Color Reactions of Proteins

I. Protein Isolation

1. Casein in Skimmed Milk Isoelectric precipitation IpH of casein = 4.6, acidic protein

White curd, noncrystalline solid, insoluble in water

Conjugated protein phosphoprotein

Other proteins found in milk Lactalbumin & Lactoglobulin

2. Gluten in Wheat Flour

Mixture of two proteins Glutenin & Gliadin

Yellowish-white solid, tough, elastic, sticky, insoluble in water

In bread-making, it traps the CO2 produced by the reaction of flour & yeast

3. Legumelin in Mongo Beans

Precipitating agent CH3COOH, acts as a dehydrating agent

White amorphous solid, soluble in water

II. Hydrolysis of Proteins

1. Chemical Hydrolysis complete hydrolysis

a. Acidic Hydrolysis

Little or no racemization

Not all amino acids are recovered

W is destroyed

Partial destruction of S, T, C

Hydrolysis of N to D & Q to E

b. Alkaline Hydrolysis

Racemization of all amino acids

W is stable

Not all amino acids are recovered

R decomposes to ornithine and urea

Partial destruction of C, C-C, S, T

Hydrolysis of N to D & Q to E

2. Enzymatic Hydrolysis partial hydrolysis

a. Presence of proteolytic enzymes, only cleave peptide bonds at specific sites

b. Amino acids are not affected

c. Process require certain temperature & pH conditions for optimum activity of enzymes

III. Color Reactions of Proteins

Color ReactionsReagentsPrinciple InvolvedVisible (+) Result

1. Biuret TestAlkaline CuSO4Complexation reactionBlue-violet to Violet solutiona. For peptide containing 2 peptide linkage

b. General test for proteins

2. Ninhydrin TestTriketohydrinenehydrate in 95% ethanolOxidative deamination followed by decarboxylationBlue/Purple/Blue-violet solutiona. Free -amino group

b. Nonspecific for amino acids

3. Xanthoproteic TestConc. HNO3 & conc. NaOHNitration of phenyl groupYellow to orange solution(+) Tyrosine & Tryptophan

4. Millons TestHgSO4 in H2SO4Complexation reaction between the phenolic group & HgOld Rose/Flesh/Red precipitate (+) Tyrosine

5. Hopkins-Cole TestGlyoxylic acid & conc. H2SO4Indole group condenses with glyoxylic acid & H2SO4Violet ring at the interphase(+) Tryptophan

6. Sakaguchi Test-napthol, NaOBr, UreaGuanido group reacts with -napthol with an oxidizing agentRed or Orange solution(+) Free or intact arginine

7. Pc(Ac)2 TestNaOH & Pb(Ac)2Fusion followed by Ionic interactionBlack Precipitate(+) Cysteine & Methionine

Color ReactionsIntact ProteinHydrolyzate

AcidAlkalineEnzymatic

1. Biuret Test

2. Ninhydrin Test

3. Xanthoproteic Test

4. Millons Test

5. Hopkins-Cole Test

6. Sakaguchi Test

7. Pb(Ac)2 Test