Spectrophotometric Determination of Total Soluble
Proteins in Blood Serum
Submitted by: Carillo, Dorish Faith M.
ABSTRACT
The human blood serum contains different types of proteins
which play a central role in biological processes. The most
abundant of which is the albumin. In this study, the
concentration and absorbance of the different protein
solutions with Bovine Serum Albumin and with human
serum are measured spectrophotometrically using the
Biuret method. This method is based on the complexation
of Cu2+ to functional groups in the protein’s peptide bonds.
A violet-purplish color is produced when cupric ions (Cu2+)
interact with peptide bonds under alkaline conditions.
INTRODUCTION
Proteins are the main and most abundant constituents of
the blood serum or plasma. They serve many different
functions, including transport of lipids, hormones, vitamins,
and minerals in activity and functioning of the immune
system. Proteins have a specific intramolecular structure
and amphoteric nature, containing the balanced portions of
hydrophilic and hydrophobic groups. They are
macromolecules built from one or more unbranched chains
of amino acids linked by peptide bonds.
Human blood serum contains two major types of proteins,
the albumin, and the globulins.
Serum albumin accounts for 55% of blood proteins.
It functions primarily as a carrier protein for steroids, fatty
acids, and thyroid hormones in the blood, and keep the
blood from leaking out of blood vessels by contributing to
oncotic pressure.
Bovine serum albumin (BSA) is a serum albumin protein
isolated from cows. It has no great effect in many different
biochemical reactions as it does not affect other enzymes
that do not need it for stabilization, therefore it is often
used as a protein concentration standard in lab experiments
as well as in numerous other biochemical applications. In
this experiment, Bovine serum albumin is used to determine
the quantity of other proteins, by comparing an unknown
quantity of protein to known amounts of BSA using a graph
generated by the computer.
Determination of total proteins is widely used in several
areas such as: clinical analysis, food science, food
technology, biochemistry, physiology, protein chemistry,
and medical research. The most widely used analytical
technique to assess the concentrations of total proteins is
the biuret method. This method is
based on colorimetric principle, in which the copper ions
from the biuret reagent react with the amide groups from
the proteins at strong alkaline pH, creating a violet color.
METHODOLOGY AND MATERIALS
Preparation of Standard Calibration Curve Using Standard
Bovine Serum Albumin (BSA) Solution and Biuret Reagent
Standard BSA Solution with a concentration of 10 mg/ml
was prepared together with the Biuret
Solution for this experiment.
Six (6) clean test tubes were prepared. Five test tubes were
labeled using a marker with the following: 0.2mL, 0.4mL,
0.6mL, 0.8 mL, and 1.0 mL respectively. The remaining one
test tube was for Blank.
A series of BSA Solution concentration volumes were
prepared from 02.mL up to 1.0mL. Using a 200-microliter
pipette, 0.2mL BSA solution was pipetted to test tube 1,
0.4mL was pipetted to test tube 2, 0.6 mL was pipetted to
test tube 3, 0.8mL was pipetted to test tube 4, and 1.0mL
was pipetted to test tube 5. No BSA solution was
transferred to the blank test tube.
The standard BSA in the test tubes were diluted to 2mL
using distilled water. 1.8mL distilled water was transferred
to test tube 1, 1.6mL distilled water was transferred to test
tube 2, 1.4 ml distilled water was added to test tube 3, 1.2
mL distilled water was pipetted to test tube 4, and 1.0mL
distilled water was transferred to test tube 5. In the test
tube that served as blank, 2mL distilled water was pipetted.
Two (2) mL of Biuret reagent was added to each test tube.
After that, the test tubes were left to stand at room
temperature for 5 minutes.
The solutions were then transferred into a separate cuvette
for spectrophotometric measurements. It was noted that a 10 (𝑚𝑔/𝑚𝐿)(0.4 𝑚𝐿)
Test tube 2: 𝑐 = = 1 𝑚𝑔/𝑚𝐿
graduation of color was observed among the test tubes. The 4 𝑚𝐿

absorbance of each test tube was determined using a pre-

10 (𝑚𝑔/𝑚𝐿)(0.6𝑚𝐿)
warmed spectrophotometer. Basic ATC (Absorbance, Test tube 3: 𝑐 = = 1.5 𝑚𝑔/𝑚𝐿
4 𝑚𝐿
Transmittance, and Concentration) was selected where
wavelength was set at 540 nm. Each test tube was put into 10 (𝑚𝑔/𝑚𝐿)(0.8 𝑚𝐿)
Test tube 4: 𝑐 = = 2.0 𝑚𝑔/𝑚𝐿
the cell holders. Lid was tightly closed to prevent stray light 4 𝑚𝐿
from entering. Absorbance was measured for each of the
test tube. Standard Calibration curve was determined by 10 (𝑚𝑔/𝑚𝐿)(1.0 𝑚𝐿)
Test tube 5: 𝑐 = = 2.5 𝑚𝑔/𝑚𝐿
4 𝑚𝐿
plotting the concentration and absorbance of each test tube
with graphical
10 (𝑚𝑔/𝑚𝐿)(0 𝑚𝐿)
display of R2, y-intercept and slope. Blank: 𝑐 = = 0 𝑚𝑔/𝑚𝐿
4 𝑚𝐿

Determination of Protein Concentration in serum sample

An exact amount of blood sample was extracted from a

volunteer to provide enough serum for the experiment. It
was placed into a test tube and was centrifuged for 10-15
mins. After the centrifugation, 200 uL of the separated
serum was collected and transferred into a clean test tube.
It was diluted to 2ml with distilled water. Then, it was added
with 2 ml Biuret reagent. The mixture was incubated at
room temperature for 5 minutes and was then placed into
the Spectrophotometer to measure its absorbance.

RESULTS AND DISCUSSION Chart 1.0: Bovine Serum Albumin (BSA) Standard Calibration
Curve without the serum sample
Results
To calculate the final concentration of each standard
B. Computation for the concentration of unknown
solution which has different volume of the stock standard
Serum sample
BSA (10 mg/ml) reagent, the C1V1 = C2V2 formula was
To compute for the unknown concentration, the following
used.
formula was used, which was derived from the standard
calibration curve.
Test Standard Total Concentration Absorbance
y= 0.289828571x + 0.026047619
tube BSA volume (mg/mL) (nm)
solution (mL) Where:
(mL) y= Absorbance of unknown (0.590 nm)
T1 0.2 4 0.5 0.168 x= concentration of the unknown
T2 0.4 4 1 0.335
T3 0.6 4 1.5 0.490 Through computation, we were able to arrive with the
T4 0.8 4 2.0 0.632 concentration of the unknown, which is 1.946 mg/mL. This
T5 1.0 4 2.5 0.705 is the total concentration of soluble proteins in the
Blank 0 4 0 0.000 blood serum using biuret method assay.
Table 1.0 Give variables for the computation of the
concentration.
Discussion
The biuret reaction for determining the amount of soluble
A. Computation of Concentration from each test
protein in a solution provides a simple and precise method
tube.
of measurement. The copper sulfate of the biuret reagent
reacts with peptide bonds of the proteins and changes color
(10 𝑚𝑔/𝑚𝐿)(0.2 𝑚𝐿)
Test tube 1: 𝑐 = = 0.5 𝑚𝑔/𝑚𝐿 when it does so. The spectrophotometer was then used to
4 𝑚𝐿
measure the intensity of the color produced. The more
protein presents the darker the color.

Constructing a standard curve was necessary to

quantitatively determine how much protein is represented
by a particular absorbance reading. In this experiment, from
the graph we can see that the highest concentration of
protein was in sample 5 and the lowest was in sample 1.

CONCLUSION

From this experiment we were able to measure the

different concentrations and absorbance of the standards
that were made with Bovine serum albumin as well as of
the unknown human serum sample using the
spectrophotometric (biuret method).

REFERENCES

Ken-ichiro Kanaya & Keitaro Hiromi (1987) Determination of

Low Concentrations of Protein by the Biuret Method Using
the “Stopped-flow Time Difference Analysis” Technique,
Agricultural and Biological Chemistry, 51:7, 1885-1892, DOI:
10.1080/00021369.1987.10868279

Layne, E. Spectrophotometric and Turbid Metric Methods

for Measuring Proteins. Methods in Enzymology 10: 447-
455. 1957.

Moman RN, Gupta N, Varacallo M. Physiology, Albumin.

[Updated 2021 Aug 9]. In: StatPearls [Internet]. Treasure
Island (FL): StatPearls Publishing; 2021 Jan-. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK459198/

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