Cheat Sheet (Bio) Combine

8/10/2019 Cheat Sheet (Bio) Combine

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What makes a strong acid?

HCL is a much stronger acid than acetic acid: ↔ 3+ −  Ka = 107

MeCOOH ↔ 3+ −  Ka = 1.74 x 10-5

This is to do with the strength (stability) of the conjugate base, Cl- is not strong enough to deprotonate H3O+, butacetate is. In other words, the chloride ion is inherently more stable than t he acetate ion.• An acid’s pKa depends on the stability of its conjugate base.- The stronger the acid HA, the weaker its conjugate base A-- The stronger the base A-, the weaker the conjugate acid HA.• For example: - HI with pKa of -10, is a strong enough acid to protonate most functional groups. It’s conjugate base, I - is not really basic.- Methyl lithium is a powerful base, which behaves as CH3-. The conjugate acid is CH 4, which isn’t acidic with pKa = 48.Peptide Bond Formation

•Condensation rxn• Between –NH2 of n residue and –COOH of n+1 residue. • Rigid, inflexible. • Loss of 1 water molecule.The peptide bond has a barrier to rotation. The resonance structure explains this, and bond length comparisons areconsistent with partial double bond character. As a consequence, t he atoms are all constrained to lie in the same plane.The peptide bond is planar. But the planar conformation can be accommodated in two alternate forms denoted as trans

and cis. The more stable trans form. The cis form is less stable because of its greater steric repulsion between the Cα

atoms and their attached groups.Peptides and Proteins

• Peptides and protein made up from long chains of amino acids via peptide bonds.• There are two types of protein structures: fibrous (elongated proteins not soluble in water and providing structuralsupport), and globular (spherical proteins soluble in water and have specific function in the immune system andmetabolism). The structural proteins

Primary Structure • The sequence of amino acids • The peptide bond is rigid and can not move due to its partial doublebond character of C-N bond. • To write peptide and protein always from N-terminal to C-terminal.Secondary Structure • Regular elements such as α -helices and β- sheets, which are formed between relatively small partsof the protein sequence. • They are determined by the local conformation of the polypeptide backbone. α-helix • Mostabundant; ~35% of residues in a protein • Repetitive secondary structure • 1.5 Å rise in 100 rotation • C=Oof i forms Hbonds with N-H of residue i+4 • Intra-strandH bonding • C=O groups are parallel to the axis; side chains point away fromthe axis • Polar ends present at surfaces • Amphipathic • All N-H and CO are H-bonded, except first N-H and last COβ-sheet• Other major structural element • Basic unit is a β-strand • Usually 5-10Residues • Can be parallel or anti-parallel based on the relative directionsof interacting β-strands • “Pleated” appearance Another impt interaction is the formation of hydrogen bondsbetween the carbonyl oxygen and amide hydrogens on adjacent regionsof the peptide backbone:β-sheet (with primary structure)(a) antiparallel and (b) parallel β-sheet. Blue and whitebeads represent the positively charged (Arg) andhydrophobic residues, respectively, and the polarresidue (Tyr) and Gly residues are denoted by greenbeads. Solid lines indicate the disulfide bonds betweenCys residues, and dotted lines indicate the backbonehydrogen bond (H-bond).β-pleated sheetThe chains are folded so that they lie alongside each other. All that means is that next-door chains are heading inopposite directions. Given the way this particular folding happens, that would seem t o be inevitable.Some of the amino acids have hydrophobic side chains; others have hydrophilic side chains. The different AA like tointeract with each other, and the protein chain folds to maximize these interactions. Also one impt way a protein folds is

such that the hydrophobic AA will be in the interior of the folded protein, and the hydrophilic AA will be on the surface. Inaddition to the backbone hydrogen bonds that permit the formation of secondary structures, other interactions betweenthe side chains of the various AA govern the overall the folded structure of t he protein.

Tertiary Structure

• Describe the complete three-dimensional structure of whole polypeptide chain . • Include the relationship of differentdomains formed by the proteins’ secondary structure and the interactions of the amino acid substituent R group.• The specific folding of a protein is only thermodynamically stable within a restricted range of environmental

parameters, e.g., Temperature, pH, ionic strength

Quaternary Structure

• Quaternary structure is the 3-Dimensional arrangement of multiple folded protein or coiling protein molecules in amulti-subunit complex by hydrogen bond, electrostatic attraction and sulfide bridge.

When functional unit consist of two or more structural domains, we speak of the “quaternary structure” of the protein. It is the linear sequence of amino acids in a protein that determines the 3 -dimensional folded structure of that protein.

Another way to state this concept is to say that “proteins fold to their thermodynamically most stable state”; ie, eachparticular folded protein has maximized all its particular combinations of possible hydrogen bonds, electrostaticinteractions, hydrophobic interactions, etc, in its final folded shape:

To demonstrated that the linear sequence of amino acids in a protein determines the folded structure of that protein.Using a chemical called urea, he unfolded a protein called Ribonuclease-A, and then reduced its internal disulfide bondswith mercaptoethanol. (Disulfide bonds stabilize the folded protein in its original shape.) When the urea andmercaptoethanol is removed, the ribonuclease “renatured” back, and regained full enzymatic activity. Based onexperiments, the information for the complete, correct folding of a protein is in t he linear AA sequence of that protein.One of the impt functions of proteins is t o serve as catalysts for chemical reactions necessary for life. Proteins thatfunctions as chemical catalysts are called “enzymes.” The complex surface of a folded protein creates crevices that can

bind other molecules. The interior of these crevices is lined with the chemically reactive side chains of the various AA.Consequently, proteins are excellent and very specific chemical catalysts. By stablilizing the transition state of thereaction, enzymes lower the activation energy.Synthesis of proteins

• Transcription • Translation • Post-translational modification: phosphorylation, acetylation, methylation, glycosylationPost-Translational Modifications

Proteins are involved in cellular signaling and metabolic regulation. They are subject to biological modifications. Almost allprotein sequences are post-translationally modified and 200 types of modifications of amino acid residues are known.The dynamic nature of the proteome

The proteome of the cell is changing. Various extra-cellular, and other signals activate pathways of proteins. A keymechanism of protein activation is post-translational modification These pathways may lead to other genes beingswitched on or off MS is key to probing the proteome and detecting PTMSDegradation of Proteins

• Proteins are hold tgt by H bonding, electrostatic attraction and sulfide bridges, which are very sensitive to its chemicaland physical environment. • The change of temperature, pH or ionic strength disrupts these interactions, causing proteindenaturation • Protein loses its activity once its normal shape is lost.

Disease Caused by Mutation • Cancer . Point mutations in multiple tumor suppressor proteins cause cancer.• A novel assay, Fast parallel proteolysis (FASTpp), might help swift screening of specific stability defects ofspecific proteins in individual cancer patients. •  FASTpp measures the quantity of protein that resistsdigestion under various conditions.• A thermostable protease is used, which cleaves specifically atexposed hydrophobic residues.•The FASTpp assay combines the thermal unfolding, specificity of athermostable protease for the unfolded fraction with the separation power of SDS-PAGE .•  Due to thiscombination, FASTpp can detect changes in the fraction folded over a large physico-chemical range ofconditions including temperatures up to 85°C, pH 6-9, presence or absence of the whole cytosolic proteome.Specific diseases caused by insertions/deletions •  Tay-Sachs Disease. Tay-Sachs Disease is a fatal diseaseaffecting the central nervous system. • Symptoms do not appear until approximately 6 months of age. Thechild becomes blind, deaf, unable to swallow, atrophied, and paralytic. • Mutations in the β-hexosaminidase A(Hex A) gene are known to affect the onset of Tay-Sachs. •Cancer Insertion/deletion mutations causecolorectal cancer and other cancers with microsatellite instability. • While environmental factors contributeto the progression of prostate cancer, genetic component also will. •  There are over 500 mutations onchromosome 17 that seem to play a role in the d evelopment of breast and ovarian cancer in the BRCA1 gene,many of which are Insertion/deletion.SNP and DISEASE • One study even identified two genes in which particular variants can slow the onset ofAIDS, demonstrating the potential of this approach for understanding why people vary in their susceptibilityto infectious diseases. • New technologies that are slashing the costs of sequencing and genome analyses willmake possible the simultaneous genome-wide search for SNPs and other DNA alterations in individuals.Proteomics• Proteomics is the large-scale study of proteins, particularly their structures and functions.• Proteins are vital parts of living organisms, as they are the main components of the physiological metabolicpathways of cells. • The proteome consists of the entire complement of proteins, including the modificationsmade to a particular set of proteins, produced by an organism or system. • This will vary with time and distinctrequirements, or stresses, that a cell or organism undergoes.Number of Proteins in Human • Analyzing genome sequences alone will not lead to new therapies to fighthuman diseases. • The human genome has approximately 35,000 genes and theoretically the ability to encodeup to 35,000 corresponding proteins. •  The occurrence of alternative RNA splicing and PTM, such asphosphorylations, acetylations, and glycosylations, or protein cleavages may increase the expression ofproteins to 500,000 –1,000,000.• The proteins reflect more accurately the intrinsic genetic mechanisms of thecell and their impact on the microenvironment, as they are the effectors and characterizeProteomics in Biomedical Research • Biomarkers are biomolecules that is associated with an in creased risk ofthe disease and serve as indicators of biological and pathological processes or physiological andpharmacological responses to a drug. •Proteins that are impt indicators of physiological or pathological statesmay contribute to the early diagnosis of disease, which may provide a basis for identifying the underlyingmechanism of disease development. • These differentially expressed proteins in serum have become an imptin monitoring the state for disease. • Comprehensive proteome of human serum fluid with high accuracy andavailability has the potential to open new doors for disease biomarker discovery and for disease diagnostics.Proteomics in Cancer Diagnostics • Allied to genomics, proteomics technologies is valuable for identifyingnew markers that improve screening, early diagnosis, prognosis and prediction of therapeutic response ortoxicity, as well as the identification of new therapeutic targets. • Studies on the proteome in cancer haveused tissue samples and biological fluids including serum, plasma, saliva, and cerebrospinal fluid in search forthe detection of diagnostic, predictive, and prognostic biomarkers. •  Among the proteomics tools, massspectrometry (MS) is one of the most used techniques for identifying unknown proteins. The massspectrometer is an analytic instrument capable of converting neutral molecules into gaseous ions andseparating them according to their mass-to-charge (m/z) ratio by using an electromagnetic field.Tandem mass spectrometry (MS) offers info about specific ions. In this approach, distinct ions are selectedbased on their m/z from the first round of MS and are fragmented by a number of methods of dissociation,such as colliding the ions with a stream of inert gas, as in collision-induced dissociation or higher energycollision dissociation. Other methods of ion fragmentation include electron-transfer dissociation and electron-capture dissociation .These fragments are then separated based on their individual m/z ratios in another round of MS. MS/MS is

commonly used to sequence proteins and oligonucleotides, as the fragments can be used to match predictedpeptide or nucleic acid sequences that are found in databases. These sequence fragments can then beorganized in silico into full-length sequence predictions.A sample is injected into the MS, ionized and accelerated and then analyzed by MS1. Ions from the MS1spectra are then selectively fragmented and analyzed by MS2 to give the spectra for the ion fragments.Sugars, AA and nucleotides can polymerize to form macromolecules called polysaccharides, proteins andnucleic acids. Sugars, AA and nucleotides polymerize to release water. In hydrolysis, a water molecule reactswith the bond linking the monomers. A monomer is broken off, resulting in a shorter polymer. Sugars aredefined by the presence of an carbonyl group and multiple hydroxyl groups. Sugars like glucose can exist inboth linear and ring forms. Like many organic molecules, sugars are “chiral” molecules- they can exist as right-handed (“D”) or left handed (L”) isomers. Right-handed(‘D’) forms predominate in cells.

When glucose forms a ring, the hydroxyl groupattached to the number 1 carbon is locked into one oftwo alternate positions: either below the plane of thering, or above it. These two ring forms of glucose arecalled alpha (α) (down) and beta (β) (up), respectively:Examples of sugar polymers: Starch is polymerizedglucose, in which α-glucosemonomers are polymerized via a 1-4linkage. Cellulose, on the other hand, is polymerized glucose, inwhich β-glucose monomers are polymerized via a 1-4 linkage.(Animals don’t  have enzymes to catalyze the hydrolysis of theβ-glycosidic link in cellulose!)Starch Structure: Starch is made from chains of α-glucose

molecules. These are linked by glycosidic bonds. Starch is found in many parts of a plant as starch grains.Why is starch a good molecule for storage in plants?

It is insoluble, so doesn’t  draw water into cells by osmosis. Wont easily diffuse out of cells because it is

insoluble, It can be stored in a small space because the tight coils make it compact, Can be easily hydrolyzedto give α-glucose , which can be used in respiration, They are a reserve form of sugar for times when freesugar In diet is absent. Starches called amylose(an unbranched α-glucose polymer) and pectin(a branchedpolymer) are the storage polysaccharides found in plants.Significance of Starch•Green plants use starch as their energy store. An exception is the family Asteraceae,where starch is replace by fructan inulin. Photosynthesis, plants use light energy to produce glucose from CO2.Starch •The glucose is stored mainly in the form of starch granules, in plastids such as chloroplasts andespecially amyloplasts.• Toward the end of the growing season, starch accumulates in twigs of trees near thebuds.• Fruit, seeds, rhizomes, and tubers store starch to prepare for the next growing season.

From Glucose to Starch• Glucose is soluble in water, binds with water and then takes up muchspace and is osmotically active .• Glucose in the form of starch, is not soluble, therefore osmoticallyinactive and can be stored much more compactly .•Glucose molecules are bound in starch by theeasily hydrolyzed alpha bonds. The same type of bond is found in the animal reservepolysaccharide glycogen.•  This is in contrast to many structural polysaccharides such as chitin,cellulose and peptidoglycan, which are bound by beta bonds and are much more resistant tohydrolysis.Production of glucose 6-phosphate •  Glucose 6-phosphate is produced by phosphorylation ofglucose on the sixth carbon. • This is catalyzed by the enzyme hexokinase in most cells, and, inhigher animals, glucokinase in certain cells, most notably liver cells. One molecule of ATP isconsumed in this reaction. •  The reason for the immediate phosphorylation of glucose is toprevent diffusion out of the cell. The phosphorylation add a charged phosphate grp so the glucose6-phosphate cannot easily cross the cell membrane.Two Forms of Starch

 Around 30%, tightly

 packed structure,

more resistant to

Digestion. Amylose can

Exist in Helical Forms.

 Around 70%,highly

branched structure,

being formed of 2,000

to 200,000 glucose

units can be quickly

degraded  Amylopectin on the other hand is a branched-chain polysaccharide where in addition t o the α-1,4-glycosidic bonds there is the occasional α-1,6-glycosidic bonds. Branching occurs about every 24-30 glucose units. Helical structure of amylopectin is disrupted by branching.Glycogen• A multibranched polysaccharide of glucose that is a form of energy storage in animalsand fungi. • In humans, glycogen is made and stored in the cells of the liver and the muscles, and

functions as the secondary longterm energy storage (primary energy stores being fats).•Glycogenis the analogue of starch, having a similar structure to amylopectin, but more branched andcompact.• Glycogen is found in the form of granules in the cytoplasm in many cell types, and playsan impt role in the glucose cycle.• Glycogen forms an energy reserve that can be quickly mobilizedto meet the need for glucose, but is less compact than the energy reserves of triglycerides.Glycogen is a branched biopolymer consisting of linear chains of glucose residues with furtherchains branching off every 10 glucoses. Glucoses are linked together linearly by α(1→4) glycosidicbonds. Branches are linked to the chains and are branched off by α(1→6) glycosidic bonds.Cellulose Made of β-glucose. To form glycosidic links, each β-glucose molecule is rotated 180o compared to the one next to it. Has straight, unbranched chains that run parallel to one another.Hydrogen bond links the chains. The β-glycosidic link between glucose molecules in celluloseresults in a polymer that forms a long linear strand. The hydroxyl groups of one cellulose moleculeare free to H bond with the hydroxyls of adjacent molecules. In plants, the long strands of cellulosebundle together to form microfibrils. Bundles of microfibrils form plant cell walls.• So many hydrogen bonds help to strength cellulose• This makes cellulose a good structural material, hence its use in plant cell walls to aid rigidity• cellulose does this by grouping together to form microfibrils• Cellulose prevents cell bursting, so they are turgid when full with water. This helps support stemsOther impt structural polysaccharides are chitin and peptidoglycanBoth composed of polymers of“amino  sugars, such as N-acetyl-glucosamine (chitin) or [N-acetyl-glucosamine plus N-acetyl-muramic acid] (Peptidoglycan). A mesh of peptidoglycan chains, crosslinked by covalent bonds,

make up the tough and flexible bacterial cell wall. (antibiotics poison the bacterial enzymes thatsynthesize cell wall)Chitin (mono monomer) (Parallel strands joined by hydrogen bonds) • Chitin is a long-chainpolymer of a Nacetylglucosamine, a derivative of glucose. • The main component of the cell wallsof fungi, the exoskeleton of arthropods such as and insects, the radulae of molluscs, and the beaksand internal shells of cephalopods . •The structure of chitin is comparable to the polysaccharidecellulose, forming crystalline nanofibrils. In terms of function, it may be compared to t he proteinkeratin. •It form covalent β -1,4 linkages (similar linkages between glucose units forming cellulose).• Chitin is cellulose with one hydroxyl group replaced with an acetyl amine group.Peptidoglycan (Parallel strands joined by peptide bonds) • also known as murein, is a polymerconsisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane ofmost bacteria, forming the cell wall . • The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. Attached to the N-acetylmuramic acidis a peptide chain of three to five amino acids. The peptide chain can be crosslinked to the peptidechain of another strand forming the 3D mesh-like layer . • Peptidoglycan serves a structural role inthe bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure ofthe cytoplasm. • peptidoglycan helps maintain the structural strength of the cell. •Peptidoglycan isalso involved in binary fission during bacterial cell reproduction. •The peptidoglycan layer issubstantially thicker in Gram-positive bacteria than in Gram-negative bacteria, with theattachment of the S-layer . • Peptidoglycan forms around 90% of the dry weight of Gram-positivebacteria but only 10% of Gram-negative strains.Meso-diaminopimelic acid (DAP) for Gram Positive  

Nucleic Acids

• Two classes of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)  • Cells use DNA to determine and control the synthesis of proteins with the help of mRNA.  • mRNA dictates the synthesis of protein from amino acids delivered by transfer RNA.  • Made up from three components: nucleobases, sugars and phosphoric acid.If U were used in DNA, then when the C in a G:C base pairdeaminated to become U, the G:C base pair would becomea G:U base pair. A G:U base pair is detected by the ongoingDNA-repair enzymes. Since U is not used in DNA, any Uformed can be recognized as illegitimate and have to comefrom mutated a C; it is cut out by repair enzymes and replacedwith C.The nucleotide (Base + sugar + phosphate)

Note: to distinguish between sugar and base, positions in thesugar are designated with a prime ( ’)  

A strand of DNA is made by attaching one nucleotide onto asecond one, and then a third one on the second one, etc. DNAis synthesize in beginning at the 5’ end and progressing towards

the 3’ end. Consequently, the convention when writing out the

nucleotide sequence of a nucleic acid is to begin with the 5’ nucleotide on the left and end with

the 3’ nucleotide on the right  

Other shorthand notation for DNA sequence: 5’- TCA – 3’ 

Two ‘complementart strands of DNA can specifically pair with each other, beacuse the bases form

specific hydrogen bond.Double helix structure

DNA contains major and minor grooves and many DNA-binding, gene regulatory proteins prefer tobind nucleotides located in the major groove.1. DNA molecule consists of two polynucleotide chains in a double helix configuration.2. The two strands are anti-parallel.3. The sugar-phosphate backbone is on the outside of the helix, bases are on the inside.4. A always pairs with T; G always pairs with C. The sequence of one strand (5’ → 3’) dictates the sequence of the other strand.5’ GCATGCAATGCCGAATG 3’ 3’ CGTACGTTACGGCTTAC 5’ 5. 2nm wide diameter: perfect for purine-pyrimidine bond.6. Base pairs are 3.4 Å apart: a complete 360º turn of the helix is 34 Å, which equals 10 base pairs.7. The helix has a major groove and a minor groove.8. When heated or when deviating from physiological conditions, hydrogen bonds between t hetwo DNA strands are cleaved and the strands are separated from each other t o form single stringDNA (ssDNA).RNA

The structure of RNA is similar to that of DNA except:1. The nucleotide subunits have ribose, rather than deoxyribose as t he sugar2. Uridine is substituted for thymidine3. RNA is generally found as a single-stranded molecule in cells.3-D structure of RNA

• GCAU instead of GCAT  • Due to the additional –OH group on the ribose sugar, steric hindrance is too great to allow forthe formation of a double strand. So, RNA exists as a single stranded molecule.• RNA can loop back to form internal self base- paired structures, called “stem-loop structures” Transfer RNA Contains a Modified Base Ψ from Uridine

It is found in tRNA , found with thymidine and cytosine in the TΨC arm and is one of the invariant

regions of tRNA. It is expected to play a role in association with aminoacyl transferases during theirinteraction with tRNA, and hence in the initiation of translation. Recent studies suggest it mayoffer protection from radiation.RNA molecules can form complex structures with pockets and clefts on their surface. Also thepurine and pyrimidine nitrogenous bases contain chemically reactive functional groups that cancatalyze chemical reactions.Proteins Proteins are synthesized beginning with the ‘amino terminal’ amino acid and finishingwith the ‘carboxy terminal’ amino acid. And when writing out the amino acid sequence of a

protein, the convention is the amino t erminus on the left , the carboxy terminus on the right.

The generalized structure of an amino acid: Amino acids are chiral molecules (can exist as right or

left handed forms). But whereas in the case of sugar, the right-handed form predominates in cells,in the case of amino acids, it is the left-handed form that is found in cells.Amino Acids

• Names for amino acids are abbrevi ated to either three symbol or a one symbol short form. • 20 amino acids found in living organisms. • Building blocks of peptide and protiens• Linear chain of amino acids forms peptide/protein. • Peptides - Small peptides with fewer than about ten amino acids are called oligopeptides

• and peptides with more than ten amino acids are termed polypeptides.• Proteins – Chain of amino acids with molecular weights of more than 10,000 (50 –100 aminoacids) are usually termed proteins.• R group varies, thus, can be classified based on R-group.• Glycine is the simplest amino acid. Side chain R=H. • Unique because Gly α-carbon is achiral.Chiral: when a molecule is not superimposable on its mirror imageZwitterionic character, pK and pI

• At the pH under physiological conditions (pH 6 -7), the amino group (pK 8.7~10.7) is ionized toNH3+ and the carboxyl group (pK 1.8~2.5) is ionized to  –COO-. So, at physiological pH,. amino acidsare zwitterionic• pK is the dissociation constant for H+.• pI (isoelectric point) - It is a specific pH value at which aa exhibits no net charge.• It can be estimated via the Henderson- Hasselbalch equation pI = ½ (pK NH3++pKCOOH), where pKiand pKj are the dissociation constants of the ionization groups involved.• At its isoelectric point, amino acid remains stationary under an applied electric field.  Acid-Base Properties

pH and pKa

• The pH of a solution is a measure of the acidity of the solution. It is defined as = 10   3+  Where [3+] is the concentration of hydronium ions in the solution.• Consequently, the pH of a solution depends on two things -The concentration of the solution  – if we have two solutions of the same acid, the moreconcentrated solution will have more free H3O+ ions and therefore a lower pH.-The acid in question – if we have two equally concentrated solution of acid, the solution of astrong acid will have a lower pH than that of a weak acid, because it is fully dissociated andtherefore produces more H3O+ ions. HCL for example, is completely dissociated.Therefore, we see that pH does not measure the strength of an acid, but the acidity of a givensolution.• The pH of water is 7. This means that a solution of pure water has 10 -7 mol/dm3 of hydroniumions. This can only happen through the autoprotolysis of water:

2 ↔ 3+ − This mean that in water,   3+ =

  −  • To be clearer about what a strong and weak acid is, we look at the reaction: 

  ↔ − 3+ 

The position of the equilibrium is measured by the equilibrium constant  = 

Now, in dilute solutions of acid, 3+  stays roughly constant at about 56 mol/dm3. we therefore

define a new equilibrium constant  – the acidity constant  = 

This is also expressed in logarithmic form are as follows:  = 10  Because of the minus sign, the lower the pKa the higher the Ka and the stronger the acid.• It turns out that the pKa of an acid is the pH at which it is exactly half-dissociated. This can beshown by re-arranging the expression for Ka:

3

+ =

 × 

 −

=   

 − 

Clearly, when [AH] = [A-], pH = pKa• This information is rather useful: o At a pH above the pKa, t he acid exist as A- in water, and will therefore be fairly soluble.o At a pH below the pKa, the acid exists mostly as HA in water, and will probably be less soluble.

Amino Acids: Classification

based on R group

• Basic amino acids• Acidic amino acids• Aliphatic amino acids• Aromatic amino acids

• Hydroxyl containing aminoacids

• Sulfur containing amino acids• Secondary amino acids 

Level Description Stabilized by:

Primary The sequence of amino acids Peptide bonds

Secondary Formation of a-helices and b-pleated sheets H-bonding between peptide groups along the peptide backbone

Tertiary Overall three-dimensional shapeof a polypeptide

Bonds and other interactions between R-groups, or between R-groups and the peptide backbone

Quaternary Shape produced by combination ofpolypeptides

Bonds and other interactions between R-groups, and betweenpeptide backbones of different polypeptides

8/10/2019 Cheat Sheet (Bio) Combine

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Modes of Detection • Fluorescence • Radioactivity Excitation Light Source • Arc and Incandescent Xenon lamp • Pulsed Xenon lamp • Low-pressure Hg and Hg-Ar lamps • Ion Lasers and solid state lasers  Xenon arc lamp • An electric light that produces light by passing electricity through ionized xenon gas at high

pressure. • It produces a bright white light that closely mimics natural sunlight. Argon Ion Lasers

Fluorescein • A common dye for labeling DNA and protein.Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group.How to capture the fluorescence emission? Photomultiplier Tube (PMT) • Constructed from a glass envelope

with a high vacuum inside, which houses a photocathode, several dynodes, and an anode. • Incident photons

strike the photocathode material, which is present as a thin deposit on the entry window of the device, withelectrons being produced as a consequence of the photoelectric effect. • These electrons are directed by the

focusing electrode towards the electron multiplier, where electrons are multiplied by the process ofsecondary emission. • The electron multiplier consists of a number of electrodes called dynodes. Each dynode

is held at a more positive voltage than the previous one. • The electrons leave the photocathode, having the

energy of the incoming photon. • Upon striking the first dynode, more low energy electrons are emitted, and

these electrons in turn are accelerated toward the second dynode.Charge Coupled Device (CCD) Camera • Sensors used in digital cam and video cam to record still and moving

images. • It captures light and converts it to digital data that is recorded by the cam.  Photodiode• A type of photodetector capable of converting light into either current or voltage. • The

traditional solar cell used to generate electric solar power is a large area photodiode. • When a photon of

sufficient energy strikes the diode, it excites an electron, creating a free electron.Radioactive Isotope • The principle of using radioactive tracers is that an atom in a chemical compound is

replaced by another atom. • The substituting atom is a radioactive isotope. • Radioactive decay is much more

energetic than chemical reactions. • Therefore, the radioactive isotope can be present in low concentration

and its presence detected by sensitive radiation detectors such as Geiger counters and scintillation counters.

• A radioactive isotope is introduced into DNA or Protein for quantization through the radioactive decay.Radioisotopes Are Commonly Used to Detect Very Small Amounts of DNA/RNA/Protein

• Allows for detection of amounts in the femtogram to picogram levels • Different radioisotopes used, hereare most common:  – 32P (DNA & RNA detection) – 35S (DNA sequencing)  – 3H (DNA & RNA detection) – 125I(protein detection) Detect radioisotopes by:  – Exposing to x-ray film (autoradiography)  – Liquid scintillationCounting (accurate quantification) determines counts per minute (cpm)Autoradiography and Quantification by Densitometry

• Expose gel to film • Develop film - dark bands indicate radioactive signal – darker band =more signal – fainter band=less signal • Scan x-ray film with light and detector to determine intensity of band=densitometryAutoradiography and Quantification by Densitometry GE of DNA fragments in three parallel lanes on a gel. Atthis point the DNA bands are invisible, but their positions are indicated here with dotted lines. Place a piece ofx-ray film in contact with the gel and leave it for several hours, or even days if the DNA fragments are onlyweakly radioactive. Develop the film to see where the radioactivity has exposed the film.Instrumentation Platform for our Biomolecular Targets

Electrophoresis• Used in fundamental research and diagnostic settings for the isolation and identification ofhigh mw biomolecules. • The separation is based upon the mobility of charged macromolecules under the

influence of an electric field. • Mobility is a fundamental property of a macromolecule, and its value depends

on the magnitude of its charge, its mw, and its tertiary or quaternary structure, and its isoelectric point. •

Because most biopolymers are charged, they can be separated and quantitated by electrophoretic methods.Different modes of electrophoresis • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) •Isoelectric focusing (IEF) • 2D-gel electrophoresis (2D-EP) • Capillary zone electrophoresis (CZE) • Capillaryisoelectric electrophoresis (CIEF) • Micellar electrokinetic chromatography (MEKC) • Capillary GE (CGE)Principle and Theory of Electrophoresis• An electrophoretic separation occurs in an intervening medium thatseparates two electrodes. • At one end of the medium is the positively charged anode, and at the other is thenegatively charged cathode. • The intervening medium may be as short as 10 cm or as long as 1 m. •

Throughout this medium, positively charged species will migrate toward the cathode and negatively charged

species will move toward the anode.• Difference in chargeand size lead to different mobilities and separationof different sample components. • Electrophoretic separations can be performed in free solution or solutioncontaining a non-conductive matrix such as agarose or polyacrylamide gel . • For free solution, the separationof ions occurs due to differences in mobility • The separation of analytes in a gel is also based on differencesin mobility, additionally, the gel has sieving effect. Large compounds are retarded more than smallercompounds. So two compounds with same charge to size ratio can be separated as long as they are differentin size. • The efficiency of an electrophoretic separation is governed by two main factors : • Theelectrophoretic mobility (μep) of the analytes and the electroosmotic flow (EOF) of bulk solution.Electrophoretic Mobility Electrophoresis is the process in which sample ions move under the influence of anapplied voltage. The ion undergoes a force that is equal to the product of the net charge and the electric fieldstrength. It is also affected by a drag force that is equal to the product of  f , the translational frictioncoefficient, and the velocity. This leads to the expression for electrophoretic mobility: μEP = q / f = q / (6πηr)

Electrostatic Force: Fef = q·E Drag Force: Fef = Ffr = f νep= 6·π·η·r·νep where f is given by the Stokes’ law; η is the viscosity of the solvent, and r is the radius of t he molecule.Electrical Force equal to Frictional Force The rate at which these ions migrate is dictated by t he charge tomass ratio. The actual velocity of the ions is directly proportional to E , the magnitude of the electric field andcan be determined by the following eqn: νep= μEP * E = (q / f ) * E

Once force equilibrium is reached, the velocity of the ion is constant. The electric field will dictate the velocity.During the turn-on of electric field, the ions accelerate from zero to v at acceleration of Force/Mass.Electroosmotic Flow (EOF) Many of materials used for electrophoretic separation exhibit surface charges. Atthe surface of capillary, an electric double layer is formed. The negative surface charges are compensated by +ions from the buffer solution to form t he Stern layer. A diffuse layer of mobile cations is formed next to theStern layer (zeta-potential). The potential drop inside the diffuse layer is exponential.Electrical Double Layer consists of a region near an interface in which the net charge density is nonzero. Ascompared to the bulk solution, the counter ions (ions with charge opposite the wall) are present at higherconcentration, while the coion (ions with charge of same sign as the wall) are present at lower concentration.Electroosmotic Flow (EOF) Equation• Upon application of an electric field, the cations in the diffuse layer (+ )move towards the cathode and drag the bulk solution with them. • This movement of the bulk solution iscalled electroosmotic flow (EOF). νEOF= ε·ζ·E/(4·π·η) μEOF=νEOF/E• For most biomolecular separations, the analyte ions are negatively charged and will be dragged to theanode, whereas the EOF is directed to the cathode.In capillary, the EOF can be controlled.1. low pH (<4)—surface charges are neutralized by protonating the silanol group.2. Chemical surface modification. (eg. Coating of the capillary walls with polymer layer)3. Using additives to change the viscosity η and the zeta-potential ζ. E.g., Organic solvent methanol and acetonitrile used to reduce or increase the viscositySeparation Efficiency and R esolution• Efficiency and resolution of an electrophoretic separation areinfluenced by the electrophoretic motion as well as the EOF . • The apparent mobility is the sum ofelectrophoretic mobility and the electroosmotic mobility • In simplified terms, μapp = μep + μEOF The migration velocity of an analyte under an electric field E, is determined by the electrophoretic mobility ofthe analyte and the electro-osmotic mobility of the buffer inside the capillary. The electrophoretic mobility ofa solute (μep) depends on the characteristics of the solute and those of the buffer in which the migrationtakes place. The electrophoretic velocity (vep) of a solute, assuming a spherical shape, is given by the

equation:  = × = 

6πηr×

When an electric field is applied, a flow of solvent is generated inside the capillary, called electro-osmoticflow. The velocity of the electro-osmotic flow depends on the electro-osmotic mobility(μeo) which in turndepends on the charge density on the capillary internal wall and the buffer characteristics. The electro-

osmotic velocity (Veo) is given by: = × = 

 ×

The velocity of the solute (v) is given by: v= vep+veo• The electrophoretic mobi lity of the analyte and the electro-osmotic mobility may act in the same directionor opposite directions, depending on the charge of the solute. • In normal capillary electrophoresis, anions ( -)will migrate in the opposite direction to the electro-osmotic flow and their velocities will be smaller than the

electroosmotic velocity. • Cations (+) will migrate in the same direction as the electro-osmotic flow and theirvelocities will be greater than the electro- osmotic velocity. • Under conditions in which there is a fast electro-osmotic vel with respect to the electrophoretic velocity of the solutes, both cations and anions can beseparated in the same run.Resolution: The time taken by the solute to migrate the distance (l) from the injection end of the capillary to

the detection point is given by : = 

+ =

  ×

(+)× 

Theoretical plate: After introduction of the sample, each analyte ion of t he sample migrates withinthe background electrolyte as an independent zone, according to its electrophoretic mobility. Zonedispersion, that is the spreading of each solute band, results from different phenomena. Underideal conditions the sole contribution to the solute-zone broadening is molecular diffusion of thesolute. In ideal case the efficiency of the zone, expressed as the number of t heoretical plates (N):

=  (+)××

××  • Plates do not really exist. • They serve as a way of measuring column

efficiency, either by stating the number of theoretical plates in a column, N (the more plates thebetter). • Other phenomena such as heat dissipation, sample adsorption onto the capillary wall,mismatched conductivity between sample and buffer, length of the injection plug, detector cellsize and unlevelled buffer reservoirs can also significantly contribute to band dispersion (Nbecomes smaller). • Separation between 2 bands, a and b (expressed as the resolution, Rs) can beobtained by modifying the electrophoretic mobility of the analyt es, the electro-osmotic mobilityinduced in the capillary and by increasing the efficiency for the band of each analyte, according to

the equation: =

  (−)

4(+) (Higher the better)

GE • Separation takes place in an electrically nonconductive hydrogel medium such, containing anelectrolyte buffer. • The pores of gel serve as a molecular sieve and retard the migrating moleculesaccording to their size. • The gels act as anti-convective support medium, reducing the bandbroadening. NO ELECTROOSMOTIC FLOW • Only analytes with a net charge can be separated.Instrumentation Separation can be performed vertically or horizontally. It flows from – to +.Usually for DNA. • Applied potential 200-500V • Buffer pH chosen such that the analytes arenegatively charged• After gel electrophoresis, the analyte bands are visualized, usually by stainingChoice Gel media • Agarose or page• The gel pore size is an impt parameter for electrophoresisseparation. • In restrictive gels, pore acts as molecular sieves. • In non-restrictive gels, the poresare too large to impede the sample movement: migration t depends on the mobility of the sample.

Agarose • Pore sizes are larger with agarose than wit h polyacrylamide gels. • So that nucleic acidsand proteins too large to be separated on polyacrylamide gels can be separated and quantitatedusing agarose gels. • Agarose gels contain charged groups —mainly sulfate and some carboxylategroups. •. These charged groups interact with charged groups on proteins, and lead to ion-exchange effects; they may also lead to significant EOF. • The pretreatment of agarose in alkalinesolution leads to the hydrolysis of these groups, and improves the sieving characteristics of thegels. • The physical properties of agarose gels, especially the viscosity, are very sensitive totemperature fluctuations, so that strict control of temperature during electrophoresis is essential.• Due to the large pores in agarose gels, their area of application is in DNA separation and analysis.Polyacrylamide gel • Polyacrylamide gels are prepared by the reaction of acrylamide (monomer)with N,N’-methylenebis(acrylamide) (cross-linker) in the presence of a catalyst and initiator. •

Initiators include ammonium persulfate and potassium persulfate, where the S2O8 2-- dianion

decomposes into two SO4-. radicals, while the commonly used catalyst istetramethylethylenediamine [TEMED, (CH3)2N(CH2)2N(CH3)2], which reacts with the sulfateradical anion to produce a longer lived radical species. • The pore size of a polyacrylamide gelcontrols the mobility and resolution of components because of the sieving effect of the pores onmacromolecular species.• The pore size may be controlled by varying the total concentrations of

monomer and cross-linker, and by varying their ratio.• Gel compositions are defined by two

parameters, their %T and %C values, is the total and crosslinker contents.These parameters are defined by following Eqs. • %T= (weight in grams of monomer pluscrosslinker)/ 100mL • %C= 100 x (grams cross-linker)/(grams monomer plus cross-linker) • Thehigher the %T, the more restrictive the gel . • Polyacrylamide gels are generally restrictive and actsas molecular sieves. High molecular compounds (MW>800 kDa) can not be run on it.Sample preparation and Buffer systems • Sample should not contain solid particle. • Salt

concentration in sample should be low, <50 mM.• The buffer must be chosen such that the

analyte molecules are charged, stable and soluble. Typically high pH buffer used such as pH 9.1Tris-glycine and pH 8.3 Tris-borate with concentration 50 mM.• Additives used to increase

solubility.•Proteins are treated with the denaturing detergent SDS which coats the protein with -charges, hence SDS-PAGE. • Disulfide bonds are reduced by adding b-mercaptoethanol.Visualization and detection• Bands are visualized by staining.• Different staining methods have

different sensitivity ranging from 100 ng to 1 μg. silver staining is more sensitive with detectionlimit < 1ng. • DNA/RNA are stained with EtBr which fluoresces under UV light .• Protein stainedwith Coomassie Blue or Silver, colloidal gold .• Quantification can be achieved by densitomertry.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

• The separation principle of SDSPAGE is solely based on the difference in protein size/molecular

weight. • The protein is denatured in the presence of anionic detergent SDS with binding ratio of

1.4 g SDS to 1g protein. • SDS -protein complex is rod shape with the large negative charges of SDSmasking the intrinsic charge of the protein. • Separation totally depends on the molecular sieving

effect of the gels. The larger the molecular weight, the slower the protein migrates.Joule Heating • When an current passes through the conductive buffer, this causes ohmic heating,also referred to as Joule heating. • Due to heat transfer, a temperature gradient is formed across

the capillary diameter or gel cross section, resulting in band broadening and loss of separationresolution. • There are several ways to minimize Joule Heating. --Applying a low electric field anddecreasing the conductivity of the buffer. -- Improving the dissipation of heat by using smalldiameter capillary or thin gel. -- Using a thermostatically controlled environment.Isoelectric Focussing (IEF) • Separates proteins on basis of isoelectric point (pI)• If the pH equals to

the pI of protein, the protein is not charged and hence, it does not move in the electric fieldanymore.• Basic (positively) charged proteins have a high pI, acidic (negatively) charged proteinshave a low pI.• IEF has a high resolution. • Bands as narrow as 0.001 pH units can be obtained. Definition of Isoelectric Point • An amino acid carries simultaneously: a carboxylic acid function -COOH, which is a weak acid (2 < pKa< 2.5) • An amine function -NH2, which is a weak base (9<pKa<9.5). • In solution as well as in the solid state, the proton of the carboxylic acid group is

transferred onto the amine to give a neutral entity, called zwitterion. • Under these form, amino

acids can be considered as being salts of the weak acid -COO- and the weak base  –NH3+ andtherefore they behave as amphoteric particles. • Amino acids, under their zwitterionic formbehave as amphoteric particles; the pH of their solutions is given by: • This pH is called isoelectric

pH (pI) because the zwitterion is overall neutral. pH=1/2(pKa1+ pKa2) In acidic is – and basic is +.Ampholytes in IEF • A stable pH gradient with constant conductivity is very important and isachieved by carrier ampholytes or immobilized pH gradients. • Carrier ampholytes are amphoteric,so that they reach an equilibrium position along the separation medium, and are possessing bothionic conductivity, to carry current, and buffering capacity, to carry pH. • Ampholytes are used togenerate stable pH gradients in the presence of the electric field.Principle of Ampholytes • Generating pH gradients in IEF gels relies on carrier ampholytes.• Carrier

ampholytes are small, soluble, amphoteric molecules with a high buffering capacity near their pI.•Commercial carrier ampholyte comprise hundreds of individual polymeric species with pI spanninga specific pH range.• When a voltage is applied, the carrier ampholytes with the lowest pI (mostnegative charge) move towards the anode (+).• The carrier ampholytes with the highest pI movetoward the cathode (-).• The other carrier ampholytes align themselves between the extremes,according to their pI, and buffer their environment to the corresponding pH result in a continuouspH gradient. •IEF can be run in either a native or a denaturing mode.• Native IEF is a convenientoption, as precast native IEF gel are available in a variety of pH gradient. •This method is preferredwhen native protein is required, when activity staining is to be employed. •The use of native IEF,however, is limited by the fact that many proteins are not soluble at low ionic strength or have lowsolubility at their isoelectric point. • Hence, denaturing IEF is employed. Urea is used, as thisuncharged compound can solubilize many proteins not soluble under IEF conditions. • Detergents

and reducing agents are used for more-complete unfolding. Urea is not stable in aqueous solution,so precast IEF gels are not manufactured with urea. • Dried precast gels are a convenient

alternative they can be rehydrated with urea, carrier ampholytes, and other additives before use.•Carrier ampholytes have limitations. Because the carrier ampholyte –generated gradient is

dependent on the electric field, it breaks down when the field is removed. • The pH gradients arealso susceptible to gradient drift, in which there is a gradual decrease in pH at the cathodic end ofthe gel and a flattening out of the pH at the anodic end. • For this reason it is impt to not over-focus the protein, because cathodic drift will increase over time. • There can be significant

variations in the properties of carrier ampholytes, which limits the reproducibility of focusingexperiments. • Problem with carrier ampholytes is their tendency to bind to the sample proteins,which may alter the migration of the protein and render the separation of carrier ampholytes fromthe focused protein difficult.

Immobilized pH gradients (IPG) • Produced by incorporating substances called immobilines in the gelpolymerization process. • Immobilines are not zwitterionic; they are either acidic or basic. • The pHdepends on the ration of these immobilines . • Controlled mixing during gel casting is required to obtaina good pH gradient. • Once cast, these gels can be prepared in quantity, dried, and stored, so that arehydration step just prior to use with reproducible gradients.Properties of IPG • Acrylamido buffers are an alternative means to form pH gradients that circumventmost of the limitations of carrier ampholytes. • Chemically, they are acrylamide derivatives of simplebuffers and do not exhibit amphoteric behavior . • The acrylic function of an acrylamido buffercopolymerizes with the gel matrix. • By pouring a gel that incorporates an appropriate gradientof acrylamido buffers, an immobilized pH gradient (IPG) is formed. Creating an immobilized pHgradient. • (A, B, C) A gradient of acrylamido buffers in an acrylamide solution is cast into a slab gel thatis crosslinked to a plastic support film. • (D) The gel is washed to remove polymerization byproducts. •

(E) The gel is dried for storage. • (F) The pH at any point in the gel is determined by the mixture of

buffers crosslinked into the gel at that site.Advantages of IPG • The protein sample can be applied immediately. • The pH gradient is stable anddoes not drift in an electric field. • Additionally, the gels are not susceptible to cathodic drift, becausethe buffers that form the pH gradient are immobilized within the gel matrix . • Individual Immobilinespecies with a specific pK value are available, suitable for casting gradients from pH 3 –10. • Becausereproducible linear gradients with a slope as low as 0.01 pH units/cm can separate proteins with pIdifferences of 0.001 pH units . • The resolution possible with immobilized pH gradient gels is 10 –100times greater than that obtained with carrier ampholyte –based IEF. • IEF is best performed in a flatbedelectrophoresis apparatus. This type of apparatus allows very effective cooling, which is necessary dueto the high voltages. • A variety of precast gels for IEF, including ready to use carrier ampholyte gels,dried IPG gels, and dried acrylamide gels . • These gels are ready for reswelling in a mixture of carrierampholytes and any other additives desired, such as detergent and denaturants.2D Gel Electrophoresis • Two modes of electrophoresis are combined on a single gel . • Usually,proteins are separated by IEF in 1 dimension, based on pI . • Followed by SDS-PAGE in a perpendiculardirection, based on size. • Mixtures of thousands of proteins can be separated. • The result can becompared to electronic databases. This method resolves few 1000 protein spots.Capillary Electrophoresis (CE) • Separation method carried out in a buffer-filled capillary tube that istypically 20 to 100 μm in internal diameter and 10 to 100 cm in length. • The tube extends betweentwo buffer reservoirs that also hold Pt electrodes. • The sample is introduced into one end of thetubing, and a dc potential in the 10 to 30 kV range is applied between the two electrodes throughoutthe separation. • The separated analytes are observed by a detector at the end of the capillary oppositethe end where the sample was introduced. • Charged analytes migrate in the presence of an electricfield. Separation is based on differential rates of migration.Hydrodynamic injection can be performed in: Pressure injection or vacuum injection, gravity flowinjection. The anodic end is removed from the buffer reservoir and placed in the sample solution. Thecapillary end is then raised so that the liquid level in the sample vial is at a height h above the level ofthe cathodic buffer, and is held in this position for a fixed time t.Electrokinetic injection involve drawing sample ions into the capillary interior with an applied potential.A high voltage is applied over the capillary between the sample vial and the destination vial for a giventime. This causes the sample to move into the capillary according to its apparent mobility, μapp.Problem: discrimination occurs between different components in the sample.Order of Elution with EOF: small cations, large cations, neutral, large anions, small anions.Capillary Electrophoresis • Separation based on electrophoretic mobility • Primary applications inbioanalysis – DNA sequencing  – DNA fragment analysis • Multiple modes for improved selectivity ofneutrals -- Capillary Zone electrophoresis (CZE) -- Micellar electrokinetic chromatography (MEKC)DNA capillary electrophoresisDuring capillary electrophoresis, the products of the cycle sequencingreaction are injected electrokinetically into capillaries. High voltage is applied so that the negativelycharged DNA fragments move through the polymer in the capillaries toward the + electrode.Detection Options • UV-absorption detection – Most common mode  – μM limit of detection

 – Peptide λ=210nm, protein and DNA at 260 or 280nm • Laser-induced Fluorescence – Very sensitive – Limited to fluorescent species - Mass SpectrometryModes of CE • (CZE) • (MEKC) –Separates compounds with micelles • Capillary Gel Electrophoresis  –

Size exclusion using sieving gels • Capillary Isoelectric FocusingCZE: + ions move faster than the  – ions. Relative to the EOF, the + ions moved ahead but  – ions movedbackwards. Solution is electrically neutral since anions and cations are surrounded by buffercounterions. Buffer systems don’t interfere with the detection of analytes.Principles of MKEC • In MEKC, separation takes place in an electrolyte solution which contains asurfactant at a concentration above the critical micellar concentration (cmc).• The solute molecules aredistributed between the aqueous buffer and the pseudo-stationary phase composed of micelles,according to the partition coefficient of the solute . • The technique can therefore be considered as ahybrid of electrophoresis and chromatography . • It is a technique that can be used for the separationof neutral and charged solutes, maintaining the efficiency, speed and instrumental suitability ofcapillary electrophoresis. • Widely used surfactants in MEKC is the anionic surfactant sodium dodecylsulphate, although other surfactants, for ex. cationic surfactants such as etyltrimethylammonium salts.Mechanism• At neutral and alkaline pH, a strong electro-osmotic flow is generated and moves theseparation buffer ions in the direction of the cathode. • If sodium dodecyl sulphate is employed as thesurfactant, the electrophoretic migration of the anionic micelle is in the opposite direction, towards theanode. • As a result, the overall micelle migration velocity is slowed down compared to the bulk flow ofthe electrolytic solution. • In the case of neutral solutes, since the analyt e can partition between themicelle and the aqueous buffer, and has no electrophoretic mobility . • The analyte migration velocitywill depend only on the partition coefficient between the micelle and the aqueous buffer.Result • In the electropherogram, the peaks corresponding to each uncharged solute are alwaysbetween that of the electro-osmotic flow marker and that of the micelle (the time elapsed betweenthese two peaks is called the separation window). • For electrically charged solutes, the migrationvelocity depends on both the partition coefficient of the solute between the micelle and the aqueous

buffer, and on the electrophoretic mobility of the solute in the absence of micelle.Summary of blotting techniques •Southern - Restricted DNA on gel →denature and trf to filter→hybridize with probe = labelled DNA • Northern -- RNA on gel → transfer to filter →hybridize with

probe = labelled DNA • Western -- protein on gel → transfer to filter → react with probe(fluroscence,hemiluminescence, colorimetric detection) = antibody. • Use GE to separate the target • Usesequence-specific or shape-specific molecular recognition between probe-target • Label the probe anddetect the targetMolecular Recognition-Bioassay • Most importantly immunoassay, is an analytical method using

antibody as reagents to quantitate specific antigen. • rely on the highly specific reaction between AB

and AG.• Very sensitive • used in bioanalytical chemistry, especially for diagnosis and management ofdiseases, pregnancy test and anthraxBioassays • AB and AG have recognition sites, called paratope and epitope respectively. • Epitope - amolecular region on the surface of an antigen capable of eliciting an immune response and ofcombining with the specific antibody produced by such a response –antigenic determinant • When

paratope and epitope match with each other, Ab- Ag complex is formed. • This kind of binding has very

high affinity. • That explains the high sensitivity and low limits of detection obtained with bioassays. •

To detect the assay product, it usually labels either the AB or AG with fluorescent, luminescent,radioactive, an enzyme or an electrochemically active group. • It can be performed in a large variety

formats, in solution or on a solid support, with limited reagent or an excess of reagent.Antibody• AB is a protein complex used by the immune system t o identify and neutralize bacteria andviruses. • Each AB recognizes a specific AG unique to its target. • Produced in living organisms viaimmune response, in response to immunogen. • AB, also referred to as immunoglobulins (Ig), consist

of four subunits: two identical light chains (~25KDa) and two identical heavy chains (~50KDa) • These

subunits are associated via disulfide bonds and non-covalent interactions t o form a Y-shapedsymmetric dimer. • Five types of Ig determined by five types of heavy chains: IgA, IgD, IgE, IgG (mostcommon AB), IgM • Two types of AB can be distinguished: monoclonal AB and polyclonal AB. •

Polyclonal AB are isolated from serum. • The resulting antiserum will contain a mixture of AB that bind

to different epitopes of AG. This may result in significant cross-reactivities, or interferences, whenemployed in immunoassays. • Monoclonal AB are a homogeneous population of identical AB

molecules, having identical paratopes and affinity for a single antigenic epitope.Structure of AB Have the same basic structure. •4 polypeptide chains linked by disulfide bonds.• Twolight chains • 2 heavy chains • AB have two AG binding regions. • A hinge region which confers

flexibility on the Molecule • A model of an ig molecule. • The heavy chains are coloured dark red and

dark blue•The light chain are light red and light blue•C  mean “crystallizable” and ab mean “AG binding” 

Useful characteristics of antibodies • They are specific to the substances used to generate them. • They areimmunogenic themselves (i.e. it is possible to raise antibodies to antibodies). • The FC portion can be modified withoutaffecting the specificity or affinity of binding. • They bind the AG with high affinity (makes the assay more sensitive).Antigen • An AG is a molecule capable of inducing an immune response when entering the body . • Two classes of AG canbe distinguished: complete and incomplete AG. • Complete AG can induces an immune response by themselves. •

Incomplete AG , also called hapten, has to attach to protein carries to trigger the production of antibodies . • The bindingsite of the AG, the epitope, makes up a small area of the t otal AG structure. Epitopes can be continuous or discontinuous.Ab-Ag Complex • The complex formation is reversible and depends on the interplay of several forces. • Electrostaticinteraction between the positively charged amino group and negatively charged carboxyl group. • H bonds betweenhydroxyl, amino and carboxyl groups • Van der Waals-forces • The binding strength between a single epitope andparatope is referred to as their affinity, which can be quantified by the equilibrium constant (Keq): Keq=[Ab-Ag]/[Ab][Ag]Immunoassay Formats • Limited (competitive) or excess reagents (non- competitive) • Homogenous or heterogeneous• Labelled or unlabelled Limited reagent Immunoassay (Competitive)The AG molecules with the sample compete with a fixed amount of labelledAG for the limited amount of AB binding sites. Only a fraction of the ABbinding sites are bound with labeled AG.Excess reagent Immunoassay (Non-Competitive)The AG sample is added to an excess of AB reagent leading to fractionaloccupancy of AB binding sites. Secondary AB with a label is added andsandwich complex is formed allowing detection.Home Pregnancy Test – Detect the Glycoprotein hormone human chorionic gonadotropin (hCG) – A few days afterconception, hCG appears in the urine and its concentration increase rapidly during the first week of pregnancy.  – Thestrip component is composed of an adsorbent material. – Once the urine sample is applied, the liquid moves along thestrip by capillary action and the assay-reactions are carried out in flow.Enzyme-linked immunosorbent assays (ELISA) Enzymes are labels – Reaction of enzyme with colorless substrateproduces colored product. • Indirect ELISA detects antibodies in serum • Sandwich ELISA used to detect AGMolecular Recognition-Biosensor • Biosensor- is an analytical device that combines a biological sensing element (such asan AB, enzyme or whole cell) with a transducer to produce a signal proportional to the analyte concentration.• Biosensor’s signal is originated from a change in proton concentration, release or uptake of gases, light emission,absorption and so forth, • The response is brought about by the reaction of biomolecule and target compound. • Thetransducer converts this signal into a measurable response such as current, potential or absorption of light throughelectrochemical or optical means . • This signal can be further amplified, processed and stored for later analysis.The enzyme is immobilized on a platinum electrode, and covered with a thin polyurethane (outer) membrane to protectthe enzyme layer, and reduce the dependence of the sensor on blood oxygen levels. Glucose oxidase, in its oxidized form,oxidises glucose entering the sensor to gluconic acid; resulting in the conversion of the enzyme to its reduced form. Theenzyme does not remain in this form for long.DefinitionsBiological receptor: A macromolecule / cell / tissue that recognizes the target analyte Transducer: Device thatconverts the biological recognition event into a measurable signal Processor: Converts the measured signal into a signalthat can be interpreted by the user, e.g., a number, a colour, a meter readout.Analytes• Microbes: E. coli, etc. • Small molecules: glucose, CO2, amino acids • Bio-macromolecules: DNA, RNA,enzymes, proteins, hormones, viruses Ways to obtain analyte sample: 1. Non-Contacting:• Electromagnetic radiation(IR/UV/Visible light, usually requires a probe) • Taking a gas sample near surface • Does not interfere with the subject2. Contacting: • Invasive/Non-invasive • Can evoke a response/ change analyte 3. Removing: • More or less traumatic(blood versus urine) • Toxic probe molecules or other additives (e.g. heparin) can be added • Most commonly usedBio receptors: Biological interactions• Weak, non-covalent interactions • Spontaneous (self-assembly) • Highly specific(single atom differences) • Complementary (lock-key) Enzymes/ substrates Most commonly used biological receptors.They are catalysts. Coupled enzyme reactions to give coloured reaction product or emitted photons.• AB/ AG Highly

selective interactions and very tight binding. AB can be raised against almost any antigen. Usually needs to be linked toother probe for detection. •Receptor proteins Many receptor proteins on surface of cells and embedded intomembranes. Applications mainly expected in detection of neuro-transmitters, hormones, neuro-active. Highly selectivebut often difficult to isolate. Use of intact biomembranes or cells. • Nucleic acids DNA, RNA diagnostic sensors in chipformat; used to detect genetic disorders and expression levels of proteins in parallel. DNA and RNA can be synthesised inthe lab. • Microorganisms E.g . genetically modified bacterial cells that light up when toxins are presented.Immobilization and surfaces Bio-receptor molecule has to be immobilized on or near the surface of the transducer . • The

immobilization is done either by physical entrapment (e.g. using a membrane) or chemical attachment. • Biological

recognition capability should not be lost!• Chemical methods of bioreceptor immobilization involve the formation ofcovalent bonds, including covalent binding, e.g., cross-linking. • Physical methods of bioreceptor immobilization, such asadsorption and entrapment.Signal transducers • The transducer converts the recognition event into a measurable signal . • The transducer can takemany forms depending upon the parameters being measured. • Electrochemical, optical, mass and thermal changes arethe most common. • Oxygen sensor: [O2] e current • pH sensor (gluconic acid): pH  voltage • Peroxide sensor: [H2O2] e current • Coupled to peroxidase rxn: [Dye]  colour change Thermal: enthalpy  T• ElectrochemicalPotentiometric -- detect changes in potential at constant current (usually zero). Amperometric --detect changes in current at constant potential. (currents generated when electrons are exchanged between a biologicalsystem and an electrode) Conductometric-- detect changes in conductivity between two electrodes. • Piezoelectriccrystals: Piezoelectric--These devices detect changes in mass . • Micromechanical systems: Cantilever transducersElectrochemical Transducers – Potentiometric Measuring cell potential at (near) zero current. The cell potential isproportional to the ion concentration. Mainly used to quantify inorganic ions, including H+. In biosensors usually enzymereactions that involve a significant pH change: penicillinase, urease, esterase. Need a reference electrode (e.g. Ag/AgCl)Electrochemical Transducers – Amperometric • A constant potential applied between a working and a referenceelectrode; the current through the cell is measured continuously. • When the oxidation potential of a molecule isreached, it is oxidised and electrons are produced: measured as a current through the cell . • Current is linearly related tothe concentration of the oxidised molecule. • Most commonly used in reactions involving REDOX enzymes, e.g., Glucoseoxidase, cholesterol oxidase, etc. The Glucose Amperometric Biosensor Why is needed? Diabetes• A chronic medical condition associated withabnormally high levels of sugar (glucose) in the blood known as hyperglycemia. • Normally, blood glucose levels aretightly controlled by insulin, a hormone produced by the pancreas which promotes cellular uptake. • Type I –  (IDDM)Pancreas undergoes autoimmune attack by the body itself. Destruction of beta cells thus renders one incapable ofmaking insulin. (10%) • Type II – (NIDDM) Cells exhibit a lack of sensitivity to insulin, thus the pancreas inadequatelyproduces larger than normal quantities in an attempt to increase cellular recognition. (90%) Diabetes leads to diseasessuch as heart disease and obesity.

The Glucose Biosensor: How is the disease managed?Home blood sugar (glucose) testing is an important part ofcontrolling blood sugar. One important goal of diabetes treatment is to keep the blood glucose levels near the normalrange of 70 to 120 mg/dl before meals and under 140 mg/dl at 2 hours after eating.Conductometric• Measuring conductance/resistance in a solution . • Useful when charges are produced duringenzymatic conversion. • Ex: detection of urea (in urine) using urease: Urea + 2H2O

Urease 2NH4 + 2HCO3

Electrochemical transducers Advantages: Easy to use, Direct interface with electronic displays, Possibility to miniaturize(faster response times) Disadvantages:Limited selectivity, Only works when biological reaction involves electron transferOptical transducers Photometric behaviour that can be exploited in biosensors : • UV/visible absorption • Fluorescence(and phosphorescence) emission • Bio-luminiscence • Chemi-luminiscence • Internal Reflection Spectroscopy (IRS)• Light scattering methods 400-700nm(UV – Infrared) Devices: photomultipliers (convert photons to current),spectrophotomers, optic fibres, etc. Advantages:• Easy to use • Can respond simultaneously to different reactants • Can

be very accurate, especially when more wavelengths are used • Can use optical fibres for efficient ‘photon transport’ •

Very high sensitivity (bio-luminiscence) Disadvantages: • Depend on availability of reactant that changes optical

properties • Dynamic range only around 102 (where Beer/Lambert law applies) • Difficult to miniaturize • Response timemay be slow: analyte diffusion • Background light interference Thermal Transducers • Making/breaking chemical bonds in enzymatic reactions results in enthalpy changes. • In additionheats of solution change especially with formation of charged species (e.g. protons). • Typically ≈10-3 K of temperaturechange, can be detected. • Reaction of interest may be coupled to reaction that produces more heat (e.g. glucoseoxidase coupled to catalase). Advantages:They work for most reactions Disadvantages:Non-selective, Low sensitivity,Difficult to miniaturizePiezo-electric transducers (Mass) • The frequency (f) of crystal oscillation depends on the mass (m) of the crystal andthat of any material adsorbed to its surface (A ). • Natural oscillation frequency decreases with adsorbed mass accordingto the Sauerbrey equation: Δf = -2.3 x 106 f2 Δm/A Piezo electric materials: Quartz (SiO2 crystals), ceramic materials,organic polymers.

8/10/2019 Cheat Sheet (Bio) Combine

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