Postlab Report on

Exercise 3.2
Determination of Protein Concentration by Spectrophotometry

Patricia Cecilia R. Peralta

CHEM 161.1 – 1L
1 Semester A.Y. 2018-2019
st

Groupmates:
Juan Miguel D. Esguerra
Bianca C. Micosa

Date performed:
September 11, 2018
Date submitted:
September 18, 2018

Laboratory Instructor:
Ms. Korina Vida G. Sinad
I. RESULTS AND DISCUSSION

The accurate measurement of a protein sample’s concentration is essential prior to further

calculations often employed in the biochemical screening of proteins. While several methods can
be used for the determination of protein concentration, the Biuret and Bradford assays are the
most common assays which are routinely used. For this experiment, the protein concentration of
an albumin isolate was determined through the Biuret assay, while the protein concentrations of
the same albumin isolate and an egg white solution were determined through the Bradford assay,
where a bovine albumin serum (BSA) solution was employed as a standard for both methods.

BSA was used as the standard for because in choosing standards for assays, the standard
must be similar to the sample. BSA is also stable in many biochemical reactions and resistant to
denaturation. However, it has its disadvantages such as being sensitive in Bradford assay, thus
affecting the concentration of the protein sample, and being able to bind to many other factors,
thus cannot be obtained at a very high purity (Johnson, 2012).

In order to be able to gather data for the quantitative or qualitative analysis of proteins,
the assay to be used must be specific for the protein of interest and highly sensitive to the
protein’s presence but still remain convenient for consistent replication of results. The
characteristics of enzymes like its ability to catalyze easily detectable products or whether it
consumes or generates acid during enzymatic reactions are taken advantage of enzyme-specific
assays. Meanwhile, proteins that are not enzymes may be assayed through their ability to bind
specific substances, through observation of their biological effects or by monitoring their
characteristic spectroscopic absorption or fluorescence (Voet and Voet, 2011).

In the Biuret method, the CuSO4 • 5H2O is the source of the Cu2+ needed for the
reaction. The potassium sodium tartrate stabilizes the Biuret complex with the copper ion once
formed. Water strengthens the complexation of the copper ion with the amino part of the peptide.
The solution was made alkaline by adding NaOH to avoid the denaturation of the protein and to
produce the color formation.

Table 3.2.1. Absorbance readings for the standard BSA solution for the Biuret assay
Test tube BSA solution concentration (mg/mL) Absorbance540
1 0.0 0.000
2 1.0 0.023
3 2.0 0.052
4 3.0 0.066
5 4.0 0.040
6 5.0 0.109

As shown by Table 3.2.1, the BSA standard stock solution with a concentration of 20
mg/mL was diluted using 0.05 M NaOH into six dilutions. These absorbance of each dilution was
then recorded and graphed against their respective BSA concentrations in order to form a
standard curve which will provide the linear equation which will be used for the determination of
the protein concentrations of the albumin isolate using the Biuret assay.

0.12
y = 0.0216x + 0.0024
0.1
R² = 0.9927

Absorbance540
0.08

0.06

0.04

0.02

0
0 1 2 3 4 5 6
BSA solution concentration (mg/mL)

Figure 3.2.1. Standard curve for protein content determination using the Biuret assay

The data gathered from test tube 4 in Table 3.2.1 was omitted in order to form a linear
standard curve for the protein content determination using the Biuret assay.

Table 3.2.2. Determination of protein content of albumin isolate through Biuret assay
Dilution of protein solution Absorbance540
1:1 0.149
1:2 0.149
1:3 0.157
1:7 0.117
1:10 0.068

The sample for the Biuret assay was prepared by dissolving 1200 mg of albumin isolate
in 0.05 M NaOH and bringing the volume to 10 mL using a volumetric flask. This prepared stock
solution was then diluted and the absorbance of each dilution was recorded in Table 3.2.1. The
absorbance at 540 nm of the solution with a 1:10 dilution was used for the calculation of the
protein concentration of the albumin isolate using the Biuret assay because it had the lowest
value among the absorbance values which can be found within the standard curve shown by
𝑚𝑔
Figure 3.2.1. A protein concentration of 3.0325 𝑚𝐿 was calculated with a percent protein value
of2.527083333%.

In the Bradford assay, the container of the reagent was covered in aluminum foil to
prevent light reactions which could result to early development of the color. This may affect the
value of the absorbance due to the change in the intensity of color. The absorbance values of the
BSA solutions were read at 595nm using a double-beam spectrophotometer against one of the
blank solution. The standard curve was then constructed to determine the concentrations of the
sample solutions.

Table 3.2.3. Absorbance readings for the standard BSA solution for the Bradford assay
BSA solution concentration
Test tube Absorbance595
(mg/mL)
1 0.000 0.000
2 0.125 0.1720
3 0.250 0.0553
4 0.500 0.0846
5 1.000 0.5490
6 1.500 0.6924
7 2.000 0.8309

As shown by Table 3.2.3, the BSA standard stock solution with a concentration of 2.0
mg/mL was diluted using distilled water into three dilutions. These absorbance of each dilution
was then recorded and graphed against their respective BSA concentrations in order to form a
standard curve which will provide the linear equation which will be used for the determination of
the protein concentrations of the albumin isolate using the Bradford assay.

1
0.9 y = 0.4x + 0.0789
0.8 R² = 0.9686
0.7
Absorbance595

0.6
0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5
BSA solution concentration (mg/mL)

Figure 3.2.2. Standard curve for protein content determination using the Bradford assay

The data gathered from test tubes 3 and 4 in Table 3.2.3 were omitted in order to form
a linear standard curve for the protein content determination using the Bradford assay.

Table 3.2.4. Determination of protein content of album isolate through Bradford assay
Dilution of protein solution Absorbance595
1:1 0.2955
1:7 0.0411
 1:10 0.0068

The first sample for the Bradford assay was the albumin isolate dissolved in distilled water
into a solution with a concentration of 10 mg/mL. This prepared stock solution was then diluted
and the absorbance of each dilution was recorded in Table 3.2.5. The absorbance at 595 nm of
the solution with a 1:10 dilution was used for the calculation of the protein concentration of the
albumin isolate using the Bradford assay because it had the lowest value among the absorbance
values which can be found within the standard curve shown by Figure 3.2.2. A protein
𝑚𝑔
concentration of 3.0325 𝑚𝐿 was calculated with a percent protein value of 5.415751335%.

Table 3.2.5. Determination of protein content of egg white through Bradford assay
Dilution of protein solution Absorbance595
1:1 0.8691
1:7 0.2935
1:10 0.0884

The second sample for the Bradford assay was a 5% v/v. This prepared stock solution
was then diluted and the absorbance of each dilution was recorded in Table 3.2.1. The absorbance
at 595 nm of the solution with a 1:7 dilution was used for the calculation of the protein
concentration of the egg white solution using the Bradford assay because it had the lowest value
among the absorbance values which can be found within the standard curve shown by Figure
𝑚𝑔
3.2.1. A protein concentration of 0.5365748092 𝑚𝐿 was calculated with a percent protein value
of 10.73149618%

Two other common methods used for the determination of protein content are the Lowry
assay and the bicinchoninic acid (BCA) assay, which are variants developed from the Biuret assay
as they are both reliant on the formation of the Biuret chromophore.

For the Lowry assay, it is reliant on two different reactions. The first reaction involves the
formation of the Biuret chromophore, where a copper ion complex with amide bonds from the
peptides present in the sample is formed which results to reduced copper in alkaline solution. The
second reaction involves the reduction of the Folin-Ciocalteu reagent, consisting of
phosphomolybdate and phosphotungstate, primarily by the reduced copper-amide bond complex
as well as by tyrosine and tryptophan residues. This reduced Folin-Ciocalteu reagent is blue and
is therefore detectable with a spectrophotometer in the range of 500 to 750 nm.

Compared to the Biuret assay, the Lowry assay is almost 100 times more sensitive as the
Folin-Ciocalteau reagent is more efficient for detecting reduced copper. However, it requires more
time compared to other assays due to its high susceptibility to many interfering compounds.
Detergents, carbohydrates, glycerol, EDTA, potassium compounds, sulfhydryl compounds,
disulfide compounds, most phenols, uric acid, guanine, xanthine, magnesium, and calcium are
substances known to interfere with the Lowry assay. These are also substances which are often
utilized for buffers needed for protein preparation, which poses a major limitation for the Lowry
assay.

The BCA assay, much like the Lowry assay, measures Cu+ and Cu2+ formed by the Biuret
complex in alkaline solution. Although it seems like the mechanism of the BCA assay is similar to
that of the Lowry assay, there are two factors which distinguish the assays from each other. The
first factor is that the reaction occurs at lower temperatures and is the result of the interaction of
copper and BCA with cysteine, tryptophan, and tyrosine residues in the protein. The second factor
is that the BCA reagent replaces the Folin-Ciocalteu reagent in the Lowry assay. The BCA reagent
forms a complex with Cu+, which has a strong absorbance at 562 nm.

BCA is advantageous in that it does not interact with as many contaminants and buffer
components as the Folin-Ciocalteu reagent, especially detergents. Components that interfere with
the BCA assay either lead to reduction of Cu2+ or copper chelators. Generally, these are not critical
components of buffers and can be easily removed or omitted prior to the assay, however the
temperature at which the assay is carried out is of high importance (Olson and Markwell, 2007).

II. SAMPLE CALCULATION

 Determination of the protein concentration of albumin isolate using Biuret assay

𝐴 = 0.002432432432
𝐵 = 0.02162162162
𝑟 = 0.9963289085
𝑦 = 0.02162162162x + 0.002432432432
0.068 = 0.02162162162x + 0.002432432432
𝑚𝑔
𝑥 = 3.0325
𝑚𝐿

 Percent protein of albumin isolate determined using Biuret assay

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛𝑡𝑒𝑟𝑝𝑜𝑙𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑎𝑠𝑠𝑎𝑦

% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑥100
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑜𝑐𝑘 𝑠𝑎𝑚𝑝𝑙𝑒
𝑚𝑔
3.0325
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑚𝐿
𝑚𝑔 𝑥100
120
𝑚𝐿
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 2.527083333%

 Determination of the protein concentration of albumin isolate using Bradford assay

𝐴 = 0.07888399582
𝐵 = 0.3999740586
𝑟 = 0.9841854395
 𝑦 = 0.3999740586x + 0.07888399582
0.2955 = 0.3999740586x + 0.07888399582
𝑚𝑔
𝑥 = 0.5415751335
𝑚𝐿

 Percent protein of albumin isolate determined using Biuret assay

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛𝑡𝑒𝑟𝑝𝑜𝑙𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑎𝑠𝑠𝑎𝑦

% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑥100
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑜𝑐𝑘 𝑠𝑎𝑚𝑝𝑙𝑒
𝑚𝑔
0.5415751335 𝑚𝐿
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑚𝑔 𝑥100
10
𝑚𝐿
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 5.415751335%

 Determination of the protein concentration of 5% v/v egg white solution using Bradford
assay
𝐴 = 0.07888399582
𝐵 = 0.3999740586
𝑟 = 0.9841854395
𝑦 = 0.3999740586x + 0.07888399582
0.2935 = 0.3999740586x + 0.07888399582
𝑚𝑔
𝑥 = 0.5365748092
𝑚𝐿

 Percent protein of albumin isolate determined using Biuret assay

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛𝑡𝑒𝑟𝑝𝑜𝑙𝑎𝑡𝑒𝑑 𝑓𝑟𝑜𝑚 𝑎𝑠𝑠𝑎𝑦

% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑥100
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑜𝑐𝑘 𝑠𝑎𝑚𝑝𝑙𝑒
𝑚𝑔
0.5365748092
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 𝑚𝐿 𝑥100
𝑣
5% 𝑣
% 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = 10.73149618%

III. REFERENCES

Johnson, M. (2012). Protein quantitation. Mater Methods. 2(1): p. 115

Olson, B.J.S.C and Markwell, J. (2007). Assays for determination of protein concentration. Curr.
Protoc. Protein Sci. 48(1): pp. 3.4.1-3.4.29
Voet, D. and Voet, J.G. (2011). Biochemistry (4th ed.). Hoboken, NJ: John Wiley & Sons, Inc.