Protein Experiments Handout

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 CO 2 produced by the reaction of flour & yeast 3. Legumelin in Mongo Beans Precipitating agent – CH 3 COOH, 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 o W is destroyed o Partial destruction of S, T, C o 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 o R decomposes to ornithine and urea o Partial destruction of C, C-C, S, T o 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

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Transcript of Protein Experiments Handout

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

o W is destroyed o Partial destruction of S, T, C o 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

o R decomposes to ornithine and urea o Partial destruction of C, C-C, S, T o 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 Reactions Reagents Principle Involved Visible (+) Result

1. Biuret Test Alkaline CuSO4 Complexation reaction Blue-violet to Violet

solution

a. For peptide containing 2 peptide linkage b. General test for proteins

2. Ninhydrin Test Triketohydrinenehydrate

in 95% ethanol

Oxidative deamination followed by

decarboxylation

Blue/Purple/Blue-violet solution

a. Free -amino group b. Nonspecific for amino acids

3. Xanthoproteic Test

Conc. HNO3 & conc. NaOH

Nitration of phenyl group

Yellow to orange solution

(+) Tyrosine & Tryptophan

4. Millons Test HgSO4 in H2SO4 Complexation reaction between the phenolic

group & Hg

Old Rose/Flesh/Red precipitate

(+) Tyrosine

5. Hopkins-Cole Test

Glyoxylic acid & conc. H2SO4

Indole group condenses with glyoxylic acid &

H2SO4

Violet ring at the interphase

(+) Tryptophan

6. Sakaguchi Test -napthol, NaOBr, Urea Guanido group reacts with -napthol with an

oxidizing agent Red or Orange solution

(+) Free or intact arginine

7. Pc(Ac)2 Test NaOH & Pb(Ac)2 Fusion followed by Ionic

interaction Black Precipitate

(+) Cysteine & Methionine