Experiment 4: Spectrophotometric Protein Assays

2011-35493, 2011-85007
Biochem 34.1 HEJ, Sir Marvin Pelovello

I. Abstract
The analysis of spectrophotometric protein assays relies on the interaction between UV and visible light (absorbed
or transmitted) and the matter of interestproteins. These assays not only determine the identity of the protein but also
quantitatively determine protein concentration. Accurate protein quantitation, either using dye-based or colored
complex measurement, is required to study various biochemical processes. Biuret assay is based on the chelation of
amide groups in peptide bonds with copper ions forming a violet complex, in alkaline solution. Lowry assay is the
amplified version of the former, with the addition of Folin-Ciocalteu reagent being reduced to molybdenum/tungsten
blue by copper-catalyzed oxidation of amino acid residues. Bradford assay is based upon the absorbance shift of
Coomassie dye from acidic conditions (red) to a stable blue colour as it binds with a protein molecule. The Bradford
assay has the highest sensitivity, followed by the Lowry, making Biuret assay with the least sensitivity. Both Biuret and
Lowry assays are linear at a wide range of concentration while Bradford is linear at a narrow range of concentration.
They are susceptible to chemical interferences though Bradford is only interfered by detergents. There is really no
absolute method for protein quantitation since all have their advantages and disadvantages.

II. Keywords: spectrophotometric protein assay, protein, Biuret assay, Bradford assay, Lowry assay

III. Introduction the amino acid composition, sequence, and even

Proteins are large biomolecules that are consisted structure.
of one or more chains of amino acid residues. Each
protein has a specific and important role in various The general objective of the experiment is the
biological processes. Understanding these protein quantification of proteins using various
structure and function can be accomplished by spectrophotometric assays. Specifically, the
studying individual proteins. Consequently, this means experiment aims to understand the principles behind
that a researcher must be able to separate and purify dye-based absorbance and colored complex
one from the others by using necessary techniques measurements. Comparison of accuracy and precision
(Nelson and Cox 2013). Isolation, purification, and between three spectrophotometric protein assays;
characterization of proteins are preceded by its general namely, Biuret, Bradford, and Lowry; as well as their
quantification and presence in nature, making it an advantages and disadvantages are also part of the
important and precedent field in biochemistry. objectives.

A wide range of these biomolecules absorb light at IV. Experimental

characteristic wavelengths. Measurement of light
absorption by a spectrophotometer is used to detect
and identify molecules and to measure their
concentration in solution (Nelson and Cox 2013).
Because of this, various spectrophotometric protein
assays have become essential in the quantification of
protein in biological samples. These techniques make
use of the ultraviolet (UV) and visible light spectra in
order to determine the concentration of these
biomolecules. Dye-based absorbance and colorimetry
(colored complex) are used in these analytical
methods. They are selected mainly due to their
accuracy, sensitivity, and convenience depending on
A. Biuret Protein Assay
Three spectrophotometric protein assays were
evaluated in this experiment. The first one is the biuret
protein assay. Preparation of the biuret reagent
included 75 mL of 10% (w/v) sodium hydroxide (NaOH)
added to 125 mL of 0.3% copper sulfate pentahydrate
(CuSO4 5H2O) and 1.2% potassium sodium tartrate
(KNaC4H6O6 4H2O) in combined solution. The
resulting mixture was diluted to 250 mL with distilled
water. Duplicates of five test tubes labeled one to five
were used for the preparation of a standard curve.
Distilled water and 10 mg/mL ovalbumin were added
according to the table.

Biochem 34.1: Spectrophotometric Protein Assays Page 1

 C. Lowry Protein Assay
Table 1. Measurements of solutions for the biuret The Lowry protein assay is the final part of the
protein assay. experiment. In here, two solutions named reagent A
dH2O ovalbumin and reagent B were prepared. In the reagent A, 1.0%
Tube # (w/v) copper sulfate pentahydrate and 2.0% (w/v)
(mL) 10 mg/mL (mL)
1 1.0 0.0 potassium sodium tartrate were diluted to 50 mL with
2 0.8 0.2 distilled water. In the other reagent, 2.0% (w/v) sodium
3 0.6 0.4 carbonate (NaCO3) and 0.4% (w/v) sodium hydroxide
4 0.4 0.6 were diluted to 50 mL with distilled water. The Lowry
5 0.2 0.8 reagent was accomplished by mixing 50 mL of reagent
A and one mL of reagent B. Similar to the Bradford
Separate duplicates of test tubes were prepared protein assay, duplicates of six test tubes were
for two unknowns. The biuret reagent was added to all prepared and labeled accordingly for the standard
test tubes at five mL and mixed thoroughly before the curve (see table 3). Two separate duplicates were
ten-minute incubation. The absorbance of each test prepared for the two unknown.
tube was measured at 540 nm to obtain a plot of
concentration against absorbance. Table 3. Measurements of solutions for the Lowry
protein assay.
B. Bradford Protein Assay dH2O ovalbumin
Tube #
(mL) 10 mg/mL (L)
The second part of the experiment is for the
Bradford protein assay. Bradford reagent was prepared 1 1.0 0.0
2 0.98 20.0
using 25 mg of Coomassie Blue G-250 dye in 12.5 mL 3 0.96 40.0
95% ethanol added to 25 mL of 85% (14.7 M) 4 0.94 60.0
phosphoric acid. The mixture was diluted to 250 mL 5 0.92 80.0
with distilled water and filtered thrice until the light 6 0.90 100.0
brown or greenish color was obtained. This time,
duplicates of six test tubes labeled one to six were Five mL of the Lowry reagent were added to each
prepared for the standard curve. Distilled water and test tube and mixed thoroughly. The test tubes were
one mg/mL ovalbumin were added based on the table incubated for 10 minutes at room temperature. One
below. Separate duplicates for the two unknowns were mL of the dilute Folin-Ciocalteu phenol reagent was
also prepared. added to each test tube, mixed well, and allowed to
stand for another 20 minutes. The absorbance was
Table 2. Measurements of solutions for the Bradford measured at 750 nm, and a plot was obtained.
protein assay.
dH2O ovalbumin V. Results and Discussion
Tube # Table 4. Protein concentrations obtained through
(L) 1 mg/mL (L)
Biuret assay.
1 500 0.00
Absorbance (540 nm) Protein
2 490 10.0
Tube # Replicate Replicate Average concentration
3 480 20.0 1 1 (mg/mL)
4 470 30.0 1(blank) - - - 0.00
5 460 40.0 2 0.117 0.113 0.115 0.333
6 450 50.0 3 0.208 0.196 0.202 0.667
4 0.308 0.312 0.310 1.00
Five mL of the Bradford reagent was added to each 5 0.348 0.365 0.357 1.33
Unknown
test tube and mixed thoroughly. The solutions were protein 1 0.064 0.084 0.074 0.146
Unknown
incubated for 10 minutes at room temperature. At 595 protein 2 0.225 0.232 0.229 0.766
nm, the absorbance was measured and recorded for
the concentration-absorbance plot.

Biochem 34.1: Spectrophotometric Protein Assays Page 2

In order to compute for the protein concentrations of
0.766
5.000
the standard solutions, the working equation used is % = 100

given by: 5.000

= 84.68%
. (/)
( )=

Table 5. Protein concentrations obtained through
Sample calculations of protein concentrations in Biuret Bradford assay.
assay: Absorbance (595 nm) Protein
Tube # Replicate Replicate Average concentration
Calculations for test tube # 2: 1 1 (mg/mL)
1(blank) - - - 0.00
( )


2 0.072 0.116 0.094 1.82 x 10-3
10 0.2 3 0.150 0.210 0.180 3.64 x 10-3
=
0.8 2 + 0.2 + 5.0 4 0.285 0.282 0.284 5.45 x 10-3
5 0.396 0.460 0.428 7.27 x 10-3
= 0.333
6 0.481 0.489 0.485 9.09 x 10-3
UK1 1.156 1.141 1.149 0.0205
Calculations for test tube # 3: UK2 1.976 1.993 1.985 0.0353

( )

Sample calculations of protein concentrations in
10 0.4

=
0.6 2 + 0.4 + 5.0
Bradford assay:

= 0.667 Calculations for test tube # 2:


( )
A standard curve was generated after plotting
1
1.0 10.0 3
concentration versus absorbance of tube numbers 1 to =
10
1
5. The equation of the standard curve was determined (490 2 + 10.0 ) 3
10
+ 5.0
through the following components:
= 1.82 103

Slope: 0.250
Y-intercept: 0.0375 Calculations for test tube # 3:
R2: 0.979 (

)

1
Equation: = 0.250 + 0.0375 1.0 20.0 3
10
=
Beers Law: = 0.250 + 0.0375 (480 2 + 20.0 ) 3
1
+ 5.0
10

Calculations for unknown protein 1: = 3.64 103

= 0.2501 + 0.0375
0.0375 0.074 0.0375 The equation of the standard curve was determined
1 = = = 0.146 through the following components:
0.250 0.250

Theoretical cUK1= 0.560 mg/mL Slope: 56.69

Y-intercept: -0.0150
0.146 0.560
% = 100 R2: 0.987

0.560

= 73.93% Equation: = 56.69 0.0150
Beers Law: = 56.69 0.0150
Calculations for unknown protein 2:
= 0.2502 + 0.0375 Calculations for unknown protein 1:
0.0375 0.229 0.0375 = 56.691 0.0150
2 = = = 0.766 + 0.0150 1.149 + 0.0150
0.250 0.250 1 = = = 0.0205
56.69 56.69
Theoretical cUK2= 5.000 mg/mL % = 96.34%

Calculations for unknown protein 2:

Biochem 34.1: Spectrophotometric Protein Assays Page 3

 = 56.692 0.0150
2 =
+ 0.0150 1.985 + 0.0150
= = 0.0353
Calculations for unknown protein 2:
56.69 56.69 = 4.202 0.0142
+ 0.0142 0.524 + 0.0142
% = 99.29% 2 =
4.20
=
4.20
= 0.128

% = 97.44%
Table 6. Protein concentrations obtained through Lowry
assay. Biuret assay is a chemical test used for detecting
Absorbance (750 nm) Protein the presence of peptide bonds (Bank n.d.). It can be
Tube # Replicate Replicate Average concentration used for the determination of protein concentration
1 1 (mg/mL)
since peptide bonds occur with the same frequency per
1(blank) - - - 0.00
amino acid in the peptide. The colour intensity of the
2 -0.009 0.001 -4 x 10-3 2.86 x 10-3
3 0.011 -0.003 4 x 10-3 5.71 x 10-3 mixture increases with the number of peptide bonds
4 0.045 0.018 0.032 8.57 x 10-3 (Thermo Scientific Pierce Protein Assay Techinical
5 0.035 0.041 0.038 0.0114 Handbook 2010). And, according to the Beer-Lamberts
6 0.034 0.043 0.039 0.0143 law, absorbance is directly proportional to the protein
UK1 0.080 0.088 0.084 0.0234 concentration. Biuret assay is based on the polypeptide
UK2 0.459 0.589 0.524 0.128
chelation of cupric ion in alkaline solution. Nitrogen
atoms of the peptide bonds react and form a
Sample calculations of protein concentrations in Lowry
assay: coordination bond with the Cu2+ ions. As a result, a
violet colored chelate complex is formed (Biuret Test
Calculations for test tube # 2: n.d.). Formation of Cu2+ protein complex requires at
(

) least three peptide bonds.

1
1.0 20.0 3
10
=
1
0.98 2 + (20.0 ) 3 + 5.0 + 1.0
The composition of the biuret reagent used in the
10
assay was already introduced in the experimental
= 2.86 103 portion. Potassium sodium tartrate is added to stabilize

the cupric ions (Gornall, Bardawill and David 1949).
Calculations for test tube # 3: The method occurs in basic solution to promote

( ) effective Cu2+ protein complex chelation by increasing

1
1.0 40.0 3
10 the nucleophilicity of the amide groups in the peptide
=
1
0.96 2 + (40.0 ) 3
10
+ 5.0 + 1.0 bonds. The reagent doesnt actually contain biuret
((H2N-CO-)2NH). It is named as such because the
= 5.71 103
interaction between amide groups and the copper ions
A standard curve was generated after plotting was first observed in the biuret molecule (Thermo
concentration versus absorbance of tube numbers 1 to Scientific Pierce Protein Assay Techinical Handbook
6. The equation of the standard curve was determined 2010).
through the following components:
The dye, Coomassie Blue G-250, is used in the
Slope: 4.20
Bradford protein assay. It exists in three forms which
Y-intercept: -0.0142
R2: 0.874 are cationic (red), neutral (green), and anionic (blue)
(Nobel Dr. 2000). When dissolved in acidic conditions,
Equation: = 4.20 0.0142 it absorbs at 465 nm, having a reddish brown colour-
Beers Law: = 4.20 0.0142 cationic. When the dye, which is negatively charged-
anionic, binds to the positively charged protein
Calculations for unknown protein 1: molecule the absorbance undergoes a shift to 595 nm
= 4.201 0.0142 with a blue colour. This shift in absorption maximum is
+ 0.0142 0.084 + 0.0142
1 = = = 0.0234 proportional to protein concentration over a narrow
4.20 4.20
range. Within the linear range (~0-2000 g/mL), the
% = 95.82% more protein present, the more Coomassie binds. The

Biochem 34.1: Spectrophotometric Protein Assays Page 4

Bradford method actually determines the presence of
basic amino acid residues arginine, lysine, and
histidine, which contributes to the formation of the
protein-dye complex (He 2011). It also determines the
presence of aromatic amino acid residues although the
interactions between the dye and the residues are very
much weaker (Boyer 2012). Furthermore, it is a
colorimetric assay hence, as the protein concentration
increases, the colour of the test sample becomes
darker (Bradford Protein Concentration Assay 2001).
Lowry assay is a more accurate version of the
biuret assay. It is based on two chemical reactions. The
first reaction is the biuret reaction, Cu2+ ions, under
alkaline conditions forms a complex with peptide
bonds in which Cu2+ ions are reduced to Cu1+ ions. The
second is the reaction of Folin-Ciocalteu reagent by the
monovalent copper ion and the radical groups of
tyrosine, tryptophan, and cystein to produce
phosphototungstic acid/phosphomolybdic acid that are
reduced to molybdenum/tungsten blue. The amount of
colour produced is proportional to the amount of
peptide bonds (G Biosciences Web site n.d.).
Biuret assay is easy to perform that it can be done
in a short time as colour development time is relatively
short. The reagents used are easily-prepared. Also, it is
marked less susceptible to chemical interference, e.g.
ammonium sulfate, than other assays. In terms of
convenience, Biuret assay has an upper hand
compared to Lowry assay. Lowry assay takes rather
very long to carry out among the three assays due to its
long incubation time and is interfered by various
materials such as ammonium sulphate, acids, EDTA,
DTT, phenol among others. Not only the colour
development is slow but the colour fades relatively
rapidly. Unlike in biuret assay, the colour intensity
remains constant for a reasonable amount of time
(Protein Concentration Determination n.d.). Between
biuret assay and Bradford assay, Bradford is less
convenient because the Bradford reagent has to be
filtered thrice to achieve a light brown colored solution.
Although, Bradford assay analysis is also fast like in
Biuret assay. Few materials interfere in Bradford assay
but the presence of detergents even at low
concentrations can hinder the method. The
interference results to the stabilization of the green
neutral dye species due to direct binding or by shifting
the pH (Nobel Dr. 2000). In addition, unlike the Biuret
Biochem 34.1: Spectrophotometric Protein Assays
and Lowry, Bradford assay is compatible with reducing
agents which are often used to stabilize proteins in
solution (Bradford Reagent 2016).
The Bradford assay dominates over the sensitivity
criteria. It is highly sensitive and able to measure 1-20
g of protein. Next to Bradford is Lowry assay which is
quite sensitive and is best use on solutions with protein
concentrations in the range 0.01- 1.0 mg/mL
(Waterborg 2002). The least sensitive is Biuret assay
that is able to measure 1-10 mg of proteins. The
obtained protein concentration of the unknown in the
Biuret assay should show the lowest amongst the three
assays due to its low sensitivity. Errors may have been
committed along the experimental process.
Analyzing the linear regression of the standard
curve of the three assays, Bradford assay gives the
most linear graph with an r2 equal to 0.987. Although,
the Bradford assay is linear over a short range only,
typically from 0-2000 g/mL, diluting the sample is
necessary before proceeding to the analysis. The least
linear graph is the Lowry assay with an r2 of 0.874. The
linearity in Biuret assay, having an r2 equals 0.979, is
in close competition with the Bradford assay. From
these data, it can be assumed that the Bradford is the
most accurate protein assay according to linear
regression analysis.
Comparing the relative errors of each method, the
results in Biuret assay are the least. Base on this,
Biuret assay is the most accurate method for the
determination of protein concentration. Compared to
other assays, Biuret assay is a reliable method for
protein quantitation because it does not depend on the
amino acid composition (Gornall, Bardawill and David
1949). The accuracy in Lowry is partially dependent on
amino acid composition especially on the presence of
readily oxidizable amino acids such as tyrosine,
cysteine, and tryptophan (Held and Hurley 2001).
Bradford assay is relatively less accurate because it
strongly depends on the sequence of protein. If the
protein doesnt contain a decent number of arginine,
then the dye will not bind to the protein as efficiently,
resulting in an underestimation of the protein
concentration. Furthermore, free amino acids, peptides
and low molecular weight proteins do not produce
colour with Coomassie dye. The number of the dye
ligands bound to each protein molecule is
Page 5
approximately proportional to the number of positive
charges on the protein. High molecular weight proteins
are required to react with the Coomassie dye (Thermo
Scientific Pierce Protein Assay Techinical Handbook
2010).
Another protein assay that is widely used (not in
the experiment) is the BCA test. It is another variant of
a copper-based assay similar to the Biuret and Lowry
assays. The difference is the addition of bicinchoninic
acid (BCA). The first reaction involves the Biuret
reaction. Then, the Cu+ is chelated by BCA, which
converts the apple-green colour of the free BCA to the
purple of the copper BCA complex. The absorbance of
the complex is read at 562 nm. Its advantages include
simplicity and compatibility with 1% detergents such as
Triton or sodium dodecyl sulfate (SDS) (Boyer 2012).
Also, the BCA Reagent is approximately 100 times
more sensitive than the biuret reagent. Though, the
downward of the method is that the presence of any of
the four amino acid residues- cysteine or cystine,
tyrosine, and tryptophan has strong influence on the
results.
VI. Conclusions and Recommendations
There are many ways to measure protein
concentration. Three particular spectrophotometric
protein assays were done, observed, and analyzed in
the experiment. They are Biuret, Bradford, and Lowry
assays. Biuret assay depends on the interaction of the
peptide bonds to the Cu2+ forming a blue to violet
complex. Lowry assay is the combination of Biuret
assay and the reduction of Folin-Ciocalteu reagent to
molybdenum/tungsten blue complex. Lastly, the
Bradford assay is based on the absorbance shift of
Coomassie Blue G-250 dye as it binds with the protein.
Theoretically, the measured absorbance is directly
proportional to the concentration of protein producing
an ideal linear plot or standard calibration curve. The
most preferred curve generates a linear response to
the standard solutions. As the calibration curve is more
linear, it will give more accurate results. Biuret and
Bradford assays provided linear regression values
closest to 1. Between these two, Biuret is the more
accurate method since it has the least relative error
that can also be a basis for accuracy.
Biochem 34.1: Spectrophotometric Protein Assays
There is no absolute protein concentration assay.
All methods have advantages and disadvantages.
There are several aspects in which the assays are
considered. First is the sensitivity of the assay. From
least sensitive to the most sensitive: Biuret, Lowry,
Bradford. Though Biuret has the least sensitivity, it is
relatively less susceptible to interferences unlike Lowry
that has several of possible interferences. Bradford has
interferences but relatively low too. Its interferences
are specifically detergents. In terms of speed, Biuret
and Bradford assays are accomplished fast unlike
Lowry. The long incubation time required for this
method is one of its disadvantages.
It is highly recommended that the reagents used
are properly handled and stored in order to prevent
reagent degradation that could affect the data results
that will be obtained. For reactions that are very much
pH dependent, it is important to maintain pH at a
suitable pH for the method to effectively occur.
Thorough mixing or using the vortex machine should be
appropriately used so that complete mixture and
dissolution of substances will be observed. The
procedure in the experiment should be meticulously
followed so that the students will not miss out on
significant steps that could affect the results. To have
further understanding and comparison on various
spectrophotometric protein assays, additional assays
are suggested to be done in the experiment.
VII. References
Bank, Eric. What Does a Biuret Test Mean in Biology?
http://education.seattlepi.com/biuret-test-
mean-biology-4659.html (accessed March
2016).
Biuret Test.
http://www.gutenberg.us/articles/biuret_test
(accessed March 2016).
Boyer, Rodney. Biochemistry Laboratory Modern
Theories and Techniques. 2nd. New Jersey:
Pearson Education, Inc., 2012.
Bradford Protein Concentration Assay. January 07,
2001.https://ww2.chemistry.gatech.edu/~lw2
6/bCourse_Information/4581/techniques/bra
dford/bradford.html (accessed March 2016).
Page 6
Bradford Reagent. 2016.
http://www.sigmaaldrich.com/life-
science/proteomics/protein-
quantitation/bradford-reagent.html (accessed
March 2016).

"G Biosciences Web site." Lowry Protein Assay.

http://www.gbiosciences.com/PDF/Protocol/4
02L_Lowry_Protein_Assay_TEACHER.pdf
(accessed March 2016).

Gornall, Allan G., Charles J. Bardawill, and Maxima M.

David. "Determination of Serum Proteins by
Means of the Biuret Reaction." The Journal of
Biology Chemistry , 1949: 751-766.

He, Fanglian. [Bio101] Bradford Protein Assay. March

20, 2011. http://www.bio-protocol.org/e45
(accessed March 2016).

Held, Ph. D., Paul, and Janet Hurley. "Determination of

Total Protein by Lowry Method ." Biotek
Website. February 20, 2001.
http://www.biotek.com/resources/docs/ELx8
08_Determining_Total_Protein_Lowry_Method
.pdf (accessed March 2016).

Nelson, David L., and Michael M. Cox. Lehninger

Principles of Biochemistry. Sixth. New York : W.
H. Freeman and Company, 2013.

Nobel Dr., Alfred. Quick Start Braford Protein Assay

Instruction Manual. California: Bio-Rad
Laboratories, Inc., 2000.

"Protein Concentration Determination."

http://www2.onu.edu/~k-
broekemeier/Chem314/ReviewProteinMethod
s.pdf (accessed March 2016).

Thermo Scientific Pierce Protein Assay Techinical

Handbook. Thermo Fisher Scientific, Inc.,
2010.

Waterborg, Jakob H. "The Lowry Method for Protein

Quantitation." In The Protein Protocols
Handbook, by J. M. Walker, 7-9. New Jersey:
Humana Press, Inc., 2002.

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