Protein Isolation and Characterization of Nestle Non fat milk

Febby Anne T. Abuzo, Celine O. Adao, Victor A. Amores,

Rudnie F. Bajalla, and Kathleen Joyce O. Boado
Group 1 2C Biochemistry Biochemistry Laboratory

ABSTRACT

INTRODUCTION
Proteins are found throughout the body. It is the polymer of amino acids. Many
amino acids attached to one another to form proteins. The function and three-dimensional
structure of a protein was determined by the sequence of amino acids. [1] This three
dimensional structure was called native conformation. If the native conformation was
disrupted there will be an alteration to the protein function.

In isolating proteins, isoelectric precipitation, salting in or salting out, and by having

selective dissolution techniques can be used. The isolated proteins can be characterized
qualitatively and quantitatively. For qualitative characterization, colorimetric reactions and
paper chromatography was used. While for quantitative characterization spectrophotometric
analysis was used.

Proteins can be described according to their large range of functions in the body. [2]
It serves a crucial function in all biological processes. It functions as a support, catalyst,
storage, receptor, they transport substance and regulate bodily activities. [3] Plants can
utilize their own amino acids while animals and humans cannot. Humans can get the
proteins by eating foods which have high protein content such as milk, egg and meat. [4]

Milk supplies the calcium for building bones. It contains water, vitamins, minerals,
proteins, sugars and lipids. There are three globular proteins found in milk; casein,
lactalbumins and lactoglobulins. [5][6] Caseins have high phosphate content which allows it
to associate with calcium. In milk, caseins it is known as the calcium caseinate. Caseins
have an isoelectric pH of 4.6; when this pH was reached, the casein become insoluble. In
cows milk, approximately 82% of milk protein was casein. Whereas, serum protein
consisting lactalbumin and lactoglobulin. This protein do not contain phosphorus. They are
soluble in water and in dilute salt solutions. However, when it was heated it coagulates;
forming white solid crystalline. [7]

In this experiment, the researchers must demonstrate commonly used isolation

techniques and explain the principles involved in each isolation technique; perform
qualitative test on the amino acids in intact and hydrolyzed proteins and explain the
principles involved in each test; execute acid, alkaline and enzymatic hydrolysis on the
isolated proteins and enumerate the advantages and disadvantages of using each type of
hydrolysis; determine the amino acid components of protein using thin layer
chromatography; and lastly, to quantitatively determine the protein concentration of given
sample using commonly used and microscale methods of total protein analysis.

EXPERIMENTAL
A. Compound tested
In this experiment, Nestle Non fat milk was used to isolate and characterize
protein.
B. Procedure

RESULTS AND DISCUSSION

Protein isolation is important in characterizing the function and structure of proteins.
In this experiment, non fat milk was subjected to different isolating techniques to isolate
casein and albumin protein.

Figure 1. The isolated casein and albumin

In isolating the casein protein, the researchers adjust the pH of the milk to 4.6 by
adding 10% acetic acid. When the pH of the milk has reached 4.6 the casein protein was
precipitated out. This technique was called isoelectric precipitation. When the pH of a
protein mixture was adjusted to its isoelectric pH its solubility was minimize thus
precipitating the protein. In figure 1, the white amorphous solid was the isolated casein. The
filtrate from the protein mixture was then subjected to heat precipitating the albumin
present in the mixture. The albumin present in the milk was soluble in water and dilute salt
solution. It will only precipitate out when it was denatured and coagulated by heat. The
isolated albumin in the non fat milk was a white crystalline solid.

The researchers were assigned to do acidic mode of hydrolysis. The appearance of

the hydrolyzed protein as shown in figure 2 was a dark brown solution.

Figure 2. The hydrolyzed intact protein

Acid hydrolysis was performed to obtain the information about the composition of the
milk. In acid hydrolysis, the water acts as a base for Brownsted Lowry acids. The water
accepts a proton from the acid after breaking the bond between the proton and acid. [8]

For qualitative characterization of proteins colorimetric reaction was used (table 1).
This technique uses different test to check the reaction of the side chains, amino acid
and carboxyl groups to different reagents. There are ten different test that was used to
characterized the proteins qualitatively. The first one is the Biuret test; this test was used to
detect the presence of peptide bonds. The positive result for this test is violet coloration of
the solution. In the experiment the intact protein solution become violet indicating that
there is a presence of peptide bond. For the hydrolyzed protein the solution turns to blue.
In this test the copper (II) ion forms a complex in an alkaline solution making it violet color.
[9]

In testing the presence of amino acid the ninhydrin test was used. The positive
result for this test was a blue violet solution. In the experiment the intact protein solution
turns into a blue violet solution, while the hydrolyzed protein turns into a dark violet
solution. The 0.1% ninhydrin solution acts as an oxidizing agent which leads to oxidative
deamination of amino groups. It also reacts with free amines from the amino acid
producing blue or purple color. [9] For the determination of aromatic groups of amino acids
the xanthoproteic test was used. The positive result for this test was an intensely yellow
color. In this test, the researchers have observed a pale-yellow solution for both intact and
hydrolyzed protein.

To test the presence of tyrosine millons test was used. The positive result for this is a pink
to dark red color. The researchers have observed a white solution with red precipitate for
both intact and hydrolyzed protein. This red coloration of the precipitate is probably due to a
mercury salt of tyrosine. In testing for the presence of tryptophan; Hopkins cole test was
used. The positive result for this test was a violet ring due to the addition of sulfuric acid.
The researchers have observed a violet ring for both intact and hydrolyzed protein. [9]
 In sakaguchi test the presence of arginine or guanidine group was determined. The
positive result for this test was a red color solution. The researchers have obtained a red
color solution for both the intact and hydrolyzed protein. The red color of the solution was
due to the reaction of guanidine group of the amino acid and the naphthol and alkaline
hypoboromite. [10] To find out the presence of sulfur containing group; the nitroprusside
test was used. The positive result for this test was a red solution. The red solution was due
to the reaction of the sodium hydroxide solution and nitroprusside solution with the thiol
group of the amino acid. Fohls test was also used to test for the presence of sulfur
containing groups. the positive result for this test was a brown solution with black
precipitate. The researcher has observed a brown solution with black sediments on the
intact protein while a pale yellow solution was observed on the hydrolyzed protein. The
brown solution with black precipitate was due to the degradation and substitution reaction
to form lead (II) sulfide. [11]

To test for the presence of asparagine and glutamine; the test for amide was used.
The change of color from red to blue litmus paper indicates that there is a presence of
asparagine and glutamine. For both intact and hydrolyzed protein the litmus turns for red to
blue. The last test for colorimetric reaction was the Paulys test. This test was used to
determine the presence of histidine and tyrosine. When the diazonium component react with
the imidazole group of histidine and phenol group of tyrosine it forms a dark red compound,
indicating the presence of both. In the experiment, the researchers have observed a white
solution on the intact protein while red orange solution on the hydrolyzed protein.

Figure 3. Colorimetric reaction set - up of intact and hydrolyzed protein