions for packing the ma-terial into columns are heeded. Cracks can occurif the material in the column is allowed to dry out.If that occurs, the only solution is to remove thematerial from the column, rehydrate and re-equilibrate it, and then repack the column.

The following subsections discuss onlythose steps where difficulties may be encoun-tered through causes other than those discussedabove. Should the enzyme lose activity or thefinal product have a specific activity signifi-cantly lower than expected, comparison of thedata for each step with those shown in Tables1.4.5 and 1.4.6 for Basic Protocol 1, or Tables1.4.7 and 1.4.8 for the Alternate Protocol,should indicate where the problem occurred.

Basic Protocol 1: purification of GDHinvolving affinity chromatography onGTP-Sepharose

Ammonium sulfate precipitation. Too rapidaddition of solid ammonium sulfate can result inhigh local concentrations of the salt, causingexcess precipitation of material that does not allredissolve. In the present procedure, homogeni-zation between additions minimizes this problem.If the solid ammonium sulfate salt containslumps, break these up using a pestle and mortarbefore using it. It is possible to perform theprecipitation by continuously stirring the enzymesolution in a beaker and adding the ammoniumsulfate to it. If this approach is used, the salt shouldbe added very slowly, over a period of 1 to 2 hr,to minimize the above problem. In either proce-


1.4.28



Table 1.4.5 Purification of Glutamate Dehydrogenase From Ox Brain According to BasicProtocol 1

Stage (seeFig. 1.4.1)

Volume(ml)

Total protein(mg)

Total activity(µmol/min)

Specific activity(µmol/min/mg)

Purification(x-fold)

Yield(%)

1 1500 75,000a 2280 0.03 1 100 2 1100 23,000a 1150 0.05 2 50 3 250 6200a 925 0.15 5 41 4 600 320b 775 2.4 80 33 5 90 12.5b 500 40 1300 22aProtein concentration determined by the biuret procedure.bProtein concentration determined from the absorbance at 280 nm.

Table 1.4.6 Purification of Glutamate Dehydrogenase From Ox Liver According to BasicProtocol 1


Volume(ml)

Total protein(mg)




Yield(%)

1 500 6000a 1800 0.3 1 100 2 450 2500a 15000 0.6 2 85 3 160 880a 1410 1.6 5 75 4 500 150b 1200 8.0 25 67 5 90 25b 1000 40 120 55aProtein concentration determined by the biuret procedure.bProtein concentration determined from the absorbance at 280 nm.

Table 1.4.8 Purification of Glutamate Dehydrogenase From Rat Liver According to AlternateProtocol 1


Volume(ml)

Total protein(mg)a




Yield(%)

1 90 2720 760 0.3 1 100 2 35 980 505 0.5 2 67 3 9.8 130 195 1.5 5 26 4 1.0 9.4 190 37 140 25aProtein concentration determined by the Lowry method.

Table 1.4.7 Purification of Glutamate Dehydrogenase From Ox Liver According to AlternateProtocol 1


Volume(ml)

Total protein(mg)a




Yield(%)

1 370 8580 1920 0.2 1 100 2 75 2010 1640 0.3 4 86 3 8.4 139 408 2.9 13 21 4 1.2 9.4 376 40 180 20aProtein concentration determined by the Lowry method.


1.4.29


dure, it is important to continue to stir for 20 to30 min after the last ammonium sulfate addi-tion, to ensure that any overprecipitated mate-rial has a chance to redissolve and that anymaterial that should precipitate at that salt con-centration does so.

The use of a homogenizer during this stage hasproven to be effective, but it is not recommendedfor ammonium sulfate fractionation of purer mate-rial, since excessive frothing can result in denatu-ration. Addition of ammonium sulfate will result ina decrease in pH. For GDH purification, it is notnecessary to adjust the pH to the starting valuebetween each addition of ammonium sulfate, al-though such readjustment is necessary for manyother enzymes.

Dialysis and concentration. Provided thatthe dialysis tubing is pretreated as described inUNIT 4.4 and APPENDIX 3B and that it has been testedfor leaks, there should be no difficulties withthese procedures. It should be stressed againthat an air-free space should be left at the topof the knotted dialysis tube, because volumeincreases during dialysis can otherwise causethe bag to rupture. The concentration proce-dures should not cause difficulties if the direc-tions given in Support Protocol 6 are followed.

Chromatography on DEAE cellulose. Com-mon difficulties with this step have been dis-cussed in the general points at the beginning ofthis section.

Chromatography on GTP-Sepharose. If theGDH enzyme does not bind to the column andthe troubleshooting points at the beginning ofthis section have been taken into account, thelikely causes are either that the affinity resinhas degraded (see Support Protocol 4) or thatthere was a problem with its preparation. In firstcase, the GTP-Sepharose should be replaced byfresh material. If there has been a problem withits preparation, a fresh batch should be preparedusing the procedure described in Support Pro-tocol 1 to check for effective GTP coupling.

Choice of reaction buffer. As indicated inBasic Protocol 1, the enzyme is unstable in theTris/acetate buffer and losses of activity canoccur if the enzyme is left in that buffer forextended periods.

Basic Protocol 3: assay of glutamatedehydrogenase activity

In all cases, it will be necessary to dilute theenzyme preparation in order to obtain a rate thatis sufficiently slow to measure with accuracy.The amount of dilution will depend on the stateof purification of the enzyme, its protein con-centation, and also the scale expansion used on

the spectrophotometer. For example, the crudehomogenate of liver GDH has a specific activityof 0.3 µmol/min/mg protein (see Table 1.4.6).

Since the production of 1 µmol of glutamatein the assay will give an absorbance change of2.073 (see Basic Protocol 3, annotation to step5) , 1 mg of this material will give an absorbancedecrease, in 1 min, of 0.3 × 2.073 ≈ 0.62. Theprotein concentration of this crude homogenateis ∼12 mg/ml (see Table 1.4.6); therefore, 50 µlof this would contain 0.6 mg protein, and wouldbe expected to give an absorbance change of∼0.373 absorbance units per min. This wouldmean that the reaction would be finished in <3min, which would be too fast for accurate in-itial-rate determinations. See, for example, Tip-ton (2002) for a fuller treatment of enzymeassays and initial-rate determinations. Dilutionof this material 20× with the assay buffer beforeadding 50 µl to the assay would give an absor-bance change of 0.01865 absorbance units permin, which would mean that it would take alittle over 8 min for the absorbance to fall by0.15 absorbance units. Similar calculationsfrom the values in Table 1.4.6 for the purifiedliver GDH show that a dilution factor of 600would be necessary so that a 50-µl volumeadded to the assay would give a rate similar tothat given by the crude enzyme.

The appropriate dilution factors for the otherstages of purification and for the brain enzymecan be estimated from the date given in Tables1.4.6 and 1.4.5, respectively. It should be notedthat these numbers are only a rough guide, sincethe degree of purification and protein concen-tration may vary for individual steps of thepurification. The addition of smaller volumesof the enzyme preparation to the assay willreduce the degree of dilution necessary.

Alternate Protocol: purification of GDHinvolving affinity precipitation with bis-NAD+

The initial steps in this procedure are thesame as those of Basic Protocol 1.

Affinity precipitation. A pilot-scale precipi-tation, as described in Support Protocol 9,should always be carried out to determine theoptimum concentration of bis-NAD+ to use.Figure 1.4.6 shows an example of a concentra-tion-precipitation relationship, but it should beremembered that the optimum bis-NAD+ con-centration may vary with protein concentration.

Failure to obtain affinity precipitation couldresult from:

1. A fault in the synthesis of the bifunc-tional ligand. Monitoring the success of eachstep in the preparatory procedure using the pro-


1.4.30



cedures outlined in Support Protocol 8 and thedata in Table 1.4.2, should reveal any problemsin this respect and where they occur.

2. Attempting to precipitate from a solutioncontaining too low a concentration of GDH (seeFig. 1.4.7). Further concentration of the enzyme-containing sample should deal with this problem.

StabilityThe enzyme, stored as described in Basic

Protocol 1, is stable for at least 6 months withoutsignificant loss of activity or alteration of itsmobility on a 13% polyacrylamide gel in thepresence SDS. This is important in view of therelatively large amounts of purified GDH that canbe prepared. However, changes can occur in itsresponses to allosteric effectors when the enzymeis stored in an atmosphere of air (Couée andTipton, 1991). These changes, which may onlybecome apparent after 2 to 3 months, are pre-vented by storing the enzyme solution under anatmosphere of nitrogen, and tubes containingstored GDH preparations should be routinelyflushed with N2 each time they are opened.

SafetyAll personnel involved should have received

training in safe laboratory procedures and in theuse of radioactive materials if Support Protocol 2(involving radiolabeled GTP) is to be used. Na-tional and local regulations for the disposal ofsuch materials as radiochemical waste and or-

ganic solvents should be followed. Several ofthe compounds used in these procedures arehighly toxic and should be handled with greatcare. Toxicity information on these compoundsis available from the manufacturers (e.g.,Sigma-Aldrich at https://www.sigma-aldrich.com) and should be consulted in advance. For theuse of animal materials, precautions against in-fectious and transmissible diseases are necessary.It is advisable that personnel be immunizedagainst such diseases where appropriate. Al-though the ox tissue samples that the authors haveused recently have been certified as being BSE-free, the initial steps of the purification are stillperformed under biohazard containment, protec-tion and disposal conditions. It is essential thatthose steps indicated in Support Protocols 1 and3 be performed under an adequately functioning,biohazard-containment level II fume hood. Safetyglasses should be worn, and, in connection withthe TLC procedure in Support Protocol 8, theseshould provide protection from UV light.

Anticipated Results

Basic Protocol 1: purification of ox brainor liver glutamate dehydrogenase involvingaffinity chromatography on GTP-Sepharose

Tables 1.4.5 and 1.4.6 summarize the resultsobtained from typical purifications of the oxbrain and liver enzymes using this procedure.

500

4

6

8

10

100 150Fraction number

4

6

2

A280 nm ( )conductivity ( )GDH Activity ( )

Con

duct

ivity

(m

S)

GD

H a

ctiv

ity (µ

mol

/min

/ml)

Figure 1.4.8 Chromatography of ox liver glutamate dehydrogenase at step 3 of Basic Protocol 1.A typical elution profile is shown. However the exact behavior may vary between preparations. Thefraction size was ∼10 ml. In the left-hand y-axis label, mS stands for millisiemens.


1.4.31


Examples of the elution profiles fromDEAE-cellulose and GTP-Sepharose areshown in Figures 1.4.8 and 1.4.9.

Alternate Protocol: purification of rat or oxliver GDH involving affinity precipitationwith bis-NAD+

Tables 1.4.7 and 1.4.8 summarize the resultsobtained from typical purifications of the oxand rat liver enzymes using this procedure.

Time ConsiderationsThe time estimates given below do not include

the time involved in pouring and equilibrating thechromatographic columns, which should be donebefore the purification procedure is started.

Basic Protocol 1: purification of ox brain orliver glutamate dehydrogenase involvingaffinity chromatography on GTP-Sepharose

The entire purification procedure can easilybe performed in 3 days.

Day 1: Extraction and ammonium sulfateprecipitation, followed by overnight dialysis.

Day 2: DEAE-cellulose fractionation andconcentration of appropriate column fractions.

Day 3: Gel filtration into the GTP-Sepharose buffer, passage through the hydraz-ide gel column, and affinity chromatographyon GTP-Sepharose.

This schedule excludes the time taken to pre-pare the GTP-Sepharose. This should take 3 daysor less, starting from CNBr-activated Sepharose.However much of this time is taken up by incu-bations, such as the 16- to 18-hr reaction ofL-glutamic acid γ-methyl ester with the activatedgel (see Support Protocol 1). The affinity resin,once made, can be used several times.

Alternate Protocol: purification of rat or oxliver GDH involving affinity precipitationwith bis-NAD+

This is a somewhat quicker procedure thanBasic Protocol 1 because it avoids the finalcolumn chromatography steps. However, it stillextends into several days. The first two of theseare the same as those for Basic Protocol 1, andthe third is for the pilot-scale precipitationstudy and the affinity precipitation itself.

The preparation of bis-NAD+ can be com-pleted in 4 days, provided that the reaction

A280 nm ( )conductivity ( )GDH Activity ( )

0

0.5

1.0

1.5

2.0

2.5

15 30 45

2

4

6

8

10

12

30

20

10

Con

duct

ivity

(m

S)

Glu

tam

ate

dehy

drog

enas

e a

ctiv

ity (µ

mol

/min

/ml)

Fraction number

A28

0

Figure 1.4.9 A typical elution profile of affinity chromatography of ox liver glutamate dehydro-genase on GTP-Sepharose at step 4 of Basic Protocol 1. The exact chromatographic behavior mayvary between preparations. The fraction size was ∼10 ml. In the label for the inset scale, mS standsfor millisiemens.


1.4.32



between iodoacetate and NAD+ is performed at37°C (see Support Protocol 7). Once prepared,the bis-NAD+ may be used for several GDHpreparations, as well as for the purification ofsome other dehydrogenases (see Irwin and Tip-ton, 1996)

Literature CitedBeattie. R.E., Buchanan, M.,. and Tipton K.F. 1987.

The synthesis of N2, N2-adipodihydrazido-bis-(N-carboxymethyl-ATP) and its use in the puri-fication of phosphofructokinase. Biochem. Soc.Trans. 15:1043-1044.

Cho, S.W., Lee, J., and Choi, S.Y. 1995. Two solubleforms of glutamate dehydrogenase isoproteinsfrom bovine brain. Eur. J. Biochem. 233:340-346.

Cohen, R.J., Jedziniak, J.A., and Benedek, G.B.1976. The functional relationship between po-lymerization and catalytic activity of beef liverglutamate dehydrogenase. II. Experiment. J.Mol. Biol. 108:179-199.

Couée, I. and Tipton, K.F. 1989a. Activation ofglutamate dehydrogenase by L-leucine. Bio-chim. Biophys. Acta 995:97-101.

Couée, I. and Tipton, K.F. 1989b. The effects of phos-pholipids on the activation of glutamate dehydro-genase by L-leucine. Biochem. J. 261:921-925.

Couée, I. and Tipton, K.F. 1990a. The inhibition ofglutamate dehydrogenase by some antipsychoticdrugs. Biochem. Pharmacol. 39:827-832.

Couée, I. and Tipton, K.F. 1990b. Inhibition of oxbrain glutamate dehydrogenase by perphenazineBiochem. Pharmacol. 39:1167-1173.

Couée, I. and Tipton, K.F. 1991. The sulphydrylgroups of ox brain and liver glutamate dehydro-genase preparations and the effects of oxidationon their inhibitor sensitivities.. Neurochem. Res.16:773-780.

Dawson, R.M.C., Elliott, D.C., Elliott ,W.H., andJones, K.M. 1989. Data for Biochemical Re-search, 3rd ed. Clarendon Press, Oxford.

Engel, J.D. 1975. Mechanism of the Dimroth rear-rangement in adenine. Biochem. Biophys. Res.Commun. 64:581 - 585.

Fahien, L.A., Strmecki, M., and Smith, S. 1969.Studies of gluconeogenic mitochondrial en-zymes. I. A new method of preparing bovine liverglutamate dehydrogenase and effects of purifica-tion methods on properties of the enzyme. Arch.Biochem. Biophys. 130:449-455.

Fang, J., Hsu, B.Y., MacMullen, C.M., Poncz, M.,Smith, T.J., and Stanley, C.A. 2002. Expression,purification and characterization of human glu-tamate dehydrogenase (GDH) allosteric regula-tory mutations. Biochem J. 363:81-87.

Flygare, S., Griffin, T., Larsson, P.O., and Mosbach,K. 1983. Affinity precipitation of dehydro-genases. Anal. Biochem. 133:409-416.

Godinot, C, Julliard, J.H., and Gautheron, D.C.1974. A rapid and efficient new method of puri-fication of glutamate dehydrogenase by affinity

chromatography on GTP-sepharose. Anal. Bio-chem. 61:264-270.

Graham, L.D., Griffin, T.O., Beattie, R..E.,McCarthy, A..D., and Tipton, K.F. 1985. Purifi-cation of liver glutamate dehydrogenase by af-finity precipitation and studies on its denatura-tion. Biochim. Biophys. Acta 828:266-269.

Hahn-Deinstrop, E. 2000. Applied Thin-LayerChromatography: Best Practice and Avoidanceof Mistakes. Wiley-VCH, Weinheim, Germany.

Halliwell, C.M., Morgan, G., Ou, C.P., and Cass,A.E. 2001. Introduction of a (poly)histidine tagin L-lactate dehydrogenase produces a mixtureof active and inactive molecules. Anal. Biochem.295:257-261.

Hiser, C., Mills, D.A., Schall, M., and Ferguson-Miller, S. 2001. C-terminal truncation and his-tidine-tagging of cytochrome c oxidase subunitII reveals the native processing site, shows in-volvement of the C-terminus in cytochrome cbinding, and improves the assay for protonpumping. Biochemistry 40:1606-1615.

Irwin, J.A., and Tipton, K.F. 1996. Affinity precipi-tation methods. In Methods in Molecular Biol-ogy 59: Protein Purification Protocols (S.Doonan, ed.) pp. 217-238. Humana Press, To-towa, N.J.

Jackson, R.J., Wolcott, R.M., and Shiota, T. 1973.The preparation of a modified GTP-sepharosederivative and its use in the purification of dihy-droneopterin triphosphate synthetase, the firstenzyme in folate biosynthesis. Biochem Biophys.Res. Commun. 51:428-435.

Larsson, P.O., Flygare, S., and Mosbach, K. 1984.Affinity precipitation of dehydrogenases. Meth-ods Enzymol. 104:364-349.

Ledent, P., Duez, C., Vanhove, M., Lejeune, A.,Fonze, E., Charlier, P., Rhazi-Filali, F., Thamm,I., Guillaume, G., Samyn, B., Devreese, B., VanBeeumen, J., Lamotte-Brasseur, J., and FrereJ.M. 1997. Unexpected influence of a C-termi-nal-fused His-tag on the processing of an en-zyme and on the kinetic and folding parameters.FEBS Lett. 413:194-196.

Lee, W.K., Shin, S., Cho, S.S., and Park, J.S. 1999.Purification and characterization of glutamatedehydrogenase as another isoprotein binding tothe membrane of rough endoplasmic reticulum.J. Cell. Biochem. 76:244-253.

McCarthy, A.D. and Tipton, K.F. 1984. The effectsof magnesium ions on the interactions of ox brainand liver glutamate dehydrogenase with ATP andGTP. Biochem. J. 220:853-855.

McCarthy, A.D. and Tipton, K.F. 1985. Ox liverglutamate dehydrogenase: Comparison of thekinetic properties of native and proteolysedpreparations. Biochem. J. 230:95-99.

McCarthy, A.D., Walker, J.M., and Tipton, K.F.1980. Purification of glutamate dehydrogenasefrom ox brain and liver: Evidence that commer-cially available preparations of the enzyme havesuffered proteolytic cleavage. Biochem. J.191:605-611.


1.4.33


McCarthy, A.D., Johnson, P., and Tipton, K.F. 1981.Sedimentation properties of native and pro-teolysed preparations of ox glutamate dehydro-genase. Biochem. J. 199:235-236.

McDaniel, H.G. 1995. Comparison of the primarystructure of nuclear and mitochondrial glutamatedehydrogenase from bovine liver. Arch. Bio-chem. Biophys. 319:316-321.

Mulcahy, P. and O’Flaherty, M. 2001. Kinetic lock-ing-on strategy and auxiliary tactics for bioaf-finity purification of NAD(P)(+)-dependent de-hydrogenases. Anal. Biochem. 299:1-18.

O’Carra, P., Griffin, T., O’Flaherty, M., Kelly, N.,and Mulcahy, P. 1996. Further studies on thebioaffinity chromatography of NAD(+)-depend-ent dehydrogenases using the locking-on effect.Biochim. Biophys. Acta 1297:235-243.

O’Flaherty, M., O’Carra, P., McMahon, M., andMulcahy, P. 1999. A “stripping” ligand tactic foruse with the kinetic locking-on strategy: Its usein the resolution and bioaffinity chroma-tographic purification of NAD(+)-dependent de-hydrogenases. Protein Expr. Purif. 16:424-431.

Peterson, P.E. and Smith, T.J. 1999. The structure ofbovine glutamate dehydrogenase provides in-sights into the mechanism of allostery. StructureFold. Des. 15:769-782.

Plaitakis, A. and Zaganas, I. 2001. Regulation ofhuman glutamate dehydrogenases, implicationsfor glutamate, ammonia and energy metabolismin brain. J. Neurosci. Res. 66:899-908.

Plaitakis, A., Metaxari, M., and Shashidharan, P.2000. Nerve tissue-specific (GLUD2) andhousekeeping (GLUD1) human glutamate dehy-drogenases are regulated by distinct allostericmechanisms: Implications for biologic function.J. Neurochem. 75:1862-1869.

Preiss, T., Hall, A.G., and Lightowlers, R.N. 1993.Identification of bovine glutamate dehydro-genase as an RNA-binding protein. J. Biol.Chem. 268:24523-24526.

Rajas, F. and Rousset, B. 1993. A membrane-boundform of glutamate dehydrogenase possesses anATP-dependent high-affinity microtubule-bind-ing activity. Biochem. J. 295:447-455.

Shashidharan, P., Michaelides, T.M., Robakis, N.K.,Kretsovali, A., Papamatheakis, J., and Plaitakis,A. 1994. Novel human glutamate dehydrogenaseexpressed in neural and testicular tissues andencoded by an X-linked intronless gene. J. Biol.Chem. 269:16971-16976

Shashidharan, P., Clarke, D.D., Ahmed, N.,Moschonas, N., and Plaitakis, A. 1997. Nervetissue-specific human glutamate dehydrogenasethat is thermolabile and highly regulated by ADP.J. Neurochem. 68:1804-1811.

Smith, T.J., Peterson, P.E., Schmidt, T., Fang, J., andStanley, C.A. 2001. Structures of bovine gluta-mate dehydrogenase complexes elucidate themechanism of purine regulation. J. Mol. Biol.307:707-720.

Tipton K.F. 2002. Principles of enzyme assay andkinetic studies. In Enzyme Assays: A Practical

Approach (R. Eisenthal and M.J. Danson, eds.)pp. 1-47. Oxford University Press, Oxford, U.K.

Tipton, K.F. and Couée, I. 1988. Glutamate dehy-drogenase. In Glutamine and Glutamate in Mam-mals. (E. Kvamme, ed.) pp.81-100. CRC Press,Baton Roca, Fla.

Wilson, K. and Walker, J. (eds.) 2000. Principles andTechniques of Practical Biochemistry, 5th ed..Cambridge University Press, Cambridge, U.K.

Yoon, H.Y., Hwang, S.H., Lee, E.Y., Kim, T.U., Cho,E.H., and Cho, S.W. 2001. Effects of ADP ondifferent inhibitory properties of brain glutamatedehydrogenase isoproteins by perphenazine.Biochimie 83:907-913.

Zaganas, I. and Plaitakis, A. 2002. A single aminoacid substitution (Gly456Ala) in the vicinity ofthe GTP binding domain of human housekeep-ing glutamate dehydrogenase markedly attenu-ates GTP inhibition and abolishes the enzyme’scooperative behavior. J. Biol. Chem. 277:26422-26428.

Key References

Glutamate dehydrogenasePlaitakis, A. and Zaganas, I. 2001. See above.

Tipton, K.F. and Couée, I. 1988. See above.

There have been few comprehensive reviews of glu-tamate dehydrogenase in recent years. The refer-ences cited above cover some fundamental aspects.

Affinity chromatographyMatejtschuk, P. (ed.) 1997. Affinity Separations: A

Practical Approach. Oxford University Press, U.K.

A collection of chapters on the theory and practiceof different aspects of affinity chromatography.

Affinity precipitationIrwin, J.A. and K.F. Tipton, K.F. 1995. Affinity

precipitation: A novel approach to protein puri-fication. Essays Biochem. 29:137-156.

Describes the theory underlying affinity precipita-tion and reviews its applications.

Contributed by Martha Motherway and Keith F. TiptonDepartment of BiochemistryTrinity CollegeDublin, Ireland

Alun D. McCarthyGlaxoSmithKlineMiddlesex, United Kingdom

Ivan CouéeCentre National de la Recherche ScientifiqueRennes, France

Jane IrwinUniversity College DublinDublin, Ireland


1.4.34



UNIT 1.5Overview of the Physical State of ProteinsWithin Cells

The word protein comes from the Greekword proteios, meaning primary. And, indeed,proteins are of primary importance in the studyof cell function. It is difficult to imagine acellular function not linked with proteins. Al-most all biochemical catalysis is carried out byprotein enzymes. Proteins participate in generegulation, transcription, and translation. Intra-cellular filaments give shape to a cell whileextracellular proteins hold cells together toform organs. Proteins transport other mole-cules, such as oxygen, to tissues. Antibodymolecules contribute to host defense againstinfections. Protein hormones relay informationbetween cells. Moreover, protein machines,such as actin-myosin complexes, can performuseful work, including cell movement. Thus,studying proteins is a prerequisite in under-standing cell structure and function.

The physical characterization of proteinsbegan well over 150 years ago with Mulder’scharacterization of the atomic composition ofproteins. In the latter half of the nineteenthcentury Hoppe-Seyler (1864) crystallized he-

moglobin and Kühn (1876) purified trypsin. Avariety of physical methods have been devel-oped over the years to increase convenience andprecision in the characterization and isolationof proteins. These include ultracentrifugation,chromatography, electrophoresis, and others.In many instances our understanding of cellproteins parallels the introduction and use ofnew techniques to examine their structure andfunction.

PROTEIN CLASSIFICATIONSAll proteins are constructed as a linear se-

quence(s) of various numbers and combina-tions of ∼20 α-amino acids joined by peptidebonds to form structures from thousands tomillions of daltons in size. Proteins are the mostcomplex and heterogeneous molecules foundin cells, where they account for >50% of thedry weight of cells and ∼75% of tissues.

Proteins can be classified into three broadgroups: globular, fibrous, and transmembrane(Fig. 1.5.1; Table 1.5.1). Globular proteins are,by definition, globe-shaped, although in prac-

Supplement 31

Contributed by Howard R. PettyCurrent Protocols in Protein Science (2002) 1.5.1-1.5.10Copyright © 2002 by John Wiley & Sons, Inc.

Figure 1.5.1 Generalclassifications of proteins. Inthese schematic representationsof globular, fibrous, andtransmembrane proteins,hydrophobic regions are shaded.Note that the disposition ofhydrophobic residues oftenreflects the protein class.

transmembrane

fibrous

globular

1.5.1


tice they can be spherical or ellipsoidal. Globu-lar proteins are generally soluble in aqueousenvironments. Examples of globular proteinsare hemoglobin, serum albumin, and most en-zymes. Fibrous proteins are elongated linearmolecules that are generally insoluble in waterand resist applied stresses and strains. Collagenis a physically tough molecule of connectivetissue. Just as collagen gives strength to con-nective tissues, intermediate filaments linkedto desmosomes give strength to cells in tissues.The third general class of proteins, transmem-brane proteins, contain a hydrophobic sequenceburied within the membrane; these proteins arediscussed more fully below (see MembraneProteins).

These protein categories are not mutuallyexclusive. For example, the nominally fibrousintermediate filament proteins also have globu-lar domains. Similarly, transmembrane pro-teins almost always possess globular domains.Thus, these definitions serve as a useful guidebut should not be rigidly applied.

HYDROPATHY PATTERNS OFTENREFLECT A PROTEIN’SCLASSIFICATION

A key physical feature of proteins is theirhydropathy pattern (i.e., the distribution of hy-drophobic and hydrophilic amino acid resi-dues). Indeed, hydrophobic interactions pro-vide the primary net free energy required forprotein folding. Figure 1.5.1 illustrates the dis-position of hydrophobic amino acids in pro-teins. In an intact globular protein, hydrophobic

amino acids are generally shielded from theaqueous environment by coalescing at the cen-ter of the molecule, with the more hydrophilicresidues exposed at its surface. However, thelinear arrangement of hydrophobic residuesfluctuates in an apparently random fashion. Theα helices within globular proteins may expressa hydrophobic face oriented toward the centerof the protein. (Within these helices hydropho-bic residues are nonrandomly positioned everythree or four amino acids to yield a hydrophobicface.) For coiled-coil α helix–containing fi-brous proteins, such as tropomyosin and α-keratin, hydrophobic residues at periodic inter-vals allow close van der Waals contact of thechains and potentiate assembly as hydrophobicresidues are removed from the aqueous envi-ronment (Schulz and Schirmer, 1979; Parry,1987). Secondarily, regularly spaced chargedgroups can also contribute to the shape of fi-brous proteins (Schulz and Schirmer, 1979;Parry, 1987). Transmembrane proteins providea rather different physical arrangement of hy-drophobic residues in which hydrophobic resi-dues are collected primarily into a series ofamino acids that is embedded within a cellmembrane.

One important means of analyzing the hy-dropathy of a sequenced protein is a hydropathyplot (Kyte and Doolittle, 1982). In this method,each amino acid residue is assigned a hy-dropathy value, an ad hoc measure that largelyreflects its relative aqueous solubility; thesevalues are plotted after being averaged. Thesuccessful interpretation of hydropathy plots

Table 1.5.1 Broad Classifications for Proteinsa

Type Location/type Examples

Globular Intracellular Hemoglobin, lactatedehydrogenase, cytochrome c

Extracellular Serum albumin,immunoglobulins, lysozyme

Fibrous Intracellular Intermediate filaments,tropomyosin, lamins

Extracellular Collagen, keratin, elastins

Transmembrane Single pass Insulin receptor, glycophorin,HLAsb

Multipass Glucose transporter, rhodopsin,acetylcholine receptor

aAdditional information regarding fibrous and transmembrane proteins can be found in Squireand Vibert (1987) and Petty (1993). Information concerning globular proteins can be found innumerous books on proteins and enzymes such as Schultz and Schirmer (1979).bHuman histocompatibility leukocyte antigens.


1.5.2

Overview of thePhysical State ofProteins Within

Cells

depends on the parameters chosen for averag-ing. The parameters are the number of residuesaveraged (amino acid interval or “window”)and how many amino acids are skipped whencalculating the next average (step size). Usingthis approach with a window of ∼10 residues,it is often possible to find the positions ofhydrophobic residues coalescing near the inte-rior of globular proteins. The method is particu-larly useful in predicting transmembrane do-mains of proteins, generally with a window of∼20 amino acids. To detect the repetitious pat-tern of coiled-coil fibrous proteins, however,windows smaller than the repeat length wouldbe required.

MEMBRANE PROTEINSIn addition to their presence in the extracel-

lular and intracellular milieus, proteins are alsofound in association with biological mem-branes. Proteins constitute one-half to three-quarters of the dry weight of membranes. Mem-brane proteins perform a broad variety of func-tions including intermembrane and intercellularrecognition, transmembrane signaling, most en-ergy-harvesting processes, and biosynthesis inthe endoplasmic reticulum (ER) and Golgi com-plex.

Membrane proteins have been traditionallycharacterized as integral (or intrinsic) or pe-ripheral (or extrinsic) on the basis of opera-tional criteria. Peripheral membrane proteinsare associated with membrane surfaces and canbe dislodged from membranes using hypotonicor hypertonic solutions, pH changes, or chela-

tion of divalent cations. Components of theerythrocyte membrane skeleton, for example,are peripheral membrane proteins. Althoughmost peripheral proteins are removed by wash-ing a sample with buffers, integral proteinscannot be removed by such treatments. To iso-late integral membrane proteins, which are em-bedded within the lipid bilayer, one must usedetergents that disrupt the bilayer and bind tothe proteins, thus solubilizing them. In general,integral membrane proteins have a portion oftheir peptide sequence buried in the lipid bi-layer whereas peripheral proteins do not. How-ever, the discovery of glycosylphosphatidyli-nositol (GPI)-linked membrane proteins addedto the ambiguity of the situation. GPI-linkedproteins are globular proteins with no mem-brane-associated peptide sequence, yet theyrequire harsh conditions for solubilization.

As the technology for studying membraneproteins improved, it became necessary to de-velop a more precise vocabulary to describemembrane proteins. Transmembrane integralmembrane proteins have at least one stretch ofamino acids spanning a membrane. Membraneproteins are classified as type I, II, III, or IVdepending on the nature of their biosynthesisand topology in membranes (Spiess, 1995; Ta-ble 1.5.2 and Fig. 1.5.2). The biosynthetic in-sertion of these proteins in membranes is, inturn, dependent on the presence or absence ofa cleavable signal peptide, the relative positionsof the hydrophobic transmembrane domain andpositively charged topogenic signals, and/or

Table 1.5.2 Definitions of Integral Transmembrane Proteins

Type Definition Examples

I An N-terminal–cleavable signal peptide is removedat the luminal face yielding a luminal N terminalduring biosynthesis. (Positive charges are found onC-terminal side of first long hydrophobic sequenceafter the signal peptide.)

LDL receptor, insulin receptor,glycophorin A, thrombinreceptor

II An N-terminal–uncleaved signal peptide leads to acytoplasmic N terminus. (Positive charges aregenerally found on N-terminal side of first longhydrophobic sequence.)

Transferrin receptor,sucrase/isomaltase, band 3

III A long N-terminal hydrophobic sequence isfollowed by a sequence of positive charges. Thisleads to a luminal N terminus in the absence of acleavable signal peptide.

β-Adrenergic receptor,cytochrome P450

IV A short C terminus is present at the luminal side ofmembrane. A large N terminus is exposed at thecytoplasmic face.

Synaptobrevin, UBC6


1.5.3


the mechanism of nascent protein delivery tothe ER.

Type I membrane proteins are synthesizedwith an amino-terminal signal sequence that isinserted into the ER membrane. When the sig-nal sequence is proteolytically removed in theER lumen, a new luminal amino terminus isexposed. A series of positively charged residuesat the C-terminal side of the first hydrophobictransmembrane domain following the signalsequence generally denotes the end of the firsttransmembrane domain (von Heijne and Gavel,1988). Although a hydrophobic transmem-brane domain followed by a positive sequenceof amino acids is sufficient to act as a stop-trans-fer signal, this motif is not required for stop-transfer events and other, less well-understoodregulatory mechanisms are also involved (An-drews and Johnson, 1996).

Membrane proteins types I, II, and III aredelivered to the ER membrane via a signalrecognition particle (SRP)-dependent mecha-nism. In contrast to type I proteins, type II andIII membrane proteins do not have a cleavableN-terminal signal sequence. Instead, they havean internal hydrophobic signal that acts as botha signal sequence for ER delivery and a trans-membrane domain in the mature protein. TypeII proteins have a cytoplasmic amino terminusand a luminal (or extracellular) carboxyl termi-nus. In this case a positively charged sequence

of amino acids at the N-terminal side of the firsthydrophobic sequence causes the amino termi-nus to be retained at the cytoplasmic face of theER membrane. Thus the internal uncleavedsignal peptide becomes the transmembrane do-main of the mature protein.

Type III membrane proteins have the sameoverall topology as type I proteins, but they areinserted into membranes by a different mecha-nism. In type III proteins the first hydrophobicsequence of amino acids is immediately fol-lowed by a series of positively charged aminoacids. Thus, the first hydrophobic sequencebecomes the transmembrane domain of theprotein, with the amino terminus at the luminalface of the membrane.

Type IV membrane proteins are charac-terized by a large, cytoplasmically exposedamino-terminal domain and a short carboxyl-terminal domain facing the lumen. Importantly,these proteins are delivered to the ER by anunknown SRP-independent mechanism.

In addition to the single-pass membraneproteins just described, integral membrane pro-teins can display zero, two, three, or moretransmembrane domains. Some membraneproteins, such as cytochrome b5, have proteinsegments buried in the hydrophobic core ofmembranes but do not cross the membrane.Membrane proteins with multiple membrane-spanning domains are classified as type I, II, or

type I type II type III type IVN

NC

C

C N

+++

+++

+++

C

N C N

cytosol

bila

yer

nontransmembrane transmembrane

exterior or lumen

Figure 1.5.2 Membrane proteins containing hydrophobic anchors. A nontransmembrane ormonotopic membrane protein is anchored to the membrane via a hydrophobic amino acid se-quence. Transmembrane proteins are classified as types I, II, III, and IV (Table 1.5.2). The firsttransmembrane segment of a multispanning membrane protein can be inserted as in type I, II, orIII proteins. This segment functions as a start-transfer peptide. Subsequent transmembranesegments will function as stop-transfer and start-transfer sequences, resulting in a multispanningmembrane topology.


1.5.4


Cells

III depending on the topogenic signals in thefirst transmembrane domain. For example, amultispan membrane protein with a cleavablesignal sequence, luminal amino terminal, anda positively charged sequence following thefirst transmembrane domain from the aminoterminal, such as the thrombin receptor, is atype I membrane protein. The remaining trans-membrane domains are inserted into the bilayerdepending on the orientation of the first trans-membrane domain. Multispanning type II andIII proteins are similarly defined according tothe properties of their single-spanning counter-parts.

In addition to hydrophobic protein se-quences acting as membrane anchors, mem-brane proteins may also carry bilayer-associ-ated hydrophobic lipid components. These hy-drophobic lipid anchors define three broadgroups of lipid-modified proteins: fatty acyl-ated, isoprenoid-linked, and GPI-linked (Fig.1.5.3). Several cytosolic transmembrane pro-teins have been identified that contain a cova-lently attached hydrophobic fatty acyl residue.For example, fatty acids, including palmitic,palmitoleic, cis-vaccenic, and cyclopropylene-

hexadecanoic, are covalently linked to theamino terminus and the amino-terminalglycerylcysteine of E. coli lipoprotein. More-over, palmitate- and myristate-labeled trans-membrane proteins have been observed in eu-karyotic cells (e.g., Schlesinger et al., 1980).

In both isoprenoid-linked and GPI-linkedproteins, globular proteins become membrane-bound due to the addition of a hydrophobiclipid moiety. Certain proteins containing con-served cysteine residues at or near the C-termi-nus are modified by prenylation, in which afarnesyl or geranylgeranyl isoprenoid tail isadded (Zhang and Casey, 1996). This hydro-phobic moiety promotes protein associationwith the cytoplasmic face of cell membranes.Notably, cytosolic G proteins and protein ki-nases that participate in signal transduction areprenylated.

GPI-linked proteins are a major class ofmembrane proteins (Cardoso de Almeida,1992; Englund, 1993). In contrast to iso-prenoid-modified proteins, GPI-linked pro-teins are attached to the luminal or extracellularface of membranes via a glycosylphospha-tidylinositol anchor of variable structure (e.g.,

exterior orlumen

cytosol

bila

yer

S

EtN

P

P

P

Cys-CH3

S

isoprenoid-linked

fatty acylated GPI-linked

C O

Figure 1.5.3 Membrane proteins containing lipid moieties. In the simplest case, fatty acids canbe covalently attached to transmembrane proteins. Hydrophobic tails are also attached to proteinsto form isoprenoid-linked proteins. A third class of lipid-attached proteins are the GPI-linked proteins.Hydrophobic regions are shaded.

Current Protocols in Cell Biology Supplement 31

1.5.5


Fig. 1.5.3). Well over 100 GPI-linked proteinshave been identified in cells, where they per-form numerous functions including acting asenzymes and receptors. The ability of GPI-linked proteins, which possess no transmem-brane or cytosolic sequences, to elicit trans-membrane signals seems paradoxical. How-ever, studies have suggested that interactionswith other proteins, including transmembraneintegrins (Petty et al., 1996), contribute to trans-membrane signaling of these proteins. In addi-tion, GPI-linked proteins can collect in micro-domains called lipid rafts within cell mem-branes (Rietveld and Simons, 1998). AlthoughGPI-linked proteins must collaborate withother membrane proteins to elicit signals, theydo possess certain functional advantages. First,GPI-linked proteins (and isoprenoid-linkedproteins as well) diffuse in membranes muchfaster than transmembrane proteins and thusrelay information faster. Second, certain cells,such as leukocytes, can rapidly shed their GPI-linked proteins, thus altering their functionalproperties in seconds. Although the importanceof lipid-linked membrane proteins has onlyrecently been appreciated, the impact of thesestructures on our understanding of cell proper-ties is growing rapidly.

ADDITIONAL FACTORSAFFECTING THE PHYSICALHETEROGENEITY OF PROTEINS

Additional factors contributing to the physi-cal-chemical heterogeneity of proteins are size,charge, chemical modifications, and assembly.A typical amino acid has a molecular mass of∼110 Da, and a small protein has a molecularmass of a few thousand daltons (e.g., for insulin,Mr = 5733). Large proteins have molecularmasses of several hundred thousand daltons.When proteins are assembled to form largemultiprotein complexes such as ribosomes,molecular masses are well into the millions.The diameters of these structures range from 4Å for an individual amino acid to ∼30 nm for aribosome.

Electrostatic charge is of major importancein protein structure and function. Charged pro-teins are more soluble than uncharged proteins.The large number of positive charges on his-tones allow them to bind DNA. The spatialarrangement of charges on cytochrome c allowsit to bind the complementary charges of itsoxidase and reductase, thereby orienting theproteins prior to electron transfer. Similarly, thearrangement of charges on the apoprotein andreceptor for low-density lipoprotein (LDL) al-

lows for lock-and-key–like interactions (Petty,1993). In addition to structural and bindingconsiderations, electrostatic interactions play aregulatory role. For example, the phosphoryla-tion and dephosphorylation of insulin receptorsalter electrostatic interations between the activesite and a regulatory loop of the kinase domain,thereby changing its three-dimensional shape(Hubbard et al., 1994). This changes the Vmax

of the kinase, thus triggering intracellular sig-nals.

In addition to the types of physical hetero-geneity listed above, >100 distinct chemicalmodifications of proteins have been observed.These include, for example, glycosylation,ubiquitin attachment, phosphorylation, acety-lation, and hydroxylation (Table 1.5.3). Thus,proteins undergo extensive physical-chemicalmodification.

PROTEIN ASSEMBLIESProteins can be assembled in a variety of

states in both aqueous media and within mem-branes. Protein assembly into complex supra-molecular structures plays vital roles in enzymeregulation, cell skeleton formation, and trans-membrane signaling. Both covalent bonds andnoncovalent bonds participate in protein as-sembly. One frequently encountered covalentmechanism of protein assembly is the forma-tion of disulfide bonds. These covalent linkagesoften form during protein maturation. They canlink two separate proteins together or two por-tions of the same protein. For example, the twochains of insulin molecules are held togetherby disulfides, as are the two chains of its mem-brane receptor. However, disulfide bond forma-tion is mostly limited to oxidative environmentssuch as the ER lumen and the exterior face ofthe cell surface.

One of the best-known examples of nonco-valent assembly is the formation of hemoglobintetramers. Polymerization is another frequentlyencountered mechanism for protein assemblyin cells. The globular protein actin polymerizesto form microfilaments in the absence of cova-lent bond formation. Intermediate filaments areformed by the polymerization of fibrous pro-teins. Under certain circumstances transmem-brane proteins polymerize as well; bacteriorho-dopsin, for example, forms two-dimensionalpseudocrystals called purple membranes. Pro-tein assemblies formed from various numbersof similar units are homodimers, homooligo-mers, and homopolymers.

Assembly of protein structures from dis-similar subunits is more common than assem-


1.5.6


Cells

bly from identical subunits. For example, het-erodimers are formed from the α and β chainsof integrins within cell membranes. Complexheterooligomeric and heteropolymeric struc-tures vary from relatively small structures suchas histone octamers, which bind to DNA in thenucleus, to large particles such as ribosomes,found both in the cytosol and attached to nu-clear and ER membranes. The signal recogni-tion particle is a relatively small heterooli-gomeric structure, composed of one RNAsubunit and six proteins, that potentiates thedelivery of secretory and most membrane pro-teins to the ER membrane. Membrane-associ-ated heterooligomeric structures have also beenobserved. One of the best examples of suchstructures is the components of the electrontransport systems in chloroplasts and mito-chondria (Petty, 1993). For example, theubiquinone-cytochrome c reductase is com-posed of eleven different subunits. Thus, pro-teins can be assembled in a variety of mannerswithin cells.

Although some protein assemblies, such asintermediate filaments, are static structures,many are dynamic structures which providefunctional flexibility. For example, microfila-ments can rapidly assemble and disassemble.In addition to the physical changes in assemblystate, compositional dynamics is also observed.

For example, interferon γ treatment alters thecomposition of proteasomes. Developmentalchanges in protein composition are also ob-served. As an example, fetal and newborn formsof a component of cytochrome c reductase areexpressed in humans. Thus, protein assembliescan be characterized by both physical and com-positional dynamics.

ALTERING THE SOLUBILITY OFPROTEINS: PROTEINEXTRACTION

The in vitro characterization of cellular pro-teins begins with their extraction from tissuesor cells into a buffer. With the exception ofglobular secretory proteins, such as those foundin plasma, proteins are generally not easilyaccessible for experimental manipulation. Forexample, many fibrous proteins are not solublein aqueous buffers. Cellular proteins are en-trapped within or on a cell and therefore mustbe extracted from the cell in a soluble form.

A variety of methods including osmoticlysis, enzyme digestion, homogenization usinga blender or mortar and pestle, and disruptionby French press and sonication have been em-ployed to disrupt cells. For a cytosolic proteinsuch as hemoglobin, no further extraction fromthe sample is necessary. However, many impor-tant cellular proteins, such as those associated

Table 1.5.3 Common Physical-Chemical Modifications of Proteinsa

Modification Example

Homodimerization Transferrin receptorHomooligomerization S. typhimurium glutamine synthetaseHomopolymerization ActinHeterodimerization IntegrinsHeterooligomerization Histones, proteasomesHeteropolymerization RibosomesProteolytic cleavage Signal peptide cleavage in ERProsthetic group addition Heme addition to cytochromes and hemoglobinOxidation-reduction Disulfide bond formation in ERGlycosylation Glycoprotein maturationPhosphorylation Regulation of protein function, such as the tyrosine kinase

activity of insulin receptorsAcetylation Blockage of N-termini of certain membrane proteinsUbiquitination Ubiquitin-dependent proteolysis via proteasomes, histonesHydroxylation Proline hydroxylation on collagenFatty acylation Insulin receptors, E. coli lipoproteinIsoprenylation G proteinsGPI addition Alkaline phosphatase, urokinase receptorsaFor details, see Freedman and Hawkins (1980, 1985), Schlesinger et al. (1980), Englund (1993), and Zhang andCasey (1996).


1.5.7


with membranes, cytoskeletal components, andDNA, remain insoluble. To further solubilizecell proteins, both nonionic (e.g., Triton X-100)and ionic (e.g., sodium dodecyl sulfate) deter-gents are often employed. Detergents are smallamphipathic molecules that interact with bothnonpolar and polar environments. Detergentsdisrupt membranes. They also bind to hydro-phobic regions of proteins, such as their trans-membrane domains, thereby replacing the un-favorable contacts between hydrophobic pro-tein regions and water with the more favorablehydrophilic domains of the detergent. Thus,instead of the hydrophobic regions of the insol-uble protein forming an aggregate in the bottomof a test tube, the protein becomes soluble andcan be employed in most in vitro analyses.

In addition to detergents, several other solu-bilization strategies are useful for the extractionand in vitro characterization of proteins (Table1.5.4). Chaotropic agents enhance the transferof nonpolar molecules to aqueous environ-ments by their disrupting influence on waterstructure. Chaotropic agents are generally largemolecular ions such as thiocyanate (SCN−),perchlorate (ClO4

−), and trichloroacetate

(CCl3COO−). Hydrophobic interactions arealso reduced by exposure to organic solventsand low salt concentrations. Electrostatic inter-actions are reduced by high salt conditions; thisdecreases the Debye-Hückel screening lengthand coulombic attraction. To disrupt hydrogenbonds, high concentrations of urea or guanidineare often employed. More vigorous methods ofsample denaturation using very low pH or harshdetergents such as sodium dodecyl sulfate arealso used to diminish intermolecular contacts.

Once proteins are extracted, their size canbe characterized by ultracentrifugation on su-crose gradients (UNIT 4.2), gel filtration chroma-tography (UNIT 8.3), SDS-PAGE (UNIT 10.1), andother methods (Table 1.5.5). The charge char-acteristics of proteins can be assessed usingisoelectric focusing and ion-exchange chroma-tography. Specific interactions, such as anti-gen-antibody and biotin-avidin interactions,can also be employed in the characterizationand isolation of proteins. These are useful inimmunoblotting (UNIT 10.10) and affinity chro-matography methods (Chapter 9).

Table 1.5.4 Physical Bases of Common Protein Extraction and/or Elution Methods

Physical property perturbed Agents

Hydrogen bonds Urea or guanidine⋅HCl, pH changesIon pair interactions High salt, pH changesHydrophobic interactions Detergents, chaotropic agents, organic

solvents, low salt

Table 1.5.5 Physical Bases of Common Protein Characterization and Isolation Methods

Physical property Method References to other units

Solubility Extraction with salts,detergents, and enzymes

Chapter 4, Racker (1985)

Size Ultracentrifugation onsucrose gradients

UNIT 4.2

Gel filtration UNIT 8.3

SDS-PAGE UNIT 10.1

Charge Isoelectric focusing UNIT 10.2

Ion-exchange chromatography UNIT 8.2

Biospecific interaction Immunoblotting UNIT 10.10

Immunoaffinitychromatography

Chapter 9

Hydrophobicity Hydrophobic chromatography UNIT 8.4

Reversed-phase HPLC UNIT 8.7


1.5.8


Cells

LIMITATIONS OF THE IN VITROMANIPULATION OF PROTEINS

The very act of isolating proteins perturbstheir physical environment. Although this is notoften a major problem, a few cautionary notesshould be made. The most primitive compart-ment of a cell, the cytosol, is a chemicallyreducing environment. Consequently, free sulf-hydryl groups are observed in the cytosol; infact, multiple cytosolic pathways help in pre-serving the proper redox conditions. On theother hand, the extracellular milieu and theluminal side of the ER are oxidative environ-ments. The oxidizing condition within the ERis presumably due to the unidirectional trans-port of glutathione and cystine. Consequently,disulfides are frequently observed in the ER andextracellular environments. Thus, to preventdisulfide formation during manipulation, sulf-hydryl blocking reagents such as iodoacet-amide are included in extraction buffers. Thecytosol is also a K+-rich and Ca2+-poor solu-tion. These parameters should be considered indesigning physiologically relevant experi-ments.

The experimental manipulation of mem-brane proteins is decidedly more difficult. Theexterior face exists in a high Na+ and Ca2+

solution that is oxidative; just the opposite istrue for the cytoplasmic face. Since no appro-priate solvent exists for such isolated proteins,experimental questions can be directed at prop-erties associated with just one face of the mole-cule. A second limitation common to all in vitrostudies of transmembrane proteins is that theymust be solubilized using detergents. In addi-tion to solubilizing a transmembrane protein,detergents can also bind to hydrophobic regionsin the globular domain(s) of the protein, thusaffecting the properties under study. One meansof countering this problem is to test severaldetergents in the hope of finding one that retainsthe full biological activity of the purified pro-tein.

Protein solubilization can also lead to lossof physiologically relevant protein-protein in-teractions. This can occur by simple dilution orby disruption of noncovalent interactionsamong proteins. For example, hemoglobin ex-ists as a supersaturated solution in vivo whichcannot be duplicated in vitro. Furthermore,protein-protein associations are generallystronger in the restricted confines of mem-branes than after solubilization into a buffer.Thus, protein assemblies found in cells maydisappear during solubilization. One means ofcountering these potential difficulties is to co-

valently cross-link protein assemblies prior todisruption and to solubilize proteins using milddetergents (e.g., Brij-58).

CONCLUSIONSStructural motifs, especially stretches of hy-

drophobic amino acids, contribute to the shapeof a protein and its classification as globular,fibrous, or transmembrane. Proteins are hetero-geneous at many different levels includingphysical attributes, covalent modifications, andsupramolecular assembly. The physical prop-erties of proteins are used to characterize andisolate these molecules. For example, the sizeof a protein is examined by sedimentation onsucrose gradients (UNIT 4.2), gel filtration (UNIT

8.3), and polyacrylamide gel electrophoresis(UNIT 10.1). Its charge is the key physical parame-ter in isoelectric focusing and ion-exchangechromatography. The units that follow containdetailed protocols describing the charac-terization of cellular proteins.

LITERATURE CITEDAndrews, D.W. and Johnson, A.E. 1996. The

translocon: More than a hole in the ER mem-brane? Trends Biochem. Sci. 21:365-369.

Ausubel, F.M., Brent, R., Kingston, R.E., Moore,D.D., Seidman, J.G., Smith, J.A., and Struhl, K.(eds.) 1998. Current Protocols in Molecular Bi-ology. John Wiley & Sons, New York.

Cardoso de Almeida, M.L. 1992. GPI MembraneAnchors. Academic Press, New York.

Englund, P.T. 1993. The structure and biosynthesisof glycosyl phosphatidylinositol protein an-chors. Annu. Rev. Biochem. 62:121-138.

Freedman, R.B. and Hawkins, H.C. 1980. The En-zymology of Post-Translational Modificationsof Proteins, Vol. 1. Academic Press, New York.

Freedman, R.B. and Hawkins, H.C. 1985. The En-zymology of Post-Translational Modificationsof Proteins, Vol. 2. Academic Press, New York.

Hoppe-Seyler, F. 1864. Über die chemischen undoptischen Eigenschaften des Blutfarbstoffs.Virchows Arch. 29:233-235.

Hubbard, S.R., Wei, L., Ellis, L., and Hendrickson,W.A. 1994. Crystal structure of the tyrosine ki-nase domain of the human insulin receptor. Na-ture 372:746-754.

Kühn, W. 1876. Über das Verhalten verschiednerorganisirter und sogenannter ungeformter Fer-mete. Über das Trypsin (Enzym des Pankreas)[Reprint, FEBS Lett. 62:E3-E7 (1976).].

Kyte, J. and Doolittle, R.F. 1982. A simple methodfor displaying the hydrophobic character of aprotein. J. Mol. Biol. 157:105-132.


1.5.9


Parry, D.A.D. 1987. Fibrous protein structure andsequence analysis. In Fibrous Protein Structure(J.M. Squire and P.J. Vibert, eds.) pp. 141-171.Academic Press, New York.

Petty, H.R. 1993. Molecular Biology of Membranes:Structure and Function. Plenum, New York.

Petty, H.R., Worth, R.G., and Todd, R.F. III. 2002.Interactions of integrins with their partner pro-teins in leukocyte membranes. Immunol. Res.25:75-95.

Racker, E. 1985. Reconstitutions of Transporters,Receptors, and Pathological States. AcademicPress, New York.

Rietveld, A. and Simons, K. 1998. The differentialmiscibility of lipids as the basis for the formationof functional membrane rafts. Biochim. Biophys.Acta. 1376:467-479.

Schlesinger, M.J., Magee, A.I., and Schmidt, M.F.G.1980. Fatty acylation of proteins in culturedcells. J. Biol. Chem. 255:10021-10024.

Schultz, G.E. and Schirmer, R.M. 1979. Principlesof Protein Structure. Springer-Verlag, New York.

Spiess, M. 1995. Heads or tails—what determinesthe orientation of proteins in the membrane.FEBS Lett. 369:76-79.

Squire, J.M. and Vibert, P.J. (eds.) 1987. FibrousProtein Structure. Academic Press, New York.

von Heijne, G. and Gavel, Y. 1988. Topogenic sig-nals in integral membrane proteins. Eur. J. Bio-chem. 174:671-678.

Zhang, F.L. and Casey, P.J. 1996. Protein prenyla-tion: Molecular mechanisms and functional con-sequences. Annu. Rev. Biochem. 65:241-269.

KEY REFERENCESRacker, 1985. See above.

A wonderful little book on membrane protein ma-nipulation which disproves the hypothesis that sci-entists can’t write.

Tanford, C. 1961. Physical Chemistry of Macro-molecules. Academic Press, New York.

A rigorous introduction to the physical properties ofproteins, which remains useful several decades later.

Tanford, C. 1980. The Hydrophobic Effect. JohnWiley & Sons, New York.

A very readable introduction to the hydrophobiceffect.

INTERNET RESOURCEShttp://www.expasy.ch

A user-friendly protein database including two-di-mensional PAGE data and 3D protein structures.

ftp://ftp.pdb.bnl.gov/

Contains protein crystallography data.

http://ww.ncbi.nlm.nih.gov

An important protein database.

Contributed by Howard R. PettyWayne State UniversityDetroit, Michigan


1.5.10


Cells

Purf Brief 1

Documents

aims of protein purification

protein science

crystalline protein

protein chemistry

target protein

purification of albumin

oneofakind purification

objectivesprotein purification