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Exercise 5.1 & 5.2

Techniques for Protein Analysis
Protein Isolation & Determination of Protein Concentration by
Spectrophotometry

Franchette Pearl M. Requino

Chem 161.1-1L
2nd Semester AY 2017-2018

Groupmate:
Justine Russel O. Molina

February 13 & 20, 2018

February 27, 2018

Ms. Rochelle P. Ibabao

I. Introduction
Proteins are large, complex molecules that consist of hundreds and thousands of
amino acids. They are essential to the human body for they do most of the work in
the cells thus, they have vital role for the structure, function, and regulation of the
body’s tissues and organs. Because of this, proteins are the most widely studied
biomolecules.

The composition and configuration of amino acids varies with each protein. Thus,
proteins have diverse properties such as its solubility, size, molecular weight, charge
and binding affinity. To determine the properties of a certain protein, analysis is done.
Analysis of proteins can be through isolation, purification, identification, and
determination of its molecular weight and concentration. In this exercise, protein
isolation and determination of protein concentration was done.

Protein isolation can be done through many ways, but the most common method
of protein isolation is through precipitation wherein the solubility of the proteins is
utilized. Two common methods of protein precipitation are salting out and isoelectric
precipitation.

Salting out of the protein involves saturating the protein with a salt such as
ammonium sulfate to decrease the solubility of the protein in solution and precipitate
it out of the solution while isoelectric precipitation is a process of protein isolation
which makes use of the pH of the protein solution. When the pH of the protein solution
is equal to its isoelectric point (pI), the charges of the protein are balanced, and the
repulsive forces are reduced while attractive forces predominate. The attractive forces
cause the proteins to aggregate and precipitate.

Another analysis of proteins is the determination of protein concentration. The

concentration of protein is used to know the specific activity of a purification process.
Determination of protein content can be done through different assays depending on
the amount of protein available for assay, protein concentration of the sample,
specificity of the assay, presence of other substances which may interfere with the
assay, convenience, and the reliability of the method.
Two of the most common assays are the Biuret and Bradford assay. Biuret assay
is a general test for peptide chains of at least three amino acids wherein the positive
result is based on the formation of a colored chelate complex between amino acid
residues and cupric ions in alkaline environment.
 Figure 1
Bradford assay, on the other hand, is a colorimetric protein assay that is based on
the absorbance shift of the dye Coomassie Brilliant Blue G-250. When the dye binds
with the protein, the maximum absorbance of the solution shifts from 465 to 595 nm
and this shift is accompanied by a color change.

Figure 2
II. Methodology
Protein isolation was first done through breaking up two medium sized eggs then
separating the egg whites from the yolk. The egg whites were then stirred to break
up the membrane of the [proteins]. Afterwards, it was filtered through a cheesecloth.
A total amount of 40.0 mL of the filtered undiluted egg white was then transferred to
a beaker. Every 1.0 mL of egg white that was transferred to the beaker, 0.1 mL of 1
M acetic acid was also added. The reaction produced a precipitate which was then
removed using a cheesecloth.
 From the 40.0 mL filtrate, 30.0 mL was obtained. Powdered ammonium sulfate
was then added portion by portion until a total of 7.26 g of ammonium sulfate was
added. {discussion explanation: to saturate to 40%]. After adding 7.26 g of
ammonium sulfate, the solution was filtered using a cheesecloth then ammonium
sulfate amounting to 3.90 g was again added portion by portion to the solution. The
solution was then allowed to stand with occasional stirring for 30 minutes in an ice
bath. After standing, it was filtered using a pre-weighed filter paper then air-dried for
a week. When the precipitate is already dry, the precipitate was weighed then stored
in a vial. The vial was then placed in a refrigerator to be used for the future exercises.
Another way of isolation is through isoelectric precipitation wherein casein was
used as the protein sample. In a volumetric flask, 0.1 g of casein, 25 mL of warm
distilled water and 5 mL of 1M NaOH was mixed. Then, 5 mL of 1 M Ch3COOH was
added to the mixture and it was mixed and cooled then filled up to 50 mL. From the
resulting mixture, 3 mL was obtained and distributed equally to three test tubes
containing 10 mL of three different buffers. The buffers are glycine-HCl buffer (pH
2.7), acetate buffer (pH 4.7) and, phosphate buffer (pH 6.7). The protein-buffer
mixture was then left to stand for 30 minutes then it was observed for cloudiness.
For the protein concentration determination, Biuret and Bradford assays were
done. For the Biuret assay, standard protein solutions of concentration 0, 0.5, 1, 2, 3
and 4 mg/mL were first prepared using 20 mg/mL BSA solution and 0.05 M NaOH.
Ovalbumin isolate amounting to 1200 mg was then dissolved in the 0.05 M NaOH and
the volume of the solution was brought up to 10 mL using the same NaOH as the
solvent. From this mixture, dilutions of 1:2, 1:3, 1:7 and 1:10 was made using 1.0 mL
of the solution and 0.05 M NaOH. From the diluted solutions, 0.50 mL was then taken
and added to 3.0 mL of the biuret reagent then it was mixed using a vortex mixer.
The solution was then allowed to stand for 10 minutes at room temperature. After
standing, the absorbance of each solution was read at 540 nm.
For the Bradford assay, standard protein solutions were again prepared using
standard BSA with a concentration of 1.0 mg/mL, distilled water and Bradford reagent.
The volumes of the reagent were as follows.
Table 1.
 After preparing the standard protein solutions, unknown protein solutions were
prepared using 250 mg of the albumin isolate. The albumin isolate was then dissolved
in 10.0 mL distilled water then transferred quantitatively in a 25 mL volumetric flask
and diluted to mark. The unknown protein solutions were then prepared using the
following volume of reagents:
Table 2.

The solutions prepared were then made to stand in the dark for 5 minutes at room
temperature. Then, the absorbance was read at 595 nm in a spectrophotometer using
test tube no.1 as the blank.

III. Results and Discussion

Protein isolation was done through salting out and isoelectric precipitation. Salting
out involves saturating the protein with a salt such as ammonium sulfate to decrease
the protein’s solubility in the solution. The solubility of the protein depends on the
ionic strength of the salt solution wherein a higher ionic strength decreases the
solubility of the protein in the solution. The ionic strength of the solution depends on
the concentration of the salt in the solution thus, a low concentration of the salt
increases the solubility of the protein while a high concentration of the salt decreases
the solubility of the protein. This is because when the concentration of salt in solution
is high, water tend to interact more with the ions from the salt thus, weakening the
protein-water interaction and precipitating out the protein.
In the exercise, ammonium sulfate (NH4)2SO4 is the salt used for based on the
Hofmeister series for effectiveness of anions in salting-out processes, ammonium
sulfate salt is a good salting out salt. The trend for of the Hofmeister series for
effectiveness of anions in salting-out processes
PO43- > SO42- > CH3COO- > Cl- > Br- > NO3- > ClO4- > SCN-
While the trend for cation effectiveness is
NH4+ > K+> Na+
Before the ammonium sulfate salt was added to the protein solution, 1M acetic
acid was first added to denature unwanted proteins while keeping the desired proteins
 intact. In the addition of ammonium sulfate (NH4)2SO4, it was observed that as the
saturation of the mixture increased, the cloudiness of the mixture also increased. This
observation was due to the fact that as the concentration of the ammonium sulfate in
the mixture increased, the protein competes to more of the salt wherein salt prevail
to stay in solution while the protein precipitated out.
Table 5.2. Data on the percentage yield of isolated albumin.

Parameters Value
Mass of isolated albumin, g 7.371
Volume of egg white, mL 40.00
Percentage yield, % 18.4275

After filtration and drying, the measured mass of the isolated albumin was 7.371 grams. The
volume of the egg white used was 40 mL thus the computed percentage yield was 18.4275%.
The isolated albumin was relatively low due to incomplete salting out or the amount of albumin
in the egg white was not really enough to have a 100% yield since the amount of protein in the
egg white is not the totality itself but rather just a fraction of itself and moreover, egg white
contains approximately 148 different proteins; therefore, we cannot expect a percentage yield of
100%.

Isoelctric precipitation,

In the next method of protein precipitation, the pH factor was used. The casein solutions prepared
were subjected to varying pH to determine in which pH, the protein precipitated out. In this case,
the pH at which cloudiness of the mixture was extensive was considered the isoelectric point
(IpH) of the solution. Proteins tend to be least soluble at their isoelectric point, the pH value at
which the sum of their positive and negative electrical charges is zero. At this pH, electrostatic
repulsion between protein molecules is minimal and they are more likely to coalesce and
precipitate out of solution (Garrette and Grisham, 2010).

Table 5.3. Observation on the isoelectric precipitation.

Reagents/ Action Observations

Casein Yellow-orange solids
Casein + dH2O +1M NaOH Dissolution of solids
After addition of 1M CH3COOH Further dissolution of other solids wherein
others were undissolved
After addition of dH2O No change in mixture
After standing for 30 minutes
Casein solution + glycine-HCl buffer (pH 2.7) Clear liquid with precipitate
Casein solution + acetate buffer (pH 4.7) Cloudy solution
Casein solution + phosphate buffer (pH 6.7) Clear liquid

As observed in the isoelectric precipitation of casein solution, the resulting mixtures of the casein
solution mixed with the varying pH buffers showed that there was no formation of cloudiness in
the mixture for casein solution with glycine-HCl buffer however there were precipitates observed
in the mixture which was mainly due to the incomplete dissolution of casein before addition of
the buffer or as observed in Figure 5.4, there is a possibility that precipitate may still form at pH
other than the IpH however not to the same extent as compared to the pH equal to the IpH. In
the casein solution mixed with the acetate buffer, there was an observed cloudiness in the
solution. In the casein solution mixed with the phosphate buffer, there was no observed
cloudiness. In determining the experimental value of the IpH, the cloudiness of the resulting
mixtures where considered. Based on the concept stated above that cloudiness occurs when the
pH of mixture is equal its IpH, it was therefore assumed that the experimental value of the IpH
is 4.7 since cloudiness was observed after the addition of the acetate buffer which has a pH of
4.7 to the casein solution. Theoretically, the IpH of the soltion should be 4.6-4.7 which means
that the experimental result was indeed in line with the theoretical result.

Figure 5.4. Relationship of solubility and the amount of precipitate produced with the pH of the
solution.