LABORATORY ACTIVITY NO.

4
SPECTROPHOTOMETRIC PROTEIN ASSAYS
Fami, Isabela N. (1), Pangilinan, Dianne G. (2)
CHEM 43 LB1A
Ms. Fatsy Cruz

I. ABSTRACT
Proteins are involved in various biological processes. To identify the proteins and determine its
quantity in biological samples, measuring its concentration is crucial. An important method of
determining such values is the use of different spectrophotometric protein assays. In this
experiment, two proteins assays – Biuret, and Bradford – are used. Concentrations of the two
unknown protein samples were determined by spectrophotometry. The values of the two
unknown samples were 4.15 mg/mL (unknown 1) and 0.89 mg/mL (unknown 2) for Biuret Test;
and 0.0245 mg/mL (unknown 1) and 0.0199 mg/mL (unknown 2) for Bradford Test. From the
experiment, the Biuret test and the Bradford test showed linearity of the relationship between
absorbance and protein concentration. Assays are essential in determining protein concentrations
more accurate and precise.

II. KEYWORDS: spectrophotometry, protein assays, Bradford reagent, Biuret reagent, protein
concentration, color intensity

III. INTRODUCTION instrument and observe the galvanometric

Proteins, long polymers of amino
acids, constitute the largest fraction (besides
water) of cells. They are perhaps the most
versatile of all biomolecules and serve crucial
functions in essentially all biological
processes. Some proteins have catalytic
activity and function as enzymes; others serve
as structural elements, signal receptors, or
transporters that carry specific substances into
or out of cells (Lehninger, et.al., 2008). They
also store other molecules such as oxygen,
provide mechanical support and immune
protection, generate movement, transmit nerve
impulses,
differentiation.
and control growth and
Every chemical compound (such as
the ones that contains proteins) absorbs,
transmits, or reflects light (electromagnetic
radiation) over a certain range of wavelength.
This is done by passing light of specific
wavelength of visible spectrum through the
solution in a photoelectric colorimeter
reading of reflection sensitizing the quantity of
light absorbed. Colorimetric reactions can be
measured on either a spectrophotometer or
colorimeter. Both of these instruments
measure the intensity of light passing through
the colored sample and convert this light
intensity to a concentration based on a stored
calibration curve. The instruments follow the
principles of the Beer-Lambert Law. The
Beer-Lambert law (referred to as Beer’s law) is
the linear relationship between absorbance
and concentration. Generally Beer’s law is
written as:
A=Ɛxbxc
Equation 1. ​Beer--Lambert’s Law
A is the measured absorbance
Ɛ is a wavelength-dependent absorptivity
coefficient
b is the cell-path length
c is the analyte concentration
Thus the variation of colour of the reaction
mixture (or system) with change of substrate
concentra​tion forms the basis of colorimetric
analysis (D, Bharati, 2019).
Spectrophotometer is the instrument
used to measure the light absorbed by the
solution. It is a more sophisticated instrument.
A photometer is a device for measuring ‘light’,
and ‘spectro’ implies the whole range of
continuous wavelengths that the light source is
capable of producing. The detector in the
photometer is generally a photocell in which a
sensitive surface receives photons; and a
current is generated that is proportional to the
intensity of the light beam, reaching the
surface. In instru​ments for measuring
ultraviolet/visible light, two lamps are usually
required: one, a tungsten filament lamp which
produces wavelengths in the visible region; the
second, a hydrogen or deuterium lamp, is
suitable for the ultraviolet (D, Bharati, 2019).
Figure 1. ​Optical Path of a Spectrophotometer
Protein assay is a spectroscopic
analytical procedure used to measure the
concentration of protein in a solution (Skoog
et. al., 2007). This method depends on the
amino acid composition of the measured
protein.
In this experiment, two protein assays
were utilized:
(1) Biuret Test is conveniently
used to detect the presence of proteins in
biological fluids. Alkaline CuSO4 reacts with
compounds containing two or more peptide
bonds to give a violet colored product which is
due to formation of coordination complex of
cupric ions with unshared electron pairs of
peptide nitrogen and O2 of water (Gaurab, K.,
2018).
(2) The ​Bradford assay​,
developed by Marion M. Bradford, a
colorimetric protein assay, is based on an
absorbance shift of the dye Coomassie
Brilliant Blue G-250 in which under acidic
conditions the red form of the dye is converted
into its bluer form to bind to the protein being
assayed (Bradford, 1976).
The objectives of this experiment
were: 1) to explain the principles behind
dye-based absorbance measurements and
colored complex measurements for protein
quantitation; 2) to quantify proteins in an
aqueous solution using various
spectrophotometric assays; 3) to compare
different spectrophotometric protein assays in
terms of accuracy and precision in determining
protein quality; and 4) to identify the
advantages and disadvantages of various
protein spectrophotometric assays for protein
quantitation in biological samples.
IV. EXPERIMENTAL
Preparation of Biuret Reagent. A ​
mixture of 75 mL 10% (w/v) NaOH and 125
mL of 0.3% copper sulfate pentahydrate
(CuSO4·5H2O) and 1.2% potassium tartrate
(KNaC4H4O6·4H2O) was diluted with distilled
water in a 250-mL volumetric flask.
Preparation of Bradford Reagent. A
mixture of 25 mg Coomassie Blue G-250 in
12.5 mL of 95% ethanol and 25 mL 85% (or
14.7 M) phosphoric acid was diluted with
distilled water in a 250-mL volumetric flask
then filtered three times, in which the resulting
color should be light brown.
Biuret Protein Assay. Two sets of test
tubes were labelled from 1-5 to prepare
standard curve and another two test tubes
were labelled as unknowns. Distilled water and
protein standard solution were pipetted in
varying volumes (in mL), with test tube 1
having ratio of 1:0, test tube 2 with ratio of The experiment has two protein
0.8:0.2, test tube 3 with ratio of 0.6:0.4, test ​ nd (2)
assays: (1) ​Biuret Protein Assay, a
tube 4 with ratio of 0.4:0.6 and test tube 5 with Bradford Protein Assay.
ratio of 0.2:0.8, all solutions, as well as the
unknown samples have a total volume of 1 mL A. Biuret Protein Assay
then 5 mL of Biuret reagent was added to all Absorbance readings obtained from
samples and mixed thoroughly. The samples the spectrophotometer for each protein
were let still for 10 minutes at room concentration for the standard curve are
temperature as it serves as incubation. The summarized in Table 1. The values were
UV-Vis spectrophotometer was set to 540 nm. plotted against each other, Figure 2, and a
Test tube 1 (blank) was transferred to a linear equation shown in Equation 2 was
cuvette to set the absorbance at 540 nm to obtained.
zero, also, the remaining solutions were
pipetted to different cuvettes and their Table 1. ​Absorbance readings for each protein
absorbances were recorded. The concentration for the standard curve of Biuret
concentration vs. absorbance of standard protein assay
solutions were plotted to determine the
Protein Concentration Absorbance
concentrations of unknown protein samples
(mg/mL)
were determined.
0 0.01
Bradford Protein Assay. Two sets of
2 0.092
test tubes were labelled from 1-5 to prepare
4 0.152
standard curve and another two test tubes
6 0.330
were labelled as unknowns. Distilled water and
8 0.397
protein standard solution were pipetted in
varying volumes (in µL), with test tube 1
having ratio of 500:0, test tube 2 with ratio of
490:10, test tube 3 with ratio of 480:20, test
tube 4 with ratio of 470:30, test tube 5 with
ratio of 460:40, and test tube 6 with a ratio of
450:50, all solutions, as well as the unknown
samples have a total volume of 500 µLL then 5
mL of Bradford reagent was added to all
samples and mixed thoroughly. The samples
were let still for 10 minutes at room
temperature as it serves as incubation. The
UV-Vis spectrophotometer was set to 595 nm. Figure 2. ​ Plot between the concentrations of
Test tube 1 (blank) was transferred to a the standard solutions and corresponding
cuvette to set the absorbance at 595 nm to absorbance in Biuret protein assay
zero, also, the remaining solutions were
pipetted to different cuvettes and their y = 0.0506x − 0.0062
absorbances were recorded. The Equation 2. ​Linear equation for the
concentration vs. absorbance of standard standardization of Biuret protein assay where
solutions were plotted and the concentrations y is absorbance and x is the protein
of unknown protein samples were determined. concentration (mg/mL)

V. RESULTS
 Using Equation 2 and the measured
absorbance readings for Biuret protein assay,
the concentrations of the unknowns were
calculated (See Appendix A) and are as
follows:

Table 2. ​Concentrations of the unknown

solutions in Biuret protein assay
Absorbance Concentration
(mg/mL)
Figure 4. ​ Plot between the concentrations of
the standard solutions and corresponding
Unknown 1 0.204 4.15
absorbance in Biuret protein assay

Unknown 2 0.039 0.89

y = 57.428x + 0.0792
Equation 3. ​Linear equation for the
standardization of Bradford protein assay
where y is absorbance and x is the protein
concentration (mg/mL)

Using Equation 3 and the measured

Figure 3. ​Unknown solutions 1 and 2, absorbance readings for Bradford protein
respectively, with Biuret reagent assay, the concentrations of the unknowns
were calculated (See Appendix A) and are as
B. Bradford Protein Assay follows:

Absorbance readings obtained from Table 3. ​Concentrations of the unknown

the spectrophotometer for each protein solutions in Bradford protein assay
concentration for the standard curve are Absorbance Concentration
summarized in Table 2. The values were (mg/mL)
plotted against each other, Figure 3, and a
linear equation shown in Equation 3 was
Unknown 1 1.488 0.0245
obtained.
Unknown 2 1.227 0.0200
Table 3. ​Absorbance readings for each protein
concentration for the standard curve of
Bradford protein assay
Protein Concentration Absorbance
(mg/mL)

0 0.09
1.82 x 10​-3 0.21
3.64 x 10​.3 0.273 Figure 5. ​Unknown solutions 1 and 2,
5.46 x 10​.3 0.330 respectively, with Bradford reagent
7.27 x 10​.3 0.509
9.09 x 10​.3 0.630
VI. DISCUSSION
The quantitation of protein content is
important and has many applications in food
industry practices and in research especially in
the field of biochemistry. The exact monitoring
of protein content in samples is a critical step
in protein analysis. The different protein assay
techniques have been developed for the
assessment of the protein concentration in a
sample (Okutucu et al. 2007). Estimation of
protein concentration is necessary in protein
purification, electrophoresis, cell biology,
molecular biology and other research
applications. Although there are a wide variety
of protein assays available, none of the assays
can be used without first considering their
suitability for the application. Each assay has
its own advantages and limitations and often it
is necessary to obtain more than one type of
protein assay. In this experiment, Biuret
protein assay - protein assay based on
alkaline copper, and Bradford protein assay -
dye-binding protein assay, were performed to
determine the protein concentration of the two
unknowns.
All of the methods performed in this
experiment used spectrophotometric
determination after treatment of different
reagents to establish standard curves. The
plot of the protein concentration against the
absorption yielded an equation of a line that
has a positive slope and was used to calculate
the protein concentration in the solution. This
was made in accordance to the Beer-Lambert
Law which states the linear relationship
between the concentration of a specific part of
the solution and the absorbance of the solution
at a certain wavelength (See Equation 1).
The traditional method for calculating
protein concentration of an unknown sample is
to use a standard curve that is generated from
known protein standards. The most reliable
protein estimation is performed using a
reference or a protein standard that has
properties similar to the protein being
estimated. Often, it is difficult to find a protein
standard with similar properties to the sample
being analyzed. As a result, it has become
acceptable to use readily available proteins
such as ​bovine serum albumin (BSA) ​and
gamma globulin ​as standards. These two are
abundant, readily purifiable, affordable,
relatively stable and offers desirable
biochemical reactivity with detectable signal.
A. Biuret Protein Assay
One of the simplest and most common
methods of protein assay is the ​Biuret Protein
Assay​. This can be used to quantify proteins in
the concentration range from 0.5 to
approximately 10 mg/mL (Layne, n.d.);
therefore, this requires more protein than other
common methods such as the BCA, Lowry
and Bradford assays.
Biuret protein assay is based on the
ability of Cu (II) ions to form a violet-colored
chelate complex with peptide bonds in alkaline
conditions. Lone pairs from ​4 nitrogen atoms
in the peptide bond coordinate a copper (II) ion
to form the chelate complex.
Figure 6. ​Chelate complex formation reaction
in Biuret protein assay
Therefore, Biuret protein assay does
not actually detect the protein itself, but rather
detects peptide bonds. Amino acids and
dipeptides do not exhibit color change due to
the fact that the Cu (II) ions forms complexes
with two adjacent amide-amide groups, in
which amino acids do not have and with
dipeptide bond only having 1 peptide bond
present.
 The chelate complex absorbs light at
540 nm. The greater the concentration of
peptide bonds, the greater the color intensity
(violet), i.e., greater absorbance. If the
concentration of peptide is low - such as when
short-chain peptides are present - the color
change from blue to pink.

By Equation 2, a concentration of 4.15

mg/mL for unknown 1 from an absorbance of
0.204 was obtained. This is reasonable
because the absorbance lies between the
absorbance readings at the concentrations 4
mg/mL and 6 mg/mL. On the other hand,
unknown 2 had a calculated concentration of
0.89 from an absorbance of 0.039.
Based on the principle of ​Biuret Test
colored complexes, and as mentioned above,
unknowns 1 and 2 both have peptide bonds of
more than two, since their colors changed to
violet. Calculated concentrations also follow
that unknown 1 should have a higher
concentration since it has a more intense
violet-colored solution compared with unknown
2. Also, obtained concentrations of the
unknowns which are 4.15 and 0.89 fall within
the concentration range of protein being
quantified by Biuret protein assay.
B. Bradford Protein Assay
The Bradford assay is based on the
use of the dye Coomassie Brilliant Blue G-250,
which is frequently abbreviated as Coomassie
G-250 or Coomassie Blue. The protein
determination method of Bradford (Bradford
M., 1976) is popular because it is rapid,
sensitive, relatively inexpensive, and specific
for protein.
Figure 7. ​ Structure of Coomassie brilliant
blue (CBBG) G-250 dye.
Under acidic conditions, Coomassie
G-250 is cationic, mainly doubly protonated,
and is red, whereas in neutral conditions the
dye is green, and the anionic form is blue. The
Bradford reagent is an acidified solution of
Coomassie G-250; the dye is thus primarily
protonated and red.
Figure 8. ​ Absorbance of Coomassie G-250 in
different conditions
Since the amount of the blue anionic
form is proportional to the amount of protein in
the sample, the quantity of protein in a sample
can be measured directly by measuring the
absorption at 595 nm.
The cationic (doubly protonated) form
of the Coomassie Blue G-250 was used in the
experiment. The strong acid phosphoric acid
(H3PO4) was responsible for the low pH of the
solution. The blue color after the addition of
Coomassie Blue G-250 to the standard protein
and protein samples was due to the binding of
the dye to the basic amino acid residues,
arginine and lysine. Because of the Bradford
protein assay’s high sensitivity, low
concentrations of standard protein were used.
At relatively high protein concentrations,
greater number of amino acid residues reacts
with the acidic Coomassie Blue G-250, and Principle of Protein-Dye Binding​,
more dye is converted into its anionic form, page 72. Retrieved from
making the absorbance at the blue region http://www.ciens.ucv.ve:8080/gener
higher. ador/sites
/lab-bioq-gen/archivos/Bradford%20
By Equation 3, a concentration of 1976.pdf
0.0245 mg/mL for unknown 1 from an
absorbance of 1.488 was obtained. On the Compton, S. J., & Jones, C. G. (1985).
other hand, unknown 2 had a calculated Mechanism of dye response and
concentration of 0.0200 from an absorbance of interference in the Bradford protein
1.227. The graph of the equation of the line for assay. Analytical Biochemistry,
Bradford Protein Assay showed a linear 151(2), 369–374.
relationship between absorbance and protein doi:10.1016/0003-2697(85)90190-3
concentration with r2 = 0.9697 and a positive
slope. However, the results may be altered by D. B.. (2019). ​Colorimetry: Principle and
the presence of interfering agents such as Instruments. Retrieved from
basic buffers, because of the ideal acidic http://www.biologydiscussion.com/c
environment of the assay (Bradford, 1976). olorimetry/colorimetry-principle-and-i
nstruments/57652
VII. CONCLUSIONS AND
RECOMMENDATIONS Gaurab, K., (2018, April 20). ​Biuret Test:
Biuret assays relied on the complexion Principle, Requirements, Procedure
of the Cu (II) ions with the peptide bonds, and Result. ​ etrieved
R from
Bradford assays, on the other hand, relied on https://www.onlinebiologynotes.com/
the spectral shift that happens when the biuret-test-principle-requirements-pr
Coomassie dye binds to the proteins. ocedure-and-result/
On the basis of linearity, both the
Biuret and Bradford test showed a linear Hach. (2018, September 28). ​What is the
relationship between the absorbance and scientific principle of colorimetry?
protein concentration of the samples. Retrieved from
It is recommended to use equimolar https://support.hach.com/app/answe
amounts of the assays since the presence of rs/answer_view/a_id/1001527/~/wha
the unbound dye in the Bradford reagent and t-is-the-scientific-principle-of-colorim
the cuprous ion affect the absorbance and the etry%3F-
sensitivity of the assays and also to work fast
in measuring the absorbance of the samples Lehninger, A., Nelson, D., & Cox, M. (2008).
especially for the Biuret assay in which it has Lehninger principles of biochemistry​.
to be done in 10 minutes. The reagents used 4th ed.
are sensitive to light, thus, it is important to
store them in amber bottled or away from Okutucu B., Dınçer A., Habib Ö., Zıhnıoglu, F.
intense light source to avoid decomposition for determination of total plasma
protein concentration. Journal of
2007. Comparison of five methods
VIII. REFERENCES Biochemical and Biophysical
Bradford, M. (1976). ​Analytical Biochemistry: A Methods [online] 70(5): 709–711.
Rapid and Sensitive Method for the [2015-09-16]. Available from:
Quantitation of Microgram http://www.sciencedirect.com/scienc
Quantities of Protein Utilizing the e/article/pii/S0165022X07001145
Skoog; et al. (2007). Principles of Instrumental
Analysis (6th ed.). Belmont, CA:
Thomson Brooks/Cole. pp.
169–173. ISBN 9780495012016.

CERTIFICATION OF CONTRIBUTION

I hereby certify that I have given substantial

contribution to this report:

_________________________
Fami, Isabela N.

_________________________
Pangilinan, Dianne G.
APPENDIX A. Calculations of Protein
Concentrations for Unknown Samples

Biuret Protein Assay

y = 0.0506x − 0.0062

Unknown Sample 1
0.204 = 0.0506x − 0.0062
x = 4.15 mg/mL

Unknown Sample 2
0.039 = 0.0506x − 0.0062
x = 0.89 mg/mL

Bradford Protein Assay

y = 57.428x + 0.0792

Unknown Sample 1
1.488 = 57.428x + 0.0792
x = 0.0245

Unknown Sample 2
1.227 = 57.428x + 0.0792
x = 0.0200