PROTEINS (Isolation, Hydrolysis, Qualitative Tests and Quantitative Determination)

1. PROTEINS (Isolation, Hydrolysis, Qualitative Tests and Quantitative Determination)

2. ISOLATION

3. CASEIN • main protein in milk • exists as the Ca salt • phosphoprotein • mixture of min of 3 similar proteins (-, - &  -casein) • 80% of protein present in milk • contains the essential amino acids (V P H MATILL) • isolated at isoelectric pH (pI), least soluble (isoelectric precipitation) • accomplished by addition of dilute acid • net charge at pI=0

4. HYDROLYSIS • bond cleavage of labile bonds simultaneous with the addition of water • needed to break amide bonds in intact proteins to produce amino acids

5. Types of Protein Hydrolysis

6. Acid hydrolysis – catalyzed by strong acids such as H2SO4, HCl, HNO3, HClO4, etc. (15 psi/5 hrs.) • total hydrolysis • does not promote racemization of a-C configuration • Trp is destroyed and converted to humin (black pigment) • Thr and Ser are destroyed • Asn and Gln are converted to Asp and Glu

7. Base/Alkaline Hydrolysis – uses strong bases such Ba(OH)2, NaOH, KOH, etc. (15 psi/5hrs.) • total hydrolysis • Trp is not destroyed • promotes racemization • Thr and Cys are lost • Arg is destroyed and converted to urea & ornithine

8. Enzymatic hydrolysis – partial cleavage/hydrolysis • regioselective and/ stereoselective • cleaves specific linkages of selected types of amino acid groups (i.e. carboxypeptidase A for aromatic AA’s)

9. QUALITATIVE CHEMICAL TESTS

10. Biuret Test – general test for intact proteins and protein hydrolyzates (at least a tripeptide!) • named after the compound, biuret • reagents: CuSO4 solution and dilute NaOH • positive result: formation of pink to violet to blue color • principle: complexation of Cu+2 with amide N atoms • NO reaction with dipeptides, urea, coagulated proteins and amino acids (except serine and threonine)

12. Ninhydrin Test – general test for compounds with free a – amino groups • one of the most sensitive color reactions known • reagent/s: ninhydrin (1,2,3 - indanetrione monohydrate) in ethanol • positive result: blue to blue violet color • principle: oxidative deamination and decarboxylation; reduction of ninhydrin • Proline, hydroxyproline, and 2-, 3-, and 4-aminobenzoic acids fail to give a blue color but produce a yellow color instead • ammonium salts give a positive test. Some amines, such as aniline, yield orange to red colors, which is a negative test

14. Xanthoproteic Test – general test for aromatic amino acids such as tryptophan, phenylalanine, histidine and tyrosine • presence of electron donating substituents enhances reaction rate • reagents: conc. HNO3 and conc. NaOH (neutralize excess acid) • positive results: formation of yellow precipitate and after addition of excess NaOH (alkaline), an orange precipitate forms • principle involved: nitration of aromatic rings (i.e. indole in tryptophan!) via electrophilic aromatic substitution

16. Hopkins-Cole Test – detects the presence of indole group in tryptophan • reagents: magnesium, oxalic acid and conc. H2SO4 • positive result: pink to violet interface • principle: reduction of oxalic acid to glyoxilic acid & acid-catalyzed condensation of 2 tryptophans with glyoxilic acid

18. Sakaguchi Test – specific for arginine (guanido group) • reagents: -napthol, NaOH and NaOBr (and urea to stabilize color and destroy excess OBr- anions) • positive result: red to red-orange color • principle: base-catalyzed condensation of -napthol with the guanido group of arginine

20. QUANTITATIVE DETERMINATION OF PROTEINS

21. Bradford Assay – simple, fast, inexpensive, highly sensitive • uses the Coomassie Brilliant Blue G-250 dye reagent ( binds electrostatically with arginine residues in anionic form and by pi-stacking interactions with aromatic AA’s) • read at 595 nm (UV spectrophotometer) • intensity of color (measured by absorbance) is directly proportional to the concentration of protein (Beer’s Law) • A = bc • unknown concentration is measured using linear regression analysis • y = mx + b • where: y = measured absorbance • m = slope • x = concentration of unknown • b = y-intercept • for standard protein preparations, use C1V1=C2V2 when dilutions are done on standard solutions.