Isolation, Hydrolysis and Characterization of Protein Objectives: -to isolate proteins (gluten, casein, albumin, and myoglobin) -to

hydrolyze proteins -to perform color reactions to intact protein and hydrolysate -to determine the concentration of protein using Bradford assay Proteins-polymers of amino acids linked head to tail, from carboxyl group to amino group, through formation of covalent peptide bonds Amino acids -building blocks of proteins -carboxylic acid group -amino group -side group R gives unique characteristics R side chain | H2H C-COOH | H Classification of Protein I. Biological function 1. 2. 3. 4. 5. 6. 7. 8. II. Structure- cellulose, keratin, collagen Catalysis- enzymes Movement/Contraction- myosin, actin Transport- hemoglobin, transferring Hormones- insulin Protection- antibodies, fibrinogen Storage- casein, ovalbumin RegulationThree-dimensional shape or conformation

1. fibrous- keratin, collagen, elastin Are organized into linear or sheetlike structures with a regular repeating folding pattern 2. globular- casein, albumin, hormones Are folded into compact, nearly spherical, globular conformation III. Composition

1. Simple- contains only amino acids and their derivatives

2. Conjugated- apart from amino acids, it may contain a carbohydrate-, lipid-, or phosphomoiety or groups Conjugated proteins Glycoproteins are proteins containing carbohydrate groups Lipoproteins are proteins that are associated with lipid molecules Nucleoproteins are proteins joined with nucleic acid Phosphoproteins contain phosphate groups Metalloproteins are protein-metal complexes Hemaproteins contain heme Flavoproteins contain riboflavin Hierarchy of Protein Structure 1. Primary structure- the sequence of amino acids 2. Secondary structure- the regular, repeating folding pattern 3. Tertiary structure- the way that segments of the protein fold in three dimensions; over all folding of domains 4. Quaternary structures- the interaction between different polypeptide chains to produce an oligometric structure Isolation of Proteins 1. Casein- isoelectric precipitation a phosphoprotein which has phosphate groups attached to some amino side chains exists in milk as calcium caseinate calcium caseinate has its isoelectric point at 4.6 Ca2Caseinate + 2HCl Casein + CaCl2 2. Albumin denaturation and coagulation by heat Are globular proteins that are soluble in water and in dilute salt solutions 3. Gluten- difference in solubility A protein found in wheat, rye, and barley Gives bread its structure, texture, and elasticity It is a composite of a prolamin and a glutelin which exist, conjoined with starch 4. Myoglobin- salt-induced precipitation ising (NH4)2 SO4 Protein Hyrdolysis Break down of peptide bonds Requires acid or base, water, and heat Gives smaller peptides and amino acids Similar to digestion of proteins using enzymes Occurs in cells to provide amino acids to synthesize other proteins and tissues Types of Hydrolysis Acidic

Basic Enzymatic

Acidic Hydrolysis Hydrolysate is neutralized with NaOH/ Ba(OH)2 Complete Hydrolysis No racemization of Amino Acids occur Tryptophan is destroyed and convented to humin Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively. Basic Hydrolysis Rxn mixture is autoclaved at 15psi for 5 hrs with Na(OH)/ Ba(OH)2 Hydrolysate is neutralized with HCl/ H2SO4 Complete hydrolysis Racemization of all AA occur Tryptophan is stable Arginine is hydrolyzed into ornithine and urea Cysteine, serine, and threonine are destroyed Enzymatic Hydrolysis Incomplete hydrolysis Exopeptidase/Endopeptidase Denaturation- Disruption of Secondary, tertiary, and quaternary protein structure by: Heat/organics-break apart H bonds and disrupt hydrophobic attractions Acids/Bases- Break H bonds between polar R groups and ionic bonds Heavy metal ions- react with S-S bonds to form solids Agitation- stretches chains until bonds break Qualitative Chemical test 1. Biuret Test Reagents: CuSO4, NaOH Relative result: violet coloration Principle involved: complexation of Cu2+ with a peptide bond 2. Ninhydrin Test Purpose: detects free alpha amino acid Positive result: blue to blue-violet color Principle involved: oxidative decarboxylation and deamination followed by condensation 3. Xanthroproteic test Reagents: conc. HNO3, conc. NaOH Purpose: test for aromatic amino acids (Y, W, and F) Positive result: yellow soln on heating; orange soln with excess NaOH

4.

5.

6.

7.

8.

9.

Principle involved: nitration of aromatic ring via electrophilic aromatic substitution Millons/Millon-Nasse test Reagents: Hg SO4 in H2SO4 Purpose: detect phenolic group in tyrosine Positive result: old rose/flesh/purple-red ppt Principle involved: complexation of nitrohydroxyphenyl derivatives with Hg3+ Hopkins-Cole test Reagents: glyoxylic adic (Mg powder, oxalic acid, acetic acid), conc. H2SO4 Purpose: specific test for indole group of tryptophan Positive result: violet interface Principle involved: acid-catalyzed condensation of two tryptophans with glyoxilic acid Sakaguchi test Purpose: specific test for guanido group of arginine Positive result: res to red-orange color Base-catalyzed condensation of napthanol with the guanido group of arginine Fohls/ Lead acetate test Reagents: Lead(II) acetate, NaOH Purpose: detects S-containing AA (methionine, cysteine) Positive result: brown or black ppt Principle involved: degradation and substitution reaction to form lead sulfide Paulys test/ Diazo reaction Reagents: NaNO2, sulfanilic acid, Na2CO3 Purpose: detects the presence of histidine and tyrosine Positive result: red color Principle involved: diazotized sulfanilic acid couples with amino phenod to form a colored azo compound in cold condition Nitroprusside test Purpose: detects the presence of SH group (cysteine) Positive result: Red ppt Principle involved: complexation