FACULTY OF APPLIED SCIENCES KUALA LUMPUR CAMPUS

Semester:​ [ / ] May [ ] September [ ] January (please “✓”)

Academic Year: ​2020
Course Code & Title: ​BABS2244 METABOLIC BIOCHEMISTRY
Programme: ​RBS 2 G1
Student’s Name (Registration Number): ​Wong Pui Mun (20WLR08576)
Submission Date: ​8/7/2020

Declaration

I confirm that I have read and shall comply with all the terms and conditions of TAR
College’s plagiarism policy.
I declare that this assignment is free from all forms of plagiarism and for all intents and
purposes is my own properly derived work.

Signature(s): ​
Name(s): Wong Pui Mun
Date: 8/7/2020
Experiment 2 ​: Determination of protein concentration using biuret assay

Objectives:
1. To learn the use of the spectrophotometer
2. To determine the concentration in a sample.
3. To analyze data from standard curves.

Introduction:
Protein assays are one of the most widely used methods in life science research. This is
because estimation of protein concentration is necessary in protein purification,
electrophoresis, cell biology, molecular biology and other research applications. Each method
of the assay has its own advantages and limitations. There few types of assay include dye
binding assays (bradford), copper ion based assays (lowry and BCA) and test strip based
protein assay. In this experiment, one of the simplest and common methods is the biuret
protein assay which is also called copper ion based assays. This method is so called biuret
because when urea is heated, a small compound of biuret is formed which causes two urea
molecules to combine. Therefore, urea molecules fuse and produce amide groups (-NH) at
the center of the molecule which then bind to copper ions at basic pH. The copper complexes
that result from this interaction produce a strong blue color that can be measured with a
spectrophotometer (Handbook & Selection Guide. 2008).

Figure 1: biuret reaction

Actually, protein also contains amide groups. This is said so as when an amino group and a
carboxyl group join to form peptide bonds, the amino group becomes an amide group.
Therefore under alkaline condition, cupric ions (Cu​2+​) chelate with the peptide bonds
resulting in reduction of cupric (Cu​2+​) to cuprous ions (Cu​+​). If the alkaline copper is in
excess over the amount of peptide bonds, some of the Cu​2+ will remain unbound to the
peptide bond and are available for detection. Thus, this assay will combine protein samples
with Biuret Reagent which contains copper ions in a basic solution. The copper ions will
complex with the amide groups in the proteins to create a blue color that will be measured
using a spectrophotometer. The amount of colour is directly proportional to the amount of
peptide bonds, size as well as the amount of protein or peptide present (​Owusu-Apenten, R.K.
2002)​.
Procedure:
2 sets of test tubes with the number of 1 to 8 were labelled. The amount of water indicated in
the table 1 below was pipetted into each test tube. Then, the appropriate amount of BSA
stock solution ( 10 mg/ml ) that had been stated in table 1 of each test tube was pipetted.
Later, 1 ml of the unknown solution was pipetted into the clean, duplicate test tubes tube of 9
and 10. Also test tubes 11 and 12 were prepared as shown in table 2 and labelled. Next, 4ml
of biuret reagent was added into each of the 12 test tubes respectively. The tubes were
covered with parafilm and were briefly vortexed to ensure that the sample and the biuret were
thoroughly mixed. All of the tubes were allowed to stand at room temperature for 20 minutes.
While waiting, the spectrophotometer was switched on and allowed to warm up. The
wavelength was adjusted to 550 nm. After 15 minutes, tube 1 was placed into the
spectrophotometer and the absorbance was set to zero. This tube served as the blank. Then,
the absorbance of the other standards was measured and recorded in the table 2. Finally, the
absorbance of the unknown was recorded. The average absorbance (ABS) for all tubes were
determined.

Results:

Table 1:
Tube BSA H​2​O BSA Biuret Absorbance Absorbance Average
Concentration (ml) (ml) reagent reading 1 reading 2 absorbance
(mg/ml) (ml) reading (ABS)

1 0 1.0 0 4 0.000 0.000 0.000

2 1 0.9 0.1 4 0.001 0.001 0.001

3 2 0.8 0.2 4 0.004 0.004 0.004

4 3 0.7 0.3 4 0.013 0.013 0.013

5 4 0.6 0.4 4 0.021 0.021 0.021

6 5 0.5 0.5 4 0.034 0.034 0.034

7 7 0.3 0.7 4 0.046 0.046 0.046

8 9 0.1 0.9 4 0.067 0.067 0.067

Table 2:
Tube Unknow H​2​O Biuret Average Concentration Average
n (ml) reagent (ml) absorbance (mg/ml) concentration of
 (ABS) unknown (mg/mL)
6.67 + 6.33 + 3.55 + 3.78
9 1 0 4 0.041 6.67 4
= 5.082
10 1 0 4 0.038 6.33

11 0.5 0.5 4 0.013 3.55

12 0.5 0.5 4 0.015 3.78

Figure 2: Graph absorbance against protein concentration

Using the regression equation above: y= 0.009x - 0.019

Y is the absorbance where x is the protein concentration.

Calculation of protein concentration of unknown in tube 9:

absorbance= 0.041
Protein concentration = (0.041 + 0.019) / 0.009
= 6.67 mg/ml

Calculation of protein concentration of unknown in tube 10:

absorbance = 0.038
Protein concentration = (0.038 + 0.019) / 0.009
= 6.33 mg/ml
Calculation of protein concentration of unknown in tube 11:
absorbance = 0.013
Protein concentration = (0.013 + 0.019) / 0.009
= 3.55 mg/ml

Calculation of protein concentration of unknown in tube 9:

absorbance = 0.015
Protein concentration = (0.015 + 0.019) / 0.009
= 3.78 mg/ml

Discussion:
The traditional method for calculating protein concentration of an unknown sample is to use a
standard curve that is generated from protein standards. The most reliable method is using a
protein standard that has the similar properties to the protein that you would like to estimate.
Since it is difficult to search for protein standards that have similar properties to the protein
that is being analyzed, so that bovine serum albumin or abbreviated as BSA and gamma
globulin are being used as standards. In this experiment, BSA was being used as the protein
standards.

Biuret Reaction is a method that can be used to determine the amount of soluble protein in a
solution. Biuret is the chemical product that forms when urea is heated to 180​o​C. In this
reaction, two molecules of urea condense to form a bi-urea molecule. In the presence of
copper ions form violet complexes. The spectrophotometer has been used to measure the
intensity of the colour produced. The more protein present in the solution, the darker the blue
color.

By using the spectrophotometer, we could not determine the concentration of the protein in a
solution only by the absorbance reading. A standard curve must be constructed in order to
quantitatively measure the concentration of protein of the unknown solution. This was done
by performing the biuret reaction on a series of prepared solutions from test tube 1 to 8 with
0,1,2 ,3, 4, 5,7 and 9 mg/ml of BSA solution in water. The absorbance readings 1 and 2
obtained from these series of test tubes were used to construct a graph of absorbance as a
function of protein concentration. The graph that was constructed in figure 2 is called the
standard curve for assay. By using the regression equation obtained from the graph, the
concentration of protein in the unknown sample could be calculated. In test tube 9, the
protein concentration was calculated to be equal to 6.67 mg/ml which is considered as the
highest concentration of protein among the unknown samples. In test tube 10, we obtain 6.33
mg/ml of protein concentration. In test tube 11 and 12, we obtained the protein concentration
by the absorbance readings of 3.55 mg/ml as the lowest concentration of protein among
samples and 3.78 mg/ml. After calculating the average of concentration of each sample, it
was found that the concentration of the unknown samples was 5.082
Other than biuret protein assay there are also some copper ion based tests that are called BCA
and lowry. In the bicinchoninic acid (BCA) assay, the copper ions generated through
interaction with protein react with BCA and then produce a strong violet color. ​The
difference however is that the formed BCA/copper complex absorbs light much more
strongly than those generated by the biuret assay alone greatly increasing the assays
sensitivity. With proper sample preparation and accurate standard curves it is possible to
assay samples accurately within the 0.001 to 2 mg/mL protein concentration range. The
lowry assay is similar to the BCA assay. It relies on the reaction of copper ions, produced
from the interaction of copper(II) sulfate with proteins present in the sample, with a further
reagent which is folin reagent. Cuprous ions (Cu+) reduction of Folin Reagent produces a
blue color that can be read at 650-750nm. The amount of color produced is proportional to
the amount of peptide bonds, i.e. size as well as the amount of protein/peptide.

Conclusion:
In conclusion, it was calculated that the average protein concentration of unknown samples is
5.082 mg/ml. This can be concluded that the protein concentration in the unknown sample is
relatively lower than the concentration of protein in the BSA solution as it can be seen clearly
in the graph. The concentration of protein is directly proportional to the absorbance. The
higher the absorbance, the higher the protein concentration. Besides, BCA has the highest
sensitivity that can assay sample within ​0.001 to 2 mg/ml among three of the copper ions
based assays.

References:
1. Berg, J., Stryer, L., Tymoczko, J. and Gatto, G. (2019). ​Biochemistry 9th ed. New
York: Macmillan Learning.
2. Handbook & Selection Guide. (2008). [online] Available at:
http://www.genotech.com/bulletins/protein-assays-introduction.pdf​. [Accessed on 7
July 2020]
3. Owusu-Apenten, R.K. (2002). Food protein analysis : quantitative effects on
processing. New York: Marcel Dekker.
4. Rocco, R.M. (2014). ​Landmark papers in clinical chemistry (R. M. Rocco, ed.).
Elsevier Science & Technology. United States.