Techniques in Protein Biochemistry Chapter 5. Problem: isolation & analysis of protein or amino acid found in cell Assumption: can somehow analyze for.

Techniques in Protein Biochemistry

Chapter 5

• Problem: isolation & analysis of protein or amino acid found in cell

• Assumption: can somehow analyze for wanted protein – Common – Colorimetric indicator (chemical rxn

color form’n; can be monitored spectrophotometrically)– Functional indicator (biological endpoint)– This example – colorimetric (breakdown of fats

purple color)• Activity assay• Use at each step of separation

Isolation of Wanted Protein from Brain Cells

• Brain cells contain wanted protein

• Open cells– Homogenization,

sonication, grinding– Maintain cold, pH,

osmolality

• Centrifugation often used Known speeds/ conditions for

different organelles

• Test each fraction for activity– Save most active fractions

• Separation of wanted protein from other types of molecules– Dialysis against physiological buffer

Separation from other proteins

A. ChromatographyAll use solid or aqueous support to which wanted protein has some affinityAll use aqueous or gaseous mobile phase; wanted protein has different affinityThis also moves molecules through/ past

support

• If wanted protein has greater affinity for support than for mobile phase, protein “adheres” to support phase

• If wanted protein has greater affinity for mobile phase than for support, protein will move with mobile phase through/away from support

Gel Filtration Chromatography (= Size Exclusion)

• Separation by MW

• Solid support = porous beads (ex: sephadex, sepharose)– Held in column– Beads have microscopic pores/pits/spaces

• Mobile phase = buffer of physio pH, ionic strength

• Sample = sol’n of wanted protein + other (unwanted) proteins; most have different MWs

• Apply sample to column• Begin slow mobile phase flow

– Smaller proteins enter spaces in beads– Larger proteins flow w/ buffer around beads (so

emerge 1st from column)

• Collect fractions; test each fraction by activity assay

Ion Exchange Chromatography

• Separation by overall charge of proteins• Solid support = resin (charged microscopic beads)

suspended in buffer• Mobile phase = buffer of particular pH, ionic strength• Sample = sol’n of wanted protein + other (unwanted)

proteinsMost have different overall + or – charges of

various strengths

• Apply sample to column, begin slow mobile phase flow– Proteins of charge opposite that of resin + of

similar strength of charge of resin: Good affinity for resin; bind electrostatically

– Proteins of the same charge or different strength of charge of resin: No good affinity for resin; flow through column quickly, so eluted first

• Result: protein similar to resin is held in column

• To elute protein held to resin in column: use buffer of higher ionic strength or stronger pH– Changes ionic environment

– Ions in new (elution) buffer “exchange” for protein (are more attractive to resin, so take the place of protein on resin)

• Collect fractions from mobile phase + elution buffer

• Test all fractions by activity assay

Affinity Chromatography

• Based on specificity of prot of interest for some molecules to which it alone will bind– Ex: Ab binds only specific Ag

• BUT binding must be reversible• Solid support = specific binding molecule

(=ligand) covalently bound to beads, etc.• Mobile phase = buffer of proper pH, ionic strength

to maintain activity of prot of interest

• Pack column; apply sample

• Begin slow mobile phase flow

• Prot of interest ONLY will bind to ligand– Types of binding (must be reversible): ionic,

H-bonds, hydrophobic interactions

• To elute, may use solution of ligand (competes w/ solid phase ligand) OR buffers of diff strength, pH that disrupt protein/ligand interactions

Electrophoresis

• Separation AND identification• Based on overall charge of protein

movement under influence of electric field• Zone

– Semisolid or gelatinous medium (plate or slab)– Spot protein mixture (w/ wanted + unwanted

proteins in solution) onto gel– Apply electric field

• Molecules migrate toward anode (+ charged)

• Distance traveled dependent on charge, size of protein

Most impt = size of protein

– Gel support acts as molecular sieve; smaller molecules go faster toward anode, so migrate further

• Also, those more strongly charged move closer to anode

• Use chemical to stain aa’s bands representing proteins of decreasing MW

• Run stds simultaneously

–  Mixture of proteins of known MW; spot on one or several lanes

– Electrophorese under same conditions as unknown protein mixture

– Stain “ladder” of bands (lowest to highest MW proteins traveling some distance under these conditions)

• Determine distance traveled for each band from origin

• Plot x = distance migrated for each std of known MW; y = log MW of stds

• Yields std curve; find distance traveled by unknown prot(s) on curve; deter MW

• Can also cut gel, dissolve to free proteins

Moving Boundary = IsoElectric Focusing (IEF)

• Separation due to charge; based on isoelectric pt of each prot

• Use gel of ampholytes (gel has regions of different pHs)

• Spot sample in middle of gel

• Apply electric field

• Each prot in mixture will migrate toward + or – electrode, according to charge

• Each prot will stop moving when it reaches pH region of gel = its isoelectric pH

• Stain aa’s of prot’s w/ chemical

• Run stds simultaneously– Mixture of proteins of known pI’s; spot in one or

several lanes

– Electrophorese under same conditions as unknown protein mixture

– Stain “ladder” of bands (lowest to highest MW proteins traveling some distance under these conditions)

– Determine distance traveled for each band from origin• Plot x = distance migrated for each std of known pI

y = pH

• Yields std curve; can find distance traveled by unknown prot(s) on curve pI                                                                    

Characterize wanted protein by aa sequence

• Once wanted protein has been isolated from all other cell molecules

• Old method: Break all peptide bonds solution of aa’s

• Analyze aa’s by chromatography– Thin Layer Chromatography (TLC) – on coated plate or

paper support; various mobile phases separate aa’s from each other

– High Pressure Liquid Chromatography (HPLC) – force sample through small column packed with various types of support; various mobile phases are forced through column by high pressure pumps to separate aa’s from each other

• Now have identified all aa’s in protein

• With original protein, use chem. rxn to label amino terminal aa

– Use various enz’s to cleave prot at partic aa’s along peptide chain peptide fragments

– Analyze fragments for overlap; use knowledge of all aa’s in protein sequence

• New method: Automated•   Chem rxn to label amino terminal aa•   Cleave amino terminal aa

– Analyze for identity of last aa

– Rest of prot now has diff amino terminal aa (second to last in original prot)

– Chem rxn to label second to last aa of amino terminal

– Cleave this terminal aa

– Analyze for identity of second-to-last aa

– Etc. etc. etc.

• Common now: identify gene for protein of interest– Isolate mRNAs (found w/in cell rich in wanted

protein) w/ gene nucleotide sequence– Use mRNAs to identify gene

• mRNA will have complimentary sequence to gene in DNA, so will pair in that region of DNA only

– Analyze gene for nucleotide sequence– Use genetic code to determine aa sequence of

wanted protein from gene which codes for it