BIOCHEM 5 – Exp 1 – Group 1 – 3ABC

COLORIMETRIC QUANTIFICATION OF BOVINE SERUM ALBUMIN

USING THE LOWRY ASSAY
Aaron, D., Addun, J., Aguilar, J., Antazo, F., Aquino, G., & Badua, A.
Department of Biochemistry, Faculty of Pharmacy
University of Santo Tomas
____________________________________________________________________________
ABSTRACT

In this experiment, different protein assay techniques were done to determine the best time to use a
certain method in measuring amount of protein in a sample. The Lowry method, which is advantageous
when low protein concentrations of protein are involved, was performed to quantify unknown protein in
the sample. The LOD of the assay was computed to be 387.99 ug/ml, which is within the actual range of
the detection limit of the Lowry assay. The Lowry assay is a great method for quantification of proteins but
it also has a few disadvantages which can deter you from using this method.
____________________________________________________________________________

INTRODUCTION Lowry protein assay was named after

Protein assays are a standout
amongst the most generally utilized
techniques in life science research.
Estimation of protein concentration is vital in
protein purification, electrophoresis, cell
biology, molecular biology and other research
applications. In spite of the fact that there are
a wide assortment of protein measures
available, none of the tests can be utilized
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 for research applications and
this is designed to help researchers select
the most appropriate assay for their
application. [8] There are several protein
assays that can be utilized and in this
experiment, the Lowry method was
employed.
the biochemist Oliver H. Lowry, who
developed the reagent in the 1940s. It is a
common method used to measure the protein
concentration in products makes use of
copper, which bonds with the peptides bonds
in proteins under alkaline conditions. The
Lowry assay functions in alkaline conditions,
and involves two steps: 1) the Biuret reaction:
based on the reduction of Cu2+which then
binds to protein forming a Cu1+ peptide
complex, and 2) subsequent reduction of the
Folin–Ciocalteu reagent by this complex. [9]
Figure 1. The interaction of copper ions with
proteins.
Since, the Lowry protein assay is a
copper-based assay, the general mechanism
of this method is that the protein solution is
mixed with alkaline solution of copper salt.
Under basic conditions, cupric ions (Cu2+)
chelate with the peptide bonds bringing about
the reduction of cupric (Cu2+) to cuprous ions
(Cu+). If the alkaline copper is in excess over
the amount of peptide bonds, some of the
cupric ions will stay unbound to the peptide
bonds and are available for detection.
Furthermore, protein assays based on
copper ions can be divided into two groups,
assays that detect reduced cuprous ions
(Cu+) and assays that detect the unbound
cupric (Cu+2) ions. Subsequently, the cuprous
ions are detected with Folin-Ciocalteu
reagent (phosphomolybdic/phosphotungstic
acid) as in the protein assays based on
Lowry method. Cuprous ions (Cu+) reduction
of Folin-Ciocalteu reagent produces a blue
color that can be read at 650-750nm. The
amount of color produced is proportional to
the amount of peptide bonds, i.e. well as the
amount of protein/peptide. In the assays
based on the detection of unbound cupric
ions, the protein solution is mixed with an
amount of alkaline copper that is in excess
over the amount of peptide bond. The
unchelated cupric ions are detected with a
color-producing reagent that reacts with
cupric ions. The amount of color produced is
inversely proportional to the amount of ions.
The amount of color produced is inversely
proportional to the amount of peptide bond. [8]
In addition, the Lowry assay has the lowest
inter- and intra-assay variation and gives the
best linearity between protein amount and
absorbance. [10]
This experiment further aims to
determine the lambda max, the dynamic
standard curve range, and the limit of
detectability of each protein assay for bovine
serum and by the use of Lowry method, the
protein concentration of the unknown sample
is to be measured.
METHODOLOGY
A. Determination of λ max for Lowry
Protein Assay
The experiment began by preparing a
500! μ g/mL standard BSA solution and
was treated with the Lowry method.
The absorbance of the treated
standard BSA solution was then read
at every 25 increment from 300 to
900nm (including 465 and 595nm).
Upon obtaining the varying
absorbance of the standard solution,
a graph was plotted against its
wavelengths in order to determine the
λ max.
B. Determination of Dynamic Standard
Curve and Limit of Detectability
A 5 0 0 0 !μ g / m L B S A s o l u t i o n
underwent a two-fold dilution in order
to prepare a set of standard BSA
solutions of varying concentrations.
Each solution was then subjected to
the Lowry method and each of their
absorbance was read from the λ max
 obtained. Each absorbance obtained
was plotted against their respective
concentrations so as to construct a
dynamic standard curve and also
obtain the limit of detectability.
C. Protein Assay: Lowry Method
A volume of 0.5 mL of the solution,
whether the standard or the sample,
was mixed with Lowry Reagent A and
was undisturbed for 10 minutes. It
was the followed by the addition of
0.25 mL of Lowry Reagent B and was
mixed immediately. Also it was
remained undisturbed for 30 minutes.
The absorbance of the solution was
then measured at the λ max obtained
from the first procedure.
RESULTS AND DISCUSSION
A. Determination of λmax for Lowry Assay
685n 750nm
Figure 2. 685 nm λmax for Lowry Assay
The phenolic group of tyrosine and
tryptophan residues in BSA protein will
produce a blue-purple complex, with
maximum absorption at 660nm wavelength,
with Folin-Ciocalteu reagent. [6] Hence, the
685 nm was the wavelength chosen for the
experiment as shown in Figure 2. However,
some use 750 nm since few other
substances absorb light at that wavelength
(less interferences). [7] The usual range of the
lowry assay is from 650 nm to 750 nm, which
is the wavelength range appropriate for the
blue color change in solution so there would
be more accurate results in determining
absorbance values and protein concentration
with the use of the spectrophotometer.

B. Determination of dynamic standard curve

The Lowry assay has two reactions. and limit of detectability for Lowry assay
The first reaction is similar as the Biuret
Lowry Assay (Two-fold dilution)
reaction, which involves the reduction of 1.8

copper ions under alkaline conditions; and 1.6

Absorbance @ 685 nm

1.4
1.2
thus, forming a complex with peptide bonds. 1
0.8
The second reaction occurs when there is 0.6
0.4

reduction of Folin-Ciocalteu reagent by the 0.2

0
0 500 1000 1500 2000 2500 3000
copper-peptide bond complex, which Concentration (ug/mL)

explains the blue coloration of the solution. [1] Figure 3. Lowry Assay using Two-fold
Dilution
 The dynamic range of the Lowry predicted y-value for each x in regression.
assay was determined by looking at the The slope is obtained from the m-value in the
graph from Figure 3. The dynamic range is linear regression line from the standard
defined to be the concentration range over curve.
which there is a measurable response to the
analyte. This can be observed before the The purpose for getting the LOD was
point at which the curve starts to plateau. to determine the minimum protein
Based from this description, the dynamic concentration that can be detected by the
range for this assay was approximated to be Lowry assay. [5] Since, this type of assay has
from 0.0910 ug/mL to 2100 ug/mL. high sensitivity, it can detect small amounts
of protein concentration. Thus, the obtained
Standard Curve (Lowry Assay) LOD (387.99 ug/mL) serves as the minimum
1.8

1.6
amount of protein which can be detected by
1.4 the assay.
Absorbance @ 685 nm

1.2

0.8
0.6
0.4
0.2
0
0 500 1000
Concentration (ug/mL) tryptophan, cysteine, histidine and
Figure 4. Standard curve for Lowry Assay
After getting the lambda max from
Figure 1, a standard curve was obtained,
which is shown in Figure 4. Using this
standard curve and the formula in Microsoft
Excel, the limit of detection (LOD) for the
Lowry assay was computed, which resulted
to 387.99 ug/mL. The formula used in
Microsoft Excel for computing the LOD is
shown below. [4]
The STEYX is described as the
function that returns the standard error of the
Most dipeptides can be detected by
y = 0.0006x + 0.0824
R² = 0.9676 the Lowry assay. The presence of any of the
five amino acid residues such as tyrosine,
1500 2000 2500 3000
asparagine in the peptide can further
enhance the amount of color produced
because they contribute additional reducing
equivalents to further reduce the
phosphomolybdic/phosphotungstic acid
complex. Other free amino acids except
tyrosine and tryptophan will not produce a
colored product with the Lowry reagent. [7]
One of the advantages of the Lowry
assay was its increased sensitivity and
accuracy compared to other protein assays.
However, it also has disadvantages. First, it
takes more time to prepare and incubate the
protein solutions which takes about 40
minutes. Incubation time is also critical in
order to obtain accurate absorbance values.
Second, this Lowry assay is also prone to
interferences which would affect variations in
protein concentration and accuracy of results.
That is why prior to measurement of protein
concentration, two-fold dilution was
performed to the sample.
Two-fold dilution is the process in
which the concentration of a solution is
reduced by a factor of two, which makes the
original concentration reduced by one half. [2]
The purpose of performing two-fold serial
dilution is to reduce the effect of interfering
substances prior to measuring the protein
concentration, which can lead to error in
readings. Some examples of interfering
substances which form precipitates could be
compounds commonly used in buffers for
protein preparation such as: EDTA, Tris,
glycerol, detergents, and carbohydrates. [3]
Other interfering substances are chelating
agents, reducing agents and thiols.
CONCLUSION
Lowry assay is an enhanced Biuret of
copper chelation chemistry. It is highly
sensitive, it can detect up to 1 µg of protein.
The principle involved is the biuret reaction
that includes the Folin-Ciocalteu reagent
which intensifies the color produced. In this
assay, an incubation time of 30 mins is
required to stabilize the initial tetradentate
copper complex, which is read at the optimal
absorbance of 750 nm. Reducing agents as
well as chelating agents can interfere with the
absorbance of the protein, which is why this
assay is not widely used for absolute
measurement. The standard curve range for
Lowry assay is from 650 nm to 750 nm, and
the experimental lambda max obtained was
685 nm. Using the limit of detectability, the
value computed for protein concentration was
387.99 µg/mL
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