DETERMINING THE PROTEIN CONCENTRATION IN SOLUTION PHOTOMETRICALLY,

USING THE BIURET AND LOWRY ASSAY TECHNIQUES.

Accurate, sensitive and rapid protein quantitation is essential in biological, biochemical and other
research sectors. Therefore, a wide array of diverse techniques was developed, with biuret and
lowry assays being the most common and simple. Biuret compound is a result of heating urea to
180°C. (sciencedirect,n.d) This method determines the presence of proteins in biological fluids and is
used widely in protein purification, electrophoresis, etc. (Katoch,n.d)  The biuret reaction takes place
in a moderately alkaline medium, where proteins and peptides react with purple copper ion (Cu2+).
(Dean,Jones,2011) It involves reducing that copper ion from cupric to cuprous form, which produces
a faint violet-blue colour. Biuret reagent is light blue. (Martin,2011) Hence the colour intensity is
directly proportional to the number of the peptide bonds in the protein molecule. Deeper purple
colour indicates more peptide-copper complexes. The coloured coordination complex is formed
between Cu2+ ion, amide nitrogen (=NH) and carbonyl oxygen (>C=O) of the peptide
bond. (Ellman,1961)  

Figure 1 – Biuret reaction process (Pazar, 2016)

The second assay technique called the Lowry assay, is based on the biuret assay, but the additional
steps and reagents were added to increase the sensitivity. The additional reagent in this method is
called Folin-Ciocalteu reagent, which consists of phosphate and tungstate molybdate. (Katoch,n.d)  It
interacts with the cuprous ions, and the side chains of tryptophan, tyrosine and cysteine. The colour
is formed because of the reaction between protein, and alkaline copper, which is followed by the
phosphomolybdate reduction performed by the phenolic groups of tyrosine and tryptophan
residues. (Kafle,n.d)  The intensity of the colour produced depends on the amount of reacting
tyrosine and tryptophan amino acid residues.

Figure 2 – The process of the Lowry reaction (Johnson,2021)

The compound formed is subsequently measured by a spectrophotometer in the visible region. (340-
750nm) The spectrophotometer is an instrument that measures the amount of light intensity in
relation to wavelength. It allows precise measurements of a particular wavelength.
(Dean,Jones,2011) Spectrophotometer is based on the beer-lambert law, and enables to quantify a
single substance in solution, providing an absorbance at a particular wavelength. This law states that
the light absorbed is directly proportional to the thickness of the analysed solution, and
concentration of the solute in the solution. (Makkar,Dawra,1982) The absorbance is a linear
function, and is expressed by the formulae : A = εbC, where A is the absorbance, ε stands for the
molar absorptivity or extinction coefficient, b means the pathlength of the cell, and C molar
concentration of the sample. The absorbance is inversely proportional to the solution transmittance.
(Swinehart,1962). The beer-lambert law would make an inaccurate measurement at high
concentration, and cause a slight non-linearity. (Martin,2011) This laboratory experiment was to
estimate the total protein concentration. It aimed to find out the absorbance of coloured albumin
samples, and prepare a standard curve to discover the unknown concentration of gelatin and
globulin. It was also great practice of accurate pipetting, use of spectrophotometer and analysing
data from standard curve.

To conduct this biuret assay experiment, a set of reagents and stock protein solutions were
prepared. It included a biuret reagent, distilled water, and solutions of albumin, gelatin, and γ -
globulin. Two pipettes with different scales, sets of pipette tips, tubes, tube holder and
spectrophotometer cuvettes were also needed. The equipment was calibrated. Test tubes with
different protein concentrations were prepared, each with the total volume of 0.5 cm 3. They were
labelled respectively from 1-10. Pipette tips were replaced after draining any liquid. The table below
presents the concentrations of the prepared tubes.

Figure 3 – Table showing the concentration of prepared test tubes (PRACTICAL & TUTORIAL
BOOKLET,2022)
 2 cm3 of biuret reagent was subsequently added to each tube, then mixed and left for 15 minutes.
The solutions from test tubes were transferred followingly, to the spectrophotometer cuvettes and
labelled as well. Afterwards, the levels of absorbance against the reagent blank at 540nm were
recorded using the spectrophotometer. Finally, the concentrations of the gelatin and γ -globulin
were estimated using the standard curve method.

The table below shows the absorbance values of samples with different protein concentration, that
were obtained with spectrophotometer at 540nm.

Figure 4 – albumin, gelatin and γ -globulin

absorbance values

This data enabled us to plot a graph, essential for determining an unknown concentration of gelatin
and γ -globulin. The diagram captioned as figure 5 shows the albumin standard curve represented by
blue, dotted line. This line is intersected by two extrapolations. Gelatin is indicated by the dark-green
line, and γ -globulin by the light-green line.

STANDARD CURVE FOR BIURET ASSAY

0.5

0.45

0.4
ABSORBANCE [540nm]

0.35

0.3

0.25

0.2GELATIN
-GLOBULIN
0.15

0.1

0.05

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

PROTEIN CONCENTRATION [mg 0.5 cm-3]

Figure 5 – Standard curve graph for biuret assay (excel generated)

To be able to plot the absorbance on the graph we needed to first calculate the average value for
the tubes 7-8, and 9-10. Calculation of the average is necessary because those tubes are duplicates
of each other, and should have the identical values.

Figure 6 – Average
absorbance calculation

Analysis of this graph enables us to find the unknown protein concentration. They are as follows: 2
(mg 0.5cm-3) for the gelatin and 1.93 (mg 0.5cm-3) for the γ -globulin.

Picture below shows the colour of our albumin solutions at different concentrations. We can clearly
observe the change of the colour, which becomes deeper purple at higher concentrations.
Figure 7 – Solutions of albumin (1-6), gelatin, and globulin prepared for spectrophotometer
analysis (photo taken in the laboratory)

The lowry assay experiment was conducted by the other group. The values are presented in the
table below.

Figure 8 – albumin, gelatin and γ -globulin

absorbance values

Figure 9 – Average absorbance calculation

Figure 10 shows the standard curve graph based on those results. We can read out the protein
concentration values, which are as follows: 24 (mg cm-3) for gelatin and 53.8 (mg cm-3) for the for
the γ -globulin.

STANDARD CURVE FOR LOWRY ASSAY

0.6

0.5
ABSORBANCE [700nm]

0.4

-GLOBULIN
0.3

0.2
GELATIN

0.1

0
0 10 20 30 40 50 60 70 80 90 100

PROTEIN CONCENTRATION [µg 0.5cm-3]

Figure 10 – Standard Curve Graph for Lowry assay (excel generated)

The biuret assay technique has many advantages including short time, only few reagents used, and
simplicity. (Dean,Jones,2011) However, its sensitivity is quite low, and a few mistakes may creep in.
Analysis of the biuret standard curve graph indicates that our pair conducted this experiment
correctly. The dots indicating absorbance against protein concentration on our graph are not aligned
perfectly, but they are increasing proportionally. However, a mistake arose during gelatin and γ -
globulin test tube preparation. The known value of protein concentration for those compounds was
2.5 (mg 0.5cm-3). Values from the graph prepared are: 2 and 1.93 (mg 0.5cm -3). The reason for this
mistake is probably inaccurate pipetting, and low specificity of the biuret assay test. A similar
mistake appeared in the lowry assay experiment.
REFERENCES

1. Biuret (n.d) https://www.sciencedirect.com/topics/chemistry/biuret

2. Dean, J. R, Jones, A. M, Holmes, D, Reed, R, Jones, A, Weyers, J. (2011) Practical Skills in
Chemistry, 2nd, Pearson
3. Ellman, L. G, (1961) The Biuret Reaction: Changes in the Ultraviolet Absorption Spectra
and Its Application to the Determination of Peptide Bonds
4. Kafle, B. P, Chemical Analysis and Material Characterization by Spectrophotometry (n.d)
Elsevier
5. Katoch, R, (n.d) Analytical Techniques in Biochemistry and Molecular Biology, Springer
6. Makkar, H. P. S, Sharma 0. P, Dawra R. K. and Neg S. S. (1982) Effect of Heating Proteins
in a Vacuum on Their Assay by the Lowry and Biuret Methods
7. Martin, H. (2011). Laboratory Measurement of Urine Albumin and Urine Total Protein in
Screening for Proteinuria in Chronic Kidney Disease. Clin Biochem.
Available: https://www.ncbi.nlm.nih.gov/pubmed/?term=Martin%2C+H.+(2011).
+Laboratory+Measurement+of+Urine+Albumin+and+Urine+Total+Protein+in+Screening
+for+Proteinuria+in+Chronic+Kidney+Disease. 
8. Onorato,P, Gratton, L,M, Polesello, M, Salmoiraghi A,Oss, S (2018) The Beer Lambert law
measurement made easy
9. Ricci,R,W, Ditzler,M,A, Nestor, L.P, (1994) Discovering the Beer-Lambert Law
10. Swinehart D. F. (1962) The Beer-Lambert law
11. Tan, H. S. and Jones W. E. (1989) Fitting of a Straight Line when Both Variables Contain
Errors Application to the Beer-lambert Law
12. Worsfold, P, Townshend, A, Poole, C. (n.d) Encyclopedia of analytical science, Elsevier

Diagrams:

- http://proteinpur.blogspot.com/2016/03/deneyin-ilkesi-lowry-method-alkali.html
- Johnson, M. (2012) https://www.labome.com/method/Protein-Quantitation.html