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Jon Ellis G. Datu, Athina Darla B. Deala, Alan Nathan D. Derige, Mary Kimberly L. Espaldon,
Ma. Theresa Angeli M. Estabillo, and Jemielle Patricia A. Estrada
Group 3 2F Medical Technology General Biochemistry Laboratory
ABSTRACT
In the experiment, the group had isolated casein and albumin from non-fat milk by isoelectric precipitation and heat
denaturation, respectively. Casein was precipitated by heating the mixture up to 40 C and adding 10% acetic acid
until the pH reached 4.6 or until a solid white curd-like substance formed. On the other hand, the albumin was
collected by subjecting the decantate to 75 C water bath for about 5 minutes. After precipitating casein and albumin,
several samples were used for the qualitative color reactions. Intact protein, acid hydrolysate and basic hydrolysate
were used in performing Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi
test, Nitroprusside test, Fohls test, test for amides and Pauly test. The intact protein, casein, yielded negative results
for Millons test and Sakaguchi test and the rest of the tests were positive. On the other hand, the intact protein,
albumin, obtained negative results after subjecting to Ninhydrin test, Xanthoproteic test, Millons test, and HopkinsCole test. Lastly, all test for amides turned the red litmus paper to blue.
INTRODUCTION
Biologically active proteins are polymers
consisting of amino acids linked by covalent
peptide bonds. Many different conformations
(three-dimensional structures) are possible for a
molecule as large as a protein. Of these many
structures, one or a few have biological activity;
these are called the native conformations. Many
proteins have no obvious regular repeating
structure. As a consequence, these proteins are
frequently described as having large segments of
random structure also referred as random
coil. The term random is really a misnomer,
because the same nonrepeating structure is
found in the native conformation of all molecules
of a given protein, and this conformation is
needed for its proper function. Because proteins
are complex, they are defined in terms of four
levels of structure namely primary, secondary,
tertiary and quaternary structures [1]. Primary
structure is the order in which the amino acids in
a protein are linked by peptide bonds [1].
Secondary structure is the arrangement in space
of the backbone atoms in a polypeptide chain and
it is dependent on hydrogen bonding [5]. The
arrangement in space of all the atoms in a
protein [1] and the overall three-dimensional
shape of an entire protein molecule is called the
tertiary structure [5]. Lastly, the quaternary
structure refers to the interaction of several
polypeptide chains in a multisubunit protein [1].
Protein isolation is a process for isolating a
single type of protein from a complex mixture.
The significance of isolating proteins is to
characterize their solubility, acid-base property,
function, structure, and interactions. Proteins can
be separated depending on their size, shape,
charge,
hydrophobicity
and
physiochemical
properties. Some of the methods that are
commonly used are isoelectric precipitation, heat
denaturation,
solubilization,
salt-induced
precipitation,
chromatography,
and
ultracentrifugation [8].
In isoelectric precipitation, the isoelectric point
must be achieved wherein the net charge of the
protein will be equal to zero. It is done by
precipitating a complex mixture until the protein
is precipitated at a certain pH level [7].
In heat denaturation, the secondary, tertiary
and quaternary structures of proteins are lost
and proteins are isolated based on their heat
sensitivity. Some proteins denature at certain
temperatures and heating will help isolate the
proteins that easily denature [6].
Specific reactions are used for the purpose of
identifying amino acids and proteins in biological
media, for qualitative and quantitative analysis.
Biuret test is used to determine peptide bonds.
Ninhydrin test is typical for -amino acids.
Xanthroproteic test is a test for the detection of
aromatic proteins in which concentrated nitric
acid reacts with the proteins to form a yellow
color that is intensified to orange-yellow by the
addition of alkali. Millons test is used to
demonstrate the presence of the amino acid
tyrosine. Hopkins-Cole test is specific for
tryptophan group. Sakaguchi test is a test for
guanidines, i.e arginine and peptides that contain
it. Nitroprusside test is a test for cystinuria. Fohls
test is used to know if sulfur-containing amino
acids are present. Test for amides is used to
detect R-groups of asparagines and glutamine
[2].
In the experiment performed, there were two
proteins that were isolated which are casein and
albumin. Casein was isolated through isoelectric
precipitation by acetic acid. Casein exists in milk
as calcium caseinate. On the other hand, albumin
was isolated through heat denaturation. Albumins
are globular proteins that are soluble in water
and in dilute salt solutions. They are, however,
denatured and coagulated by heat [2].
EXPERIMENTAL
A. Compounds tested (or Samples used)
Non-fat milk
Milk is a liquid substance produced by the
mammary glands of mammals. There are several
types of proteins in milk and the major milk
proteins are unique to milk, it is not present in
any other tissue. The primary group of milk
proteins are the caseins. All other proteins found
in milk are grouped together under the name of
whey proteins. The major whey proteins in cow
milk are beta-lactoglobulin and alpha-lactalbumin
[4].
B. Procedure
1. Isolation of Proteins
a. Casein from Non-fat Milk
Millons Test
Hopkins-Cole
Test
Sakaguchi Test
Nitroprusside
Test
Fohls Test
Test for Amide
Pauly Test
Intact Protein
(Casein)
Violet
Blue-violet
+ acid: Yellow
+ base:
Orange
Colorless
Violet ring
Intact Protein
(Albumin)
Violet
Colorless
+ acid:
Colorless
+ base:
Colorless
Colorless
Colorless
Colorless
Yellow
White-turbid
Yellow
Brown
sediment
Red-Blue
litmus paper
Red-orange
Brown
sediment
Red-Blue
litmus paper
Yellow-orange
1
7
2
3
4
8
9 10
10 9
3
2
8
1
REFERENCES
[1] Campbell, M.K., and Farrell, S.O. (2015).
Biochemistry. 8th ed. Lorong Chuan, Singapore:
Cengage Learning
[2] Crisostomo, A.C., Daya, M.L., de Guia, R.M.,
Farrow, F.L., Gabona, M.G., Liu, M.I.D., Pena,
G.T., Pena, L.L., Santiago, L.A., Santiago, M.R.,
Sarile, A.S., Torres, P.C., Vargas, A.G., and Ysrael,