Isolation and Characterization of Proteins

Isolation and Characterization of Proteins

Amino Acids

Nelson and Cox, 2004

Amino Acids

Nelson and Cox, 2004

Amino Acids

Nelson and Cox, 2004

Levels of Protein Structure

Nelson and Cox, 2004

Classification of Proteins Accdg. To Composition1. SIMPLE PROTEINS - yield only amino acids upon hydrolysis

2. CONJUGATED PROTEINSsimple proteins + non-protein substances ex. Nucleoprotein Glycoprotein Lipoprotein Phosphoprotein Hemoprotein Metalloproteins

Accdg. To Biological Function1. CATALYST2. TRANSPORT PROTEINS3. NUTRIENT and STORAGE PROTEINS4. STRUCTURAL PROTEINS5. CONTRACTILE and MOTILE PROTEINS6. DEFENSE PROTEINS7. REGULATORY PROTEINS

Accdg. to Shape1. GLOBULAR PROTEINS- polypeptide chain/s folded into spherical or globular shape-soluble in aq. system-ex. enzymes2. FIBROUS PROTEINS- polypeptide chains arranged in long strands or sheets-water insoluble-ex, keratin, collagen, fibroin

Accdg. To Solubility1. ALBUMINS- soluble in water and dilute aq. solutions2. GLOBULINS- soluble in dilute salt solutions but are insoluble or sparingly soluble in water3. GLUTELINS- soluble in dilute solutions of acids and bases- insoluble in neutral solvents4. PROLAMINS- soluble in 50-90% alcohol- insoluble in water, neutral solvents or absolute alcohol5. ALBUMINOIDS/SCLEROPROTEINS- insoluble in in most ordinary solvents

Protein DenaturationA loss of three-dimensional structure sufficient to cause of loss of function. Types: 1. Irreversible Denaturation - biological function /activity cannot be regained 2. Reversible Denaturation - biological function/activity can be regained

Denaturating AgentsProteins can be denatured by: a. Strong acids and bases b. Organic solvents c. Detergents d. Reducing agents e. Salts Salting-in Salting-out f. Heavy metals g. Temperature

Protein Hydrolysis1. COMPLETE HYDROLYSIS- uses a strong acid or base + high T- product/s: amino acids2. INCOMPLETE/PARTIAL HYDROLYSIS- uses enzymes called protease- product/s: mixture of amino acids and oligopeptides

COMPLETE HYDROLYSIS 1. ACID HYDROLYSIS- most commonly used reagent is 6N HCl- disadvantages:a. partial destruction of cys and tyrb. complete destruction of trp c. incomplete liberation of val and iled. racemization and destruction of ser and thre. asn + gln converted to asp + glu

COMPLETE HYDROLYSIS2. ALKALINE HYDROLYSIS- uses NaOH or KOH-advantages: a. trp not destroyed-disadvantages: a. arg, asn, gln, ser are destroyed

INCOMPLETE HYDROLYSIS-specific peptide bonds hydrolyzed by proteases like:TrypsinChymotrypsinPepsinBromelainPapain

Separation/Purification of Proteins

Properties of proteins being considered:1. Charge2. Molecular size, shape3. Solubility4. Affinity to a ligand5. pI

Casein- Phosphoprotein (Phosphate groups attached to OH groups of ser or thr) that exists as calcium caseinate- present as micelles in milk- Serves as a storage protein in milkIsolated from milk by isoelectric precipitationIsoelectric pH 4.6

Isolation of Casein and Albumin from Cows MilkCows milk

Milk proteins

Casein Whey proteins

alpha-s1 alpha-lactalbumin alpha-s2 beta-lactoglobulin beta serum albumin kappa immunoglobulins other proteins

Alpha-Lactalbumin- second major protein in bovine milk- metalloprotein that can bind to several metal ions like calcium and zinc- It can serve as a regulatory protein in lactose biosynthesis- isolated from whey by heat denaturation (in acidic condition)

Gladys Ilagan Gen Biochemistry

Gladys Ilagan Gen Biochemistry

Myoglobin- Small, bright red protein common in muscle cells - Stores oxygen (used when muscles are hard at work) - A hemoprotein containing a heme group at its center- Isolated by salt precipitation

Myoglobin

Gladys Ilagan Gen Biochemistry

Gladys Ilagan Gen Biochemistry

Gluten- Storage protein responsible for the elasticity and extensibility of dough- consists of gliadin and glutenin- Isolated by difference in solubility in water- Isolated gluten free of starch when (-) to iodine test

Qualitative Color Reactions1. BIURET TEST-test for presence of a peptide bond (peptide must have at least 3 amino acids)

Reagent:CuSO4 + NaOHPrinciple:Principle: formation of coordination complex of Cu2+ and four nitrogen atoms (two from each of the two polypeptide chains)(+) Result:purple color of solution

Qualitative Color Reactions2. NINHYDRIN TEST-test for a-amino acid Reagent:Ninhydrin (triketohydrindene hydrate)Principle:oxidative decarboxylation & deamination followed by condensation(+) Result:blue-violet color yellow for proline (pyrrolidine ring)

Qualitative Color Reactions3. XANTHOPROTEIC TEST-test for aromatic amino acidsReagent:HNO3 , NaOHPrinciple:nitration of aromatic rings via SEAr(+) Result:yellow color of solution with HNO3 orange color of solution with NaOH

Qualitative Color Reactions4. MILLONS TEST-test for tyrosineReagent:salt of Hg dissolved in HNO3Principle:complexation (mercuration & nitration or nitrosation/ complexation of nitrohydroxyphenyl derivatives with Hg2+)(+) Result:red color

Qualitative Color Reactions5. HOPKINS-COLE TEST-test for tryptophanReagent:glacial CH3COOH, glyoxylic acid, concd H2SO4Principle:reduction of oxalic acid to glyoxylic acid and acid-catalyzed condensation of two tryptophans with glyoxylic acid(+) Result:purple color at the interface

Qualitative Color Reactions6. SAKAGUCHI TEST-test for arginineReagent:-naphthol, NaOBr, NaOH, urea (to stabilize color & destroy excess OBr- ions)Principle:complexation (base-catalyzed condensation of -naphthol with the guanido group of Arg)(+) Result:red color

Qualitative Color Reactions7. NITROPRUSSIDE TEST-test for S-containing amino acidsReagent:NaOH, nitroprussidePrinciple:Complexation reaction(+) Result:red color

Qualitative Color Reactions8. FOHLS TEST-test for S-containing amino acids

Reagent:NaOH, (CH3COO)2PbPrinciple:degradation & substitution reaction to form PbS (+) Result:dark brown / black precipitate

Qualitative Color Reactions9. TEST FOR AMIDE- asn, gln

Reagent:NaOHPrinciple:Basic hydrolysis(+) Result:evolution of gas, presence tested using a litmus paper

Qualitative Color Reactions10. PAULY TEST-test for histidine and tyrosineReagent:sulfanilic acid in NaNO2 solution,Na2CO3Principle:Formation of azo dyes

(+) Result:Red color

Qualitative Color ReactionsColor ReactionIntact ProteinProtein HydrolysateacidicbasicenzymaticBiuretNinhydrinXanthoproteicMillonsHopkins-ColeSakaguchiNitroprussideFohlsTest for amidePauly

Paper Chromatography- Used to determine the amino acid composition of a given protein solution- Visualized using ninhydrin

- Retention factor, RFRF = distance travelled by the amino acid, cm distance travelled by the solvent, cm

Stages in Paper ChromatographySample/Standard Application: small spots of std/sample are applied to avoid overlapping and tailing during developmentDevelopment: equilibration (saturation of chamber with mobile phase) to hasten developmentVisualization: chemical visualizing agent: ninhydrin spray for amino acids and proteinsEvaluation: comparing Rf values of sample and standardsDocumentation: chromatogram

Bradford Assay-colorimetric method for determining protein concentration- involves use of Coomassie Brilliant Blue G-250 (dye), which reacts primarily to basic (especially arginine) and aromatic amino acids-measures 10-100 mg protein-standard used: bovine serum albumin (BSA)-Bradford reagent: dye dissolved in ethanol and phosphoric acid

Bradford AssaySteps in quantifying proteins using Bradford assay:1. Prepare BSA standards with different concentration - stock solution added with different amounts of water - final concentration of standard is computed using C1V1 = C2V22. Read A595 of standards and samples3. Plot standard curve Absorbance (y) vs. Concentration (x)4. Draw the best fit line5. Determine the concentration of sample from the standard curve by extrapolation

Bradford Assay

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