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ABSTRACT
The group was assigned to isolate casein from skimmed milk using isoelectric precipitation. Ten percent acetic
acid was used to isolate casein at its isoelectric pH. The caseinate was then subjected to acid hydrolysis using 6M HCl.
After autoclaving of the acid hydrolyzed caseinate, it was used in performing different tests for qualitative color
reactions. The same tests were performed with the intact protein. After the qualitative color reactions, paper
chromatography was performed for the separation and identification of amino acid standards based on the polarities of
tryptophan, arginine, proline, cysteine, serine, aspartate, histidine, glycine, and alanine.
INTRODUCTION
Protein isolation is a series of processes
intended to isolate one or a few proteins from a
complex
mixture.
It
is
done
for
the
characterization of function, structure and
interactions of the protein interest and also, to
determine their solubility and acid-base property.
Proteins can be separated depending on their
size,
shape,
charge,
hydrophobicity
and
physiochemical properties. The most commonly
used methods are isoelectric precipitation, heat
denaturation,
solubilization,
salt-induced
precipitation,
chromatography
and
ultracentrifugation.
In isolating casein from skimmed milk,
isoelectric precipitation was the method used.
Milk is a mixture of many types of proteins, most
of them present in very small amounts. Milk
proteins are classified into three main groups of
proteins on the basis of their widely different
behaviours and forms of existence and one of
these proteins is casein. Caseins are made up of
many hundreds of individual amino acids. Each
may have a positive or a negative charge,
depending on the pH of the system. At some pH
value, all the positive charges and all the
negative charges on the casein will be in balance,
so that the net charge on the protein will be
zero.
That pH value is known as the isoelectric
point (IEP) of the protein and is generally the pH
at which the protein is least soluble. For casein,
the IEP is approximately 4.6 and it is the pH
value at which acid casein is precipitated. In
skimmed milk, which has a pH of about 6.6, the
casein micelles have a net negative charge and
are quite stable. During the addition of acid to
milk, the negative charges on the outer surface
of the micelle are neutralized (the phosphate
groups are protonated), and the neutral protein
precipitates.
EXPERIMENTAL
A. Compounds tested (or samples used)
Casein, acid hyrolysate, tryptophan,
arginine, proline, cysteine, serine, aspartate,
histidine, glycine, alanine
B. Procedure
1. Isolation of Casein from Skimmed Milk
Purple solution
Brown turbid
solution
Ninhydrin
No color
change
Blue-violet
coloration in
the solution
Xanthoproteic
Very light
orange color
Light brown
turbid solution
Millons
White turbid
solution
Light brown
turbid solution
Hopkins-Cole
Purple color of
interface
Light brown
turbid solution
Sakaguchi
Red solution
Brown turbid
solution
Nitroprusside
Yellow solution
Yellowish
brown turbid
solution
Fohls
Black brown
precipitate
Brown solution
with black ppt.
Amide test
Red-Blue
litmus paper
Red-Blue
litmus paper
Pauly
Red orange
color
Reddish brown
solution
REFERENCES
From books
Crisostomo A. C., Daya, M. L., et al (2010)
Laboratory Manual in General Biochemistry
Quezon City: C & E Publishing, Inc.
From the Internet
Protein Purification. Retrieved March 3, 2015,
from
http://en.wikipedia.org/wiki/Protein_purification
Hydrolysis of casein. Retrieved March 3, 2015,
from http://www.jbc.org/content/9/3/333.full.pdf
Isoelectric Precipitation of Proteins: Casein from
Milk. Retrieved March 3, 2015, from
http://amrita.vlab.co.in/?
sub=3&brch=63&sim=158&cnt=1