BIOCHEM 1 L Experiment # 02 2A BC Group 2

ISOLATION AND CHARACTERIZATION OF CASEIN FROM NON-FAT

POWDERED MILK
Aquino, M. G. I., Badua, A. A., Belardo, A. J. S., Cabasag, A. S. D.
Cantal, I. N.,
Department of Biochemistry, Faculty of Pharmacy
University of Santo Tomas

ABSTRACT
Milk contains most of the biological molecules necessary to sustain life
including water, vitamins, minerals, proteins, sugars, and lipids. The
objectives of this experiment were to demonstrate protein isolation
techniques and explain the principles involved; to perform qualitative
tests in both intact and hydrolyzed proteins and explain the principles; to
execute acid, basic, and enzymatic hydrolysis on isolated protein and to
compare each by enumerating the advantages and disadvantages; to
determine the amino acid components by using paper chromatography;
and to quantitatively determine the protein concentration of unknown
using total protein analysis. In this experiment, the sample used is a non-
fat powdered milk (Milk Magic). Casein, which is a particular protein of
milk was isolated using isoelectric precipitation which is done by adding
10% acetic acid. The isolated protein or intact protein was subjected into
hydrolysis which was divided into three parts: acid hydrolysis, which was
done by adding 6M HCl; basic hydrolysis which used 4M NaOH; and
enzymatic hydrolysis in which meat tenderizer was added and 0.1M
phosphate buffer in order maintain pH at 7.50. Qualitative test on amino
acids were done to both intact protein and hydrolysates. Biuret test,
Ninhydrin test, Xanthoproetic test, Millons test, Hopkins-Cole test,
Sakaguchi test, Nitroprusside test, Fohls test, test for amides, and Paulys
test were performed for the color reactions. The intact casein yielded a
positive result in Biuret test, Xanthoproetic test, Fohls test, test for amide,
and Paulys test. The acid hydrolysate yielded a positive result in Fohls
test, test for amide, and Paulys test. The basic hydrolysate yielded
positive result in Biuret test, Ninhydrin test, Xanthoproetic test, test for
amide, and Paulys test. The enzymatic hydrolysate yielded a positive
result in Biuret test, Xanthoproetic test, Millons test, test for amide, and
Paulys test. To determine the amino acid components of the protein,
paper chromatography was performed. The result showed that acid
hydrolysate contains tyrosine and histidine components while tryptophan
and cysteine were present in basic hydrolysate. In enzymatic hydrolysate,
only cysteine was identified. The unknown protein concentration was
determined by using Biuret total protein assay in which the concentration
was graphed against absorbance and linear regression analysis was used.
*insert result*
INTRODUCTION mixed. After mixing, 10% acetic acid
was added dropwise until the mixture
METHODOLOGY
became curdy. The solution was
I. Isolation of Protein
filtered using cheesecloth and the
In a beaker, 20 g of non-fat milk
sample and 50 mL of water were
residue was used as the crude protein mL of distilled water. In 50-mL protein
or isolated protein for qualitative test. mixture, 0.050 g of meat tenderizer
was added directly. To maintain the pH
II. Hydrolysis of Intact Proteins of the mixture, 10 mL of 0.1M
Acid Hydrolysis of Intact Protein phosphate buffer, pH 7.5 was added.
In a hard glass test tube, 5 mL of The mixture was left overnight at a
6M HCl was added to 0.5 g of isolated room temperature to implement
protein. Cotton was used to cover the digestion of protein. The enzymatic
tube prior to autoclaving. After hydrolysate was also used in
autoclaving, 10 mL of distilled water performing qualitative analysis and
was added. The mixture was identification of amino acids.
transferred in a 250-mL beaker for
neutralization with 3M NaOH. The III. Qualitative Color Reactions
acidic hydrolysate was used in both A set of ten test tubes were
qualitative analysis and identification prepared for each sample: (a total of
of amino acids. 40 test tubes)
Intact protein solution (0.5 g
Alkaline Hydrolysis of Intact intact protein in 1 mL distilled
Protein water)
In another hard glass test tube, 10 Acid hydrolysate
mL of 4M NaOH was added to 0.5 g of Basic hydrolysate
isolated protein. The tube was covered
Enzymatic hydrolysate
with cotton and underwent
The following qualitative color
autoclaving. An amount of 10 mL of
reaction tests were performed for
distilled water was added and the
each sample:
mixture was transferred in a 350-mL
beaker. Neutralization was performed
Biuret Test
by adding 3M HCl. The basic
In the sample, 20 drops of 2.5M
hydrolysate was used both in
NaOH was added. After mixing, 2-3
qualitative analysis and identification
drops of 0.1M CuSO4 solution was
of amino acids.
added. The color of solution was
recorded.
Enzymatic Hydrolysis of Intact
Ninhydrin Test
Protein
In the diluted sample, 6-10 drops
Protein mixture was prepared by
of 0.1% ninhydrin solution was added.
mixing 1 g of isolated protein and 100
The tube was heated in a boiling water
bath. The color of solutions was was added. The tube was placed in a
recorded. boiling water bath. The positive result
Xanthoproetic Test for this test is appearance of dark
In the diluted sample, 10 drops of brown or black sediments.
conc. HNO3 was slowly added and the Test for Amides
color was recorded. After recording In 10 drops of the sample, 1 mL of
the color, 10 drops of conc. NaOH was 20% NaOH was added. The tube was
slowly added and the resulting color heated in a boiling water bath.
was also recorded. Moistened red litmus paper was
Millons Test placed over the mouth of the tube to
In the diluted sample, 5 drops of test the evolution of gas. The result
Millons reagent was added and the was recorded.
color was recorded. Paulys Test
Hopkins-Cole Test Diazo reagent was prepared by
In the sample, 20 drops of mixing 3-5 drops of 1% sulfanillic acid
Hopkins-Cole reagent was added and with 3 drops of 5% NaNO2 solution. In
mixed. The test tube was inclined and the diazo reagent, 5 drops of sample
20 drops of conc. H2SO4 was slowly and 10% Na2CO3 was added. The
added along the side. The color of the positive result for this test is
interface was recorded. appearance of red coloration.
Sakaguchi Test
In the sample, 10 drops of 10% IV. Separation and Identification
NaOH and 10 drops of 0.02% naphthol of Amino Acids by Paper
solution were added. The solution was Chromatography
mixed and 3 drops of 2% NaOBr was Filter paper was used as the
added. The solution was mixed again support of 1-butanol: acetic acid:
and the resulting color was recorded. water (4:1:5) mobile phase. A line was
Nitroprusside Test drawn across the filter paper with a
In 0.5 mL of sample, 0.5 mL of 3M 1.5-cm margin from the bottom of the
NaOH and 0.25 mL of 2% longer edge. The paper was marked
nitroprusside solution was added. If with 12 equidistant points on the line
the color turns red, the color was for the spotting of amino acid
recorded. standards: 2% tryptophan, arginine,
Fohls Test proline, cysteine, serine, aspartic acid,
In the sample, 5 drops of 30% tyrosine, histidine, and glycine, and
NaOH and 2 drops of 5% lead acetate the 3 hydrolysate samples: acid, basic,
and enzymatic. The standards were Two unknowns were used in total
applied by spotting the paper 5 times protein assay. The first unknown was
using capillary tube and letting it dry undiluted hydrolysates while the
first before application of samples 10 second was diluted hydrolysates,
times. The paper was placed inside a 1:100 of hydrolysate and water.
pre-equilibrated chamber with a
solvent that is below the origin. The Biuret Total Protein Assay
chamber was covered and the solvent After the preparation of sample
was allowed to ascend undisturbed. and protein standards, 2.50 mL of
When the solvent is 0.5 cm from the unknown 2 was transferred in another
top edge of the paper, it was removed test tube. Into each test tube, 2.5 mL
and the solvent front was immediately of Biuret reagent was added. The test
marked by using pencil. The tubes were heated at 40 C-50 C
chromatogram was air-dried and water bath for 5 minutes. The
sprayed with 1% ninhydrin reagent solutions were cooled and transferred
before it was placed in a hot plate. in a microwell plate for
The amino acid spots were encircled spectrophotometric analysis. The
by using pencil and the Rf values were wavelength of the spectrophotometer
computed. was set at 540 nm and the reading
was set to zero using the blank. The
V. Quantitative Protein Analysis absorbance of standards and
Preparation of Sample and unknowns were recorded and
Protein Standards graphed. The unknown protein
Standard protein concentrations solution was determined by using
were prepared by mixing the following graphical and linear analysis.
solutions:
Table 1. Amount of BSA standard RESULTS AND DISCUSSION
solution and distilled water for each
test tube CONCLUSION
Test Tube BSA Distilled In this experiment, casein was
(mL) Standard Water precipitated using the isoelectric
(mL) precipitation technique in which the
Blank 0 2.50 pH of the non-fat milk was adjusted to
Standard 1 0.10 2.40
Standard 2 0.50 2.00 be sufficiently acidic that the protein is
Standard 3 1.00 1.50 insoluble. Lowering the pH leads to
Standard 4 1.50 1.0
Standard 5 2.50 0 dissolution of calcium phosphate until,
at the isoelectric point of casein (pH Acid Hydrolysis
4.6), all phosphate is dissolved and Advantages:
the caseins precipitate. Complete hydrolysis of peptide
The isolated casein is insoluble in bonds
water but dissolves in alkaline and No racemization
some acidic solutions. Proteins are Disadvantages:
least soluble in water at their Destruction of tryptophan to
isoelectric points and are more soluble humin
at higher or lower pH's.
Partial destruction of cysteine
and tyrosine
The qualitative color reaction tests
Basic Hydrolysis
of both intact protein and hydrolysates
Advantages:
were characterized based on the
Complete hydrolysis of peptide
principle of chemical reaction of
bonds
reactive groups present in the amino
Disadvantages:
acids. The following are the
Arginine, asparagine,
specificities of each test:
glutamine, and serine are
Biuret test peptide bond
destroyed
Ninhydrin test alpha amino
Enzymatic Hydrolysis
group
Advantages:
Xanthoproetic test aromatic
Specific amino acids can be
amino acids
determined
Millons test tyrosine
Requires less energy and
Hopkins-Cole test tryptophan produces less by-product
Sakaguchi test arginine Disadvantages:
Nitroprussided test sulfur- Low reaction rates
containing amino acids High yield of sugar monomers
Fohls test cysteine In paper chromatography, the
Test for amide amide principle involved is polarity in order
Paulys Test histidine and to separate the components by means
tyrosine of capillary action. The presence of
The advantages and amino acids in each hydrolysate were
disadvantages of each type of determined by using this technique.
hydrolysis were realized in the The results showed that tyrosine and
experiment: histidine were present in acid
hydrolysate while tryptophan and
cysteine were identified in basic
hydrolysate sample. In enzymatic
hydrolysate, the result only showed
the presence of cysteine.
For the total protein assay, the
concentration of unknown was
determined by using linear regression
and graphical analysis. *insert result*
REFERENCES
Ex 112 Casein, Lactose, Albumin - Penn State University. (n.d.). Retrieved
March 13, 2017, from http://www.bing.com/cr?
IG=51D2B6A7037F4495A5B80E564DEE6C2A&CID=36B6BE738D9260C8
3C35B4368CA361BB&rd=1&h=09R5pg4gYCd4QOoBsKGqVtt-
ZoaaDbQbEz-b-1WWER4&v=1&r=http%3a%2f%2fcourses.chem.psu.edu
%2fchem36%2fNew%2520Syn%252036%2520pdf
%2fExp112.pdf&p=DevEx,5051.1