TITLE: Estimation of protein

ABSTRUCT
This experiment is performed in order for one to learn the principles of protein assays.
This exercise introduces students to method of determining protein concentrations. The
determination of protein concentration is an essential technique in all aspects of protein
studies and proteomics. This laboratory activity is designed to teach students the
principles behind a common protein estimation assay known as the Biuret Protein Assay
(absorbance at 540nm). Standard Biuret reagent was already prepared for us. The 2ml
of protein solution was mixed with the 3ml of prepared Biuret reagent. The solution was
mixed well and incubated at 37ºC for 10 minutes and allowed to cool then the
absorbance for each tube was read against the blank at 540 nm. The standard curve
was plotted using absorbance values obtained at 540 nm against the concentration of
the standard (mg/ml). The standard was used to determine the concentration of the
unknown protein and it was found to 2.45mg/ml.

INTRODUCTION
The determination of the protein concentration in a solution is a routine requirement
during the protein purification. In order to assess the concentration of a protein Biuret
assay method is used, in this method the protein concentration is determined from the
standard curve of the absorbance against the concentration drawn using the
absorbance obtained from the spectrophotometer and the calculated concentration of
the standards. An assay must be specific for the protein being purified and must be
convenient to use for it is being done repeatedly at every stage of the process. This
assay is the appropriate one to use since amide bonds occur with the same frequency
per amino acid in the peptide (Taylor, et al., 1997).
The Biuret reagent (copper sulphate in a strong base) reacts with peptide bonds (which
join amino acids to form proteins) and changes colour when it does so, the
spectrophotometer at 540nm is used to measure the intensity of the color produced, the
more protein present the darker the colour and the larger the absorbance. The reaction
does not occur with amino acids because the absence of peptide bonds, and also that
with di-peptide because of the presence of only one peptide bond, but do with tri-,
oligo-, and poly-peptides. Biuret reaction needs presence of at least two peptide bonds
in a molecule (Nelson and Michael, 2008).

AIM:
To approximate the molarity of an unknown protein using a calorimetric technique by
finding out its absorbance using Bovine serum albumin (BSA) as a standard solution.

MATERIALS AND METHODS

A series of dilute solutions of the (BSA) were prepared from the (BSA) solution whose
concentration was 5mg/ml.
Seven test tubes were taken and a series of dilutions were carried out as shown in the
table below.
Tube number 1 2 3 4 5 6 7
BSA(ml) 0 0.4 0.8 1.2 1.6 2
Unknown (ml) - - - - - - 2
Distilled H2O (ml) 2 1.6 1.2 0.8 0.4 0 0
Biuret reagent (ml) 3 3 3 3 3 3 3

A Biuret reagent was prepared by dissolving 3g of copper sulphate pentahydrate and 9g

of sodium potassium tartrate in 500ml of 0.2M sodium hydroxide and 5g of potassium
iodide was added up to 1 litre with 0.2M sodium hydroxide.
3ml of prepared Biuret reagent was added to each of the test tubes and mixed with the
contents, the mixtures were incubated in a water bath at 37 ◦C for about 10 minutes.
The mixtures were allowed to cool and was put inside the spectrophotometer at a
wavelength of 540nm and the absorbance values were noted.

RESULTS
ATTCHED
 0.5

0.45
f(x) = 0.09x - 0.02
0.4 R² = 0.99

0.35
Abs orbance (540nm)

0.3

0.25

0.2

0.15

0.1

0.05

0
0 1 2 3 4 5 6
(BSA) Concentration (mg/ml )

Figure 1: shows the graph of absorbance(540nm) versus

concentration(mg/ml)
Calculations of the concentration of an unknown protein using the graph’s equation.
The equation of the graph: y=0.0902 x−0.015
We know that y=mx +c
Where y= absorbance
X= to the concentration
y−c
We make x subject of the formula x=
m
0.206−(−0.015)
x=
0.0902
x=2.45 mg/ml
DISCUSSION
The Biuret reaction can be used for both qualitative and quantitative analysis of protein.
The biuret method depends on the presence of peptides bonds in proteins. When a
solution of proteins is treated with cupric ions (Cu 2+) in a moderately alkaline medium, a
purple colored (Cu2+) peptide complex is formed which can be measured quantitatively
by spectrophotometer in the visible region. So, biuret reagent is alkaline copper sulfate
solution. The intensity of the color produced is proportional to the number of peptide
bonds that are reacting, and therefore to the number of protein molecules present in the
reaction system. The reaction takes its name "Biuret Reaction" from the fact that biuret
itself, obtained by heating urea, gives a similar coloured complex with cupric ions
(Hames and Nigel, 2005).
The main objective of this experiment was to determine the concentration of the
unknown protein. This was done using seven test tubes in duplicates and it was found
that the concentration of the bovine serum albumin in each test tube is directly
proportional to intensity of the purple colour that was observed, as well as the
absorbance readings showing that there is a coherent relationship as the findings were
consistent with the experimental hypothesis. In order to quantitatively determine how
much protein of the unknown present in tube 7, it was necessary to construct a standard
curve, this was done by performing the Biuret reaction on a series of prepared solutions
of bovine serum albumin at a concentration of 0,1,2,3,4 and 5 mg/ml in water. The
absorbance readings obtained from these solutions were used to construct a graph of
absorbance as a function of protein concentration. This graph is called the standard
curve for assay, the concentration of the unknown was obtained by interpolating the
absorbance value of an unknown protein and its concentration was found to be
2.45mg/m and the R2 value from the graph was found to be 0.9879 indicating a small
dispersion of the points from the slope (Lodish, et al., 2007).

The concentration of bovine serum albumin ranging from 0mg/ml to 5mg/ml was used
since further increases in the (BSA) concentration for example beyond 10mg/ml are not
applicable because mammalian (BSA) can form gels around that concentration. The
absorbance of water was recorded as a blank since there was no analyte of interest and
we were only interested in the concentration of (BSA) for the experiment to be
standardized and also if the assay was not properly standardized, solutions would have
been subjected in the wrong quartile. A Biuret reagent was added in order to stabilize
the cupric ions (Cu 2+ ions). The reaction of the cupric ions with the nitrogen atoms
involved in the peptide bonds leads to the displacement of the peptide bond and
hydrogen atoms under alkaline conditions and a tri or tetra dentate chelation with the
peptide of nitrogen produces the biuret color but this is found with dipeptides (Mills and
Ben, 2009).
REFERENCES
 Hames D and Nigel H, 2005, Molecular cell Biology, 3rd edition, page 842-
843
 Lodish H, Arnold B, Lawerence Z, 2007, Lehninger Principles of
Biochemistry, page 64-65.
 Mills A, Ben G, 2009, students companion to stryers biochemistry, volume
1, page 150.
 Nelson L and Michael M, 2008, spectrophotometric analysis of the bovine
serum, page 103.
 Taylor D, Green O, Stout G, 1997, Biological Science 1&2, 3rd edition,page
54-58.