Isola&on

and Hydrolysis of Casein from Non-

fat Milk Color Reac&ons of Intact Casein and
Hydrolyzate Protein

Dharmatov Rahula B. Albano, Ph.D.
Department of Chemistry, College of Science


 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

•Proteins are biomolecules that contain many

amide bonds, formed by joining amino acids.

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 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

•Peptides and proteins are formed when amino

acids are joined together by amide bonds.

•A dipeptide has two amino acids joined together

by one amide bond.

•The amide bond is called a peptide bond.

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 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

•A tripeptide has three amino acids joined together

by two amide bonds.

•Polypeptides have many amino acids, while

proteins have more than 40 amino acids.
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 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

•Proteins account for 50% of the dry weight of the

human body.
•They have many functions in the body.
•Unlike lipids and carbohydrates, proteins are not
stored, so they must be consumed daily.
•Current recommended daily intake for adults is
0.8 grams of protein per kg of body weight (more
is needed for children).
•Dietary protein comes from eating meat and milk.

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Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

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Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

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Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

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 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Objectives

isolate casein from non-fat milk

hydrolyse the casein using an acid or a base

characterize the isolated casein and hydrolyzate

quantify an unknown protein using the bradford method

 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Isolation of casein from milk

Casein – major protein in milk (complete protein)

It is a globular protein.
Contains ALL special AAs (F, V, H, M, R, T, W, I, L, K)
Heating at 55 deg C – facilitates precipitation
Isolation was done using Isoelectric precipitation – process of
lowering pH up to isoelectric point (IpH)
IpH = 0 charge protein is most insoluble
IpH casein = 4.6
10% Acetic Acid – used to lower the pH and precipitate out the
casein
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Hydrolysis of the Intact Protein

Intact protein – individual AAs are linked together by
peptide bond (very strong)

Hydrolysis – breakage of the peptide bond in the

presence of H2O and catalyst (acid, base or enzyme)
Acid hydrolysis – 8 N H2SO4
Base hydrolysis – Ba(OH)2(s)
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Acid Hydrolysis (Complete) Base Hydrolysis (Complete)

• H2SO4
• Autoclaved at 15 psi for 5 h
• Neutralized with Ba(OH)2
BaSO4
• Filter (filtrate hydrolyzate
contains only AAs)
• H2SO4 was used instead of HCl:
HCl + Ba(OH)2 BaCl2(aq) + H2O
• Ba(OH)2
• Autoclaved at 15 psi for 5 h
• Neutralized with H2SO4
BaSO4
• Filter (filtrate hydrolyzate
contains only AAs)
• Ba(OH)2 was used instead of
NaOH:
NaOH + H2SO4 Na2SO4(aq) +
H2O
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Advantage of Acid Hydrolysis Advantage of Alkaline Hydrolysis

o No racemization – all AAs retain o W is not destroyed
their structural configuration ( )
Racemization – process wherein the structural configuration
is being altered (L D; D L)

Disadvantages
Disadvantages

W is destroyed, forming humin Racemization or formation of

(black color) racemic mixture
Loss of Serine and Threonine Loss of Serine, Threonine, and
Asparagine and Glutamine are Cystine
converted to Aspartic acid and Arginine is destroyed and
Glutamic acid converted to urea and ornithine
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Biuret Test
test for intact protein (presence of peptide bond)
Principle: Complexation reaction
Reagents: CuSO4·5H2O; NaOH
(+) Result: formation of violet solution – intensity of the
color depends on the length of the peptide bond (2
or more peptide bonds)
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Ninhydrin Test
test for free amino acids (presence of -AA)
Principle: Oxidative deamination & Condensation
Reagents: triketohydrindene hydrate (ninhydrin)
(+) Result: violet (or deep-blue) solution
proline & OH-proline (imino acid) – yellow soln (-)-result
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Xanthoproteic Test
test for aromatic AAs
Principle: Nitration of aromatic ring
Reagents: conc. HNO3; NaOH
(+) Result: yellow ppt orange soln
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Sakaguchi Test
test for the presence of guanido group (R)
Principle: Condensation and Complexation
Reagents: -naphthol; NaOBr; NaOH
(+) Result: red coloration w/c easily disappear
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Hopkins’-Cole Test
test to detect the presence of indole group (W)
Principle: Complexation and Condensation
Reagents: Glyoxilic acid (Mg + Oxalic acid); conc. H2SO4
(+) Result: violet interphase
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Millon’s Test
test to detect the presence of phenolic group
Complexation (Flesh/Red ppt)
Reagents: Hg(NO3)2; conc. HNO3

Lead Acetate Test

test to detect the sulfuhydril group
Degradation & Substitution (Black ppt)
Reagents: NaOH; Pb(Ac)2
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Bradford method for Protein Concentration

20 – 200 µg protein

Coomasie Brillant Blue G-250

- pinkish-brown (protein unbound)

- blue (protein bound)

- 465 nm to 595 nm

- hydrophobic and ionic interactions

 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Angelo Gonzaga, a BS Biology student, isolated a protein from Pandanus

amaryllifolius as part of his undergraduate thesis. After purification using affinity
chromatography, the protein was tested to exhibit an anti-viral property. Aside from
elucidating the correct sequence of amino acids from his protein isolate, Mr. Gonzaga
also would like to determine the concentration of the protein. The protein suspension
gave a total volume of 10 mL.

To determine the concentration of the protein, Mr. Gonzaga prepared a calibration

curve, in which the concentration of the protein stock solution is 200 µg/mL.
 Isolation and Hydrolysis of Casein from Non-fat Milk
Color Reactions of Intact Casein and Hydrolyzate
Protein Assay by the Bradford Method

Blank/ Standard
solution
1 2 3 4 5 6
mL Stock 0 2 4 6 8 9
solution
mL H2O 10 8 6 4 2 1
mL dye 5 5 5 5 5 5
Concentration
standard
(µg/mL)
Absorbance 0.0 0.1246 0.2259 0.3321 0.4611 0.4913

Calculate the concentration (in mg/mL) of the protein suspension if the absorbance
of the 10 mL protein suspension (plus 5 mL dye reagent) is 0.1563.