protein experiments handout
DESCRIPTION
dTRANSCRIPT
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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
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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