+PROTEINS Proteins are naturally occurring polypeptides of molecular weight greater than 5000.

These macromolecules show great diversity in physical properties, ranging from water-soluble enzymes to the insoluble keratin of hair and horn, and they perform a wide range of biological functions. Proteins may be classified according to: A. Three-Dimensional Shape or Conformation 1. Fibrous are organized into linear or sheetlike structures with a regular, repeating folding pattern. (e.g. keratin, collagen, elastin) 2. Globular are folded into compact, nearly spherical, globular conformation. (e.g. casein, albumin, globulin, hormones) B. Composition 1. Simple contains only amino acids and their derivatives 2. Conjugated apart from amino acids, it may contain a carbohydrate- , lipid-, or phospho- moiety or groups (e.g. phosphoprotein, lipoprotein, nucleoprotein) C. 1. 2. 3. 4. 5. 6. 7. 8. Biological Function Structure - cellulose, keratin, collagen Catalysis - enzymes Movement/Contraction myosin, actin Transport hemoglobin, transferrin Hormones insulin, erythropoietin Protection antibodies, fibrinogen Storage casein, ovalbumin, ferritin Regulation histones, non-histones

Hierarchy of Protein Structure 1. Primary Structure the sequence of amino acids 2. Secondary Structure the regular repeating folding pattern (e.g. E-helix,F-pleated sheet), stabilized by hydrogen bonds between peptide groups close together in the sequence. 3. Tertiary Structure the way that segments of the protein fold in three dimensions, stabilized by interactions between distant parts of the sequence. 4. Quaternary Structure the interaction between different polypeptide chains to produce an oligometric structure, stabilized by noncovalent bonds only.

ISOLATION OF PROTEINS 1. 2. 3. 4. Casein isoelectric precipitation Albumin denaturation and coagulation by heat Gluten difference in solubility Myoglobin salt-induced precipitation using (NH4)2SO4

I. Casein and Albumin (from milk) Casein is a phosphoprotein which has phosphate groups attached to some amino acid side chains. It exists in milk as the calcium salt, calcium caseinate. This salt has a complex structure. It consists of E, F and O caseins, which form a micelle, or a solubilized unit. Calcium caseinate has its isoelectric point at pH 4.6. The pH of milk is about 6.6. If acid (e.g. HCl) is added to milk, the phosphate groups present in casein is protonated and the neutral protein precipitates. Ca2+Caseinate + 2HCl Casein + CaCl2

Albumins are globular proteins that are soluble in water and in dilute salt solutions. Its molecular weight is about 41000. Once the casein has been removed from milk, and the solution has been made acidic, the lactalbumins can be isolated by heating the mixture to precipitate them. Lactalbumin is a major whey protein and it is a calcium metalloprotein. II. Gluten (from Wheat flour) Gluten is the composite of a prolamin and a glutelin, which exist, conjoined with starch, in the endosperm of various grass-related grains. Of the gluten proteins, gliadrin has a relative molecular mass between 30000 and 80000 Da whereas glutenin is multi-chained with relative molecular mass up to several million Da. Gluten can be isolated by washing the dough with water. Starch is partially soluble while gluten is insoluble in water. Thus, gluten can be separated from starch. Complete removal of starch can be detected using the iodine test. III. Myoglobin (from Beef muscle) Myoglobin is made up of a single polypeptide chain, globin and a prosthetic heme group, an Iron(II) protoporphyrin-IX complex. Myoglobin has been known to be a major contributor to the color of muscle, depending on its redox states. It can be isolated by ammonium sulfate precipitation from the buffered muscle extract.

QUALITATIVE CHEMICAL TESTS 1. Biuret Test it is a general test for proteins; Biuret is an organic compound produced when urea is heated above its melting point. Reagents: CuSO4, NaOH Principle: formation of a metal ion coordination complex with a peptide bond (at least a tripeptide) (+) visible result: pink to violet to blue color (the color intensity increases as the number of peptide bonds increases)

2. Ninhydrin Test used to detect free Eamino acids Reagents: 1,2,3-indanetrione monohydrate or triketohydrindene, ethanol Principle: oxidative decarboxylation and deamination followed by condensation (+) visible result: blue to blue-violet color (the color deepens as the number of amino goups increases; proline and hydroxyproline produces yellow color, asparagine yields a characteristic brown color)

3. Xanthoproteic Test a qualitative test for aromatic amino acids such as Y, W and F (F is not as readily nitrated and does not or weakly responds to this test) Reagents: conc. HNO3, conc. NaOH Principle: nitration of aromatic ring via aromatic electrophilic substitution (+) visible result: yellow solution on heating; orange solution with excess NaOH

4. Millons Test/ Millon-Nasse Test used to detect phenolic group in tyrosine Reagents: HgSO4 in H2SO4 Principle: complexation of nitrohydroxyphenyl derivatives with Hg2+ (+) visible result: purple-red ppt

5. Hopkins-Cole Test specific test for indole group in tryptophan Reagents: Glyoxylic acid (Mg powder, oxalic acid, acetic acid), conc. H2SO4 Principle: acid-catalyzed condensation of two tryptophans with glyoxylic acid (+) visible result: violet interface 6. Sakaguchi Test specific test for guanido group in arginine Reagents: E-naphthol, NaOBr, NaOH, urea Principle: base-catalyzed condensation of E-naphthol with the guanido group of arginine (+) visible result: red to red-orange color 7. Fohls Test/ Lead (II) Acetate Test used to detect S-containing amino acids (methionine, cysteine) Reagents: Lead (II) acetate, NaOH Principle: degradation and substitution reaction to form lead sulfide (+) visible result: brown or black ppt

8. Paulys Test/ Diazo Reaction used to detect the presence of histidine and tyrosine Reagents: NaNO2, sulfanilic acid, Na2CO3 Principle: diazotized sulfanilic acid couples with amino phenol and immidazole to form a colored azo compound in cold condition (+) visible result: red color

9. Nitroprusside Test detects the presence of cysteine Reagents: Na2Fe (CN)5NO in dilute NH3 Principle: comlexation (+) visible result: red ppt

HYDROLYSIS OF PROTEINS 1. Acid Hydrolysis takes place via nucleophilic acyl substitution reaction (hydrolysis of a carboxylic acid amide) - Reaction mixture is autoclaved at 15 psi for 5 hrs with 8N H2SO4. - Hydrolysate is neutralized with Ba(OH)2, precipitating as BaSO4. - Complete hydrolysis (all peptide bonds are broken yielding a mixture of L-amino acids in the hydrolysate) - No racemization of amino acids occurs - Tryptophan is destroyed and converted to humin. - Some serine and threonine are loss. - The amide group of aspargine and glutamine undergoes complete hydrolysis to give aspartic acid and glutamic acid, respectively. 2. Base Hydrolysis - takes place via nucleophilic acyl substitution reaction (hydrolysis of a carboxylic acid amide) - Reaction mixture is autoclaved at 15 psi for 5 hrs with Ba(OH)2 - Hydrolysate is neutralized with H2SO4, precipitating as BaSO4. - Complete hydrolysis (all peptide bonds are broken yielding a mixture of L-amino acids in the hydrolysate) - Racemization of all amino acids occurs. - Tryptophan is stable. - Arginine is hydrolyzed into ornithine and urea. - Cysteine, serine and threonine are destroyed. 3. Enzymatic Hydrolysis partial or selective hydrolysis of polypeptides to yield a set of short peptides. - Uses proteases/peptidases to hydrolyze peptide bonds. - Employed hydrolases present in 2% pancreatin. The reaction mixture was incubated in a water bath at 370C. - Pancreatin is a mixture of enzymes: amylase, lipase and proteases (trypsin and chymotrypsin). - Na2CO3 and Thymol were added to the reaction mixture. - Na2CO3 is used to buffer the mixture at optimum pH. - Thymol serves as preservative and disinfectant. - Incomplete hydrolysis. - Action is highly specific (regioselective and/or stereoselective).

CHROMATOGRAPHIC ANALYSIS OF THE PROTEIN HYDROLYSATE Partition paper chromatography is widely employed for the determination and separation of amino acids. The amino acids present in hydrolysate is separated based on the difference in the affinity of the solute between the mobile phase and stationary phase. The mobile phase is liquid/gas (EtOH:NH3:H2O; 8:1:1) and the stationary phase is liquid (the water molecules adsorb by the cellulose fiber); thats why its partition chromatography. Normal phase chromatography is involved since the stationary phase is polar and the mobile phase is least polar. The higher the Rf value, the greater the affinity of the amino acid to the mobile phase, the least polar the amino acid. The lower the Rf value, the greater the affinity of the amino acid to the stationary phase, the most polar the amino acid. The identity of the amino acid is determined by comparing the Rf value of the unknown component with that of the standard amino acids. Steps in paper chromatography includes: 1. Sample application spots should be as small as possible to prevent overlapping and tailing 2. Development equilibration is performed to hasten development 3. Visualization ninhydrin spray reagent is used 4. Evaluation Rf value is computed 5. Documentation chromatogram is sketched

BRADFORD ASSAY -Bradford assay is based on the color change (red to blue) of the dye Coomassie Brilliant Blue G-250 upon binding to added protein. - The absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 to 595 nm when noncovalent and Van der Waals interactions with protein occur. -The dye is anionic; thus, it is more sensitive to proteins with high Arg, Lys and His content. - Bradford assay is used to determine the protein concentration of the sample. - Beer-lamberts Law relates the absorbance of the solution to the concentration of the sample. -Absorbance is directly related to the concentration of the sample. (As the concentration of protein sample increases, the color intensity deepens, and the absorbance also increases.) - The protein concentration of the sample can be determined once the calibration curve of the standard solutions has been constructed. It can be done graphically or analyzed using the linear regression. -The equation of the line, y = mx + b is important in linear regression, where y = absorbance of the sample m = slope of the calibration curve b = y-intercept x = concentration of the protein sample (to be determined) -Algebraically, x = (y-b)/m - The standard used in the experiment is Bovine Serum Albumin (BSA).

ENZYMES - Enzymes are proteins that catalyze biochemical reactions without being consumed in the process by lowering the activation energy of the reaction. - Each enzyme is characterized by specificity for a narrow range of chemically similar substrates. - Many enzymes have small nonprotein molecules associated with or near the active site that determine substrate specificity. These are called: 1. Cofactors if they are noncovalently linked to the protein. 2. Prosthetic groups if they are covalently linked. I. Catalytic action of Invertase - Invertase is highly specific for sucrose. - It hydrolyzes the glycosidic bond between E-D-glucose and -D-fructose; thus, yields an equimolar amount of glucose and fructose. sucrose + invertase glucose + fructose

-There are factors that influence enzyme activity: 1. pH 2. Temperature 3. Substrate concentration -The optimum pH and temperature of the invertase is 5.0 and 600C, respectively at which it has its greatest activity. II. Dinitrosalicylic acid (DNS) Assay - This method tests for the presence of free carbonyl (C=O) group, the so-called reducing sugars.3,5Dinitrosalicylic acid is an aromatic compound that reacts with reducing sugars to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. - In the experiment, the sample involved is sucrose. Sucrose is a non-reducing disaccharide. But still, DNS assay can be employed because sucrose was hydrolyzed first with an acid to yield glucose and fructose. - One mole of sugar (e.g. glucose) will react with one mole of 3,5-Dinitrosalicylic acid. - It involves redox reaction. Glucose is oxidized to gluconic acid and 3,5-dinitrosalicylic acid is reduced to 3amino-5-nitrosalicylic acid.