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Exercise 4
Isolation and Quantification of Proteins

GEROLAGA, WINSTON JAKE C.

Chem 145.1 – 1
1 Semester, AY 2018 – 2019
st

Groupmate/s:

GRABATO, JEB REECE

ANTHONY SALVADOR ALBALADEJO

Laboratory Instructor
 I
INTRODUCTION

Proteins are high molecular weight polymers of 40 or more amino acids joined together by
amide linkages (Smith, 2011). Various several proteins exist in a single cell. A detailed study of
the properties of a particular protein requires a homogenous sample which only consists of only
one kind of molecule (protein). The separation and isolation, or purification, of proteins constitutes
an essential first step to further experimentation (Campbell & Farrell, 2015).

Egg white, or in technical terms albumen, comprises 60% of the whole egg by weight. It is
composed of 88% water and 10.4% proteins, and the minority of the percentage goes to
carbohydrates, minerals, and lipids (Burley & Vadehra, 1989). Egg whites usually contain about
11% of proteins, which consists of more than 40 different kinds of proteins. Among these proteins,
the major protein is ovalbumin, which constitutes about 54% of the total egg white proteins
(Yamamoto et al. 1997).

This experiment aims to isolate crude albumin from egg whites by salting out method
which uses ammonium sulfate. This experiment also aims to determine and quantify the protein
concentration in the isolated crude albumin by the Biuret method and UV-visible
spectrophotometry.

II
METHODOLOGY

A. Isolation of crude albumin from egg white by ammonium sulfate precipitation (salting out)

Two medium-sized eggs were purchased from a local sari-sari store in Miagao,
Iloilo. The egg whites were carefully separated from the yolks, and the egg whites were
beaten in a beaker. The beaten egg whites were then filtered through a cheesecloth. Then,
30.0 mL of the filtered egg whites were measured using a graduated cylinder and was
transferred into a beaker. The filtrate was gradually added with 7.26 grams of powdered
ammonium sulfate with constant stirring for the solution to be 40% saturated. The solution
was then filtered through a cheesecloth and the filtrate was transferred to a beaker.
Consequently, 3.90 grams of ammonium sulfate was again added to the filtrate gradually
with constant stirring to bring the solution to 60%. Then, the solution was allowed to stand
in an ice bath for around 30 minutes with occasional stirring. While this happened, a filter
paper was pre-weighed in an analytical balance. After 30 minutes, the solution was filtered
using the pre-weighed filter paper. The filtration setup was left inside a cool, dry place to
air dry until the next meeting of the laboratory class. After air drying, the filter paper with
the residue (ovalbumin) was weighed in an analytical balance. The amount of crude
ovalbumin isolated was calculated.

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 B. Determination of protein concentration by spectrophotometry

Preparation of Biuret reagent

In 31.25 mL of distilled water, 0.09375 grams of CuSO4•5H2O and 0.375 grams of

sodium potassium tartrate were dissolved. Then, 10% NaOH with a volume of 18.75 mL
was added to the previous solution. Then the resulting solution was diluted with enough
distilled water to make a 250-mL solution.

Preparation of standard curve

Due to the lack of bovine serum albumin (BSA) solution in the laboratory, albumin
standard was used as the standard instead. Six test tubes were prepared and solutions of 0-
10 mg albumin all dissolved in 1.0 mL of 0.05 M NaOH. All tubes were then added with 8
mL Biuret reagent. The tubes were allowed to stand for 10 minutes for full color
development. Then, each tube, from the lowest concentration to the highest, were read for
its absorbance at 540 nm using a UV-visible spectrophotometer. A standard curve (albumin
concentration vs. absorbance) was generated by plotting the data into Microsoft® Excel.

Determination of protein content in sample

A 1200-mg portion of the albumin isolate was dissolved in 10.0 mL 0.05 M NaOH
in a beaker. Five test tubes were obtained and labeled “undiluted”, “1 in 2”, “1 in 3”, “1 in
7”, and “1 in 10”. These correspond to a certain concentration such as 1 mL albumin
solution + 1 mL 0.05 M NaOH (1 in 2), and so on.

To each of the tubes, 2 mL of Biuret reagent was added. The tubes were allowed to
stand for 10 minutes to allow full color development. Then the absorbances of the sample
solutions were read at 540 nm using a UV-visible spectrophotometer. The amount of
protein in g per 100 g of the original isolate was calculated.

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 III
RESULTS

The following are the results of the experiment.

1. Percent crude albumin yield

2. Preparation of standard curve

0.12

0.1

0.08
Absorbance

0.06

0.04

0.02 y = 0.0141x + 5E-05

R² = 0.9695

0
0 2 4 6 8 10
Albumin concentration, mg/mL

Figure 1. Plot of albumin standard concentration (in mg/mL) versus

absorbance. The equation of the line and the R2 value is also shown.

3. Albumin isolate results

Table 1. Absorbance readings (at 540 nm) and protein concentrations of the albumin
isolate in different dilutions.
Protein concentration
Dilution A540
(mg/mL)*
undiluted 0.100 7.09
1 in 2 0.100 7.09
1 in 3 0.100 7.09
1 in 7 0.098 6.95
1 in 10 0.099 7.02
* Protein concentrations are not actual. For actual concentrations (with the application of the dilution factors),
please refer to Table 2.
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 4. Protein concentration calculations from Table 1

Equation of the line:

Plugging the values for absorbance (A540) in y and solving for x:

• undiluted, 1 in 2, and 1 in 3 (since the three have the same values of absorbance):

• 1 in 7:

• 1 in 10:

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 5. Actual protein concentrations from the albumin isolate

Table 2. Protein concentrations with the application of the dilution factor.

Actual protein
g protein per
Dilution Dilution factor* concentration
100 g isolate
(mg/mL)
undiluted 5 35.44 29.35
1 in 2 10 70.87 58.69
1 in 3 15 106.33 88.04
1 in 7 35 243.14 201.32
1 in 10 50 350.89 290.54
* Dilution factor calculations are found after this table.

6. Calculations for the actual protein concentrations

• undiluted:

1 𝑚𝐿

• 1 in 2:

1 𝑚𝐿 1 𝑚𝐿

• 1 in 3:

1 𝑚𝐿 1 𝑚𝐿

• 1 in 7:

1 𝑚𝐿 1 𝑚𝐿

• 1 in 10:

1 𝑚𝐿 1 𝑚𝐿

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 7. % w/w protein calculations

• undiluted:

• 1 in 2:

• 1 in 3:

• 1 in 7:

• 1 in 10:

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 IV
DISCUSSION

To isolate the albumin from the egg whites, the salting out method was used. This involves
the use of ammonium sulfate, which is a salt. Proteins have varying solubilities in polar and ionic
compounds and these proteins remain soluble because of their interactions with water. The
ammonium sulfate was added to the egg whites for it to be saturated. The addition of ammonium
sulfate takes water away from the protein, which in turn makes ion-dipole bonds with the salt
molecules. With less water available to hydrate the protein, they begin to interact each other by
hydrophobic interactions (Campbell & Farrell, 2015). This means that the more ammonium sulfate
is added, the more protein is precipitated out of the solution. The use of an ice bath is similar to
recrystallization in organic synthesis.

The percent yield of the albumin in egg whites was found out to be 25.08%. The
calculations in this part can be found at Part 1 in the Results (III) section of this paper. This
indicates a yield that is not too low nor too high.

Figure 1 depicts the plot of the albumin concentration (in mg/mL) versus the absorbance
reading. A R2 value of 0.9695 was obtained. This indicates that the obtained graph is slightly linear
only. The standard curve can be used to estimate the amount of protein (albumin) in the sample.

The sample solutions were prepared and added with Biuret reagent. The Biuret reagent is
made up of sodium hydroxide (NaOH), hydrated copper(II) sulfate, and potassium sodium tartrate,
which is added to chelate and stabilize the cupric ions in the solution. When proteins are present
in a solution containing the Biuret reagent, the solution will turn into a characteristic purple color,
which is caused by the reaction of the cupric ions with the nitrogen atoms involved in the peptide
bonds in proteins. Since in the sample preparation, the solution was made basic, the peptide
hydrogen atoms will be displaced. This will lead to chelation which produces the said characteristic
color that can be measured by spectrophotometry – since the intensity of the color developed is
proportional to the concentration of proteins in the sample. This solution is most sensitive at 540
nm (Gornall et al. 1949).

Table 1 summarizes the results of the absorbance readings for the sample. At 540 nm, it is
very noticeable that were only slight differences in the absorbances, even though there is dilution.
This might be caused by certain errors during the experiment. The spectrophotometer might have
displayed the wrong readings, since there were instances that the instrument displayed that the
value of the absorbance was outside the calibration range. This might also imply that there is a
need for the samples to be further diluted in order for the absorbances to be inside the working
range.

As seen in Table 2, the actual protein concentrations of the samples do not coincide with
the dilutions. In principle, dilution affects concentration. The more diluted a solution is, the lesser
will be its concentration. By this, it is safe to say that the results were very erroneous since more
dilution made the concentration of protein higher.

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 V
CONCLUSION

The albumin in two egg whites were isolated using salting out with ammonium sulfate.
The precipitate formed was a white, spongy, powdery solid. This gave a yield of 25.08%. The
protein concentration of the sample was determined by the Biuret protein assay method using a
UV-visible spectrophotometer. It was found out that the results were erroneous. It is recommended
that the sample solutions be diluted further in order to obtain accurate, precise, and correct results
the next time this experiment is performed.

VI
REFERENCES

Burley, R. W. & Vadehra, D. V. (1989). The albumen: Chemistry. The Avian Egg – Chemistry and
Biology. John Wiley & Sons: New York.

Campbell, M. K. & Farrell, S. O. (2015). Biochemistry (8th ed.). Cengage Learning Asia Pte Ltd:
151 Lorong Chuan, #02-08 New Tech Park, Singapore 556741.

Gornall, A. G., Bradwill, C. S., & David, M. M. (1949). Determination of serum proteins by means
of the biuret method. Journal of Biological Chemistry, 177, 751-756.

Smith, J. G. (2011). Organic Chemistry (3rd ed.). The McGraw-Hill Companies, Inc.: 1221
Avenue of the Americas, New York, NY 10020.

Yamamoto, T. Y., Juneja, L. R., Hatta, H., Kim, M. (1997). Hen eggs: their basic and applied
science. CRC Press LLC: 2000 N. W. Corporate Blvd., Boca Raton, Florida 33431.

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