Upload
peter-limjoco-david
View
48
Download
9
Tags:
Embed Size (px)
DESCRIPTION
Is
Citation preview
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
http://www.bio-rad.com