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
1. Biuret Test

2. Ninhydrin
Test
3.
Xanthoproteic
Test
4. Millons Test

5. Hopkins-Cole
Test

6. Sakaguchi
Test

7. Pc(Ac)2 Test

Reagents

Principle Involved

Visible (+) Result

Alkaline CuSO4

Complexation
reaction

Blue-violet to Violet
solution

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

Triketohydrinenehydr
ate in 95% ethanol

Oxidative
deamination
followed by
decarboxylation

Blue/Purple/Blueviolet solution

a. Free -amino group

b. Nonspecific for
amino acids

Conc. HNO3 & conc.

NaOH

Nitration of phenyl
group

Yellow to orange
solution

(+) Tyrosine &

Tryptophan

HgSO4 in H2SO4

Complexation
reaction between the
phenolic group & Hg

Old Rose/Flesh/Red
precipitate

Glyoxylic acid &

conc. H2SO4

Indole group
condenses with
glyoxylic acid &
H2SO4

Violet ring at the

interphase

-napthol, NaOBr,
Urea

Guanido group
reacts with -napthol
with an oxidizing
agent

Red or Orange
solution

NaOH & Pb(Ac)2

Fusion followed by
Ionic interaction

Black Precipitate

(+) Tyrosine

(+) Tryptophan

(+) Free or intact

arginine
(+) Cysteine &
Methionine

Color Reactions
1. Biuret Test
2. Ninhydrin Test
3. Xanthoproteic Test
4. Millons Test
5. Hopkins-Cole Test
6. Sakaguchi Test
7. Pb(Ac)2 Test

Intact
Protein

Hydrolyzate
Acid

Alkaline

Enzymatic