protein experiments
Post on 02-Nov-2015
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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
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