SEPARATION AND IDENTIFICATION OF AMINO ACIDS PRESENT IN

GLUTEN FROM WHEAT FLOUR BY PAPER CHROMATOGRAPHY AND

PROTEIN ASSAY USING THE BRADFORD METHOD
Tiffany Rae S. Espiritu, Lorenz Rey A. Esteban, Kathleen Anne E. Francisco, Ma. Casey Louisse P. Garcia and Aimee Dianne C. Hermoso

Group 4, 2E-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Paper chromatography is generally used for the separation of water-soluble organic and inorganic
compounds or highly polar compounds such as amino acids and sugars. The Bradford method, on the
other hand, is a rapid and accurate method for the estimation of protein concentration. The objectives of
this experiment are (1) to determine the amino acid components of the proteins by paper chromatography
and (2) To quantitatively determine protein concentration in a given sample through Bradford assay. In
paper chromatography, 10 standard amino acids were used to compare and determine which amino acid is
present in the protein, Gluten. Rf values were computed and it was seen that gluten has an almost the
same Rf values with cysteine and aspartic acid. Thus, gluten contains these amino acids. In the Bradford
assay method, 9 test tubes were prepared with different standard volumes and water volumes, and 2 test
tubes had diluted milk sample and an unknown sample, respectively. After subjecting to a
spectrophotometer, the absorbance of each sample was determined and plotted into a linear graph. The
concentration of the unknown sample was also computed through the line regression method and resulted
to 0.113 g/ml.

INTRODUCTION The Bradford method, on the other

Paper chromatography is generally used hand, is a rapid and accurate method for the

for the separation of water-soluble organic and estimation of protein concentration. It is

inorganic compounds or highly polar essential in many fields of protein study. This

compounds such as amino acids and sugars. The technique is simpler, faster, and more sensitive

separation of the solutes (amino acids) is based than the Lowry method. Moreover, when

on the liquid-liquid partitioning of amino acids compared with the Lowry method, it is subject

in paper chromatography. The partitioning takes to less interference by common reagents and

place between the water molecule (static phase) nonprotein components of biological samples.

absorbed to the cellulosic matter of the paper The Bradford assay relies on the binding
and the organic (mobile) phase. of the dye Coomassie Brilliant Blue G-250 to
protein. It has a high hydrophilic nature, as it has
various hydrocarbons and carbon rings that
stabilises its ionic form and causes a visible
colour change.The cationic form of the dye,
which predominates in the acidic assay reagent
solution, has a maximum wavelength of 470 nm.
In contrast, the anionic form of the dye, which
binds to protein, has a maximum wavelength of
595 nm. Thus, the amount of dye bound to
protein can be quantified by measuring the
absorbance of the solution at 595 nm.
Figure 1. Structure of Coomassie Brilliant Blue G-250
MATERIALS AND METHODS
Materials
Bradfrod Reagent (Coomasie dye), 100g/mL
Bovine Serum Albumin (BSA), evaporated milk
sample, test tubes, UV-Vis Spectrophotometer
Methods
Paper Chromatography:
A 12x15 filter paper was prepared and
drawn from the bottom of the longer edge of the
paper with a straight line 1.5 cm as the origin.
The amino acid standards were applied using the
capillary tubes 5 times and the samples 10 times.
The papers peripheral edges were stapled
together to form a cylinder, placed inside the
pre-equilibrated chamber and was covered. The
paper was removed when the solvent front was
approximately 1 cm from the top edge. The
solvent front was immediately marked with
pencil. The paper was air-dried and sprayed with
1% ninhydrin reagent and was allowed to get
dry. All the observed spots were encircled and
the Rf value of each molecule was computed.

Paper Chromatography: Bradford Assay:

A series of test tube were prepared as

Filter paper, 1000 mL-beaker, capillary tubes,
follows:
amino acid standards: 2% w/v tryptophan,
arginine, proline, cysteine, serine, aspartic acid, Tube 1 2 3 4 5 6 7 8 9
#
tyrosine, histidine, glycine, and alanine, protein mL 0 0. 0. 0. 0. 0. 0. 0. 0.
stand 10 15 20 25 30 35 40 45
samples: enzymatic, acid, alkaline hydrolysates, ard
1% Ninhydrin solution spray, 1-Butanol:acetic mL 1. 1. 1. 1. 1. 1. 1. 1. 1.
H2O 50 40 35 30 25 20 15 10 05
acid:water (4:1:5)

The milk sample was diluted to 1:500,

Bradford Assay:
1:1000 and 1:2000 with distilled water. 1.5
mL of the diluted sample was taken and Where the distance traveled by the solvent is
labeled as 10, 11 and 12, respectively. Then, measured as 10 cm.

1.5 mL of Bradford reagent was added to
each tube and was allowed to stand for 5
minutes. The samples were transferred into
individual cuvettes and were inserted to the
spectrophotometer. The absorbance was read
Table 1 shows the computed Rf values of the
samples. It is seen that the acid and alkaline
hydrolysate samples have the Rf values of 0.10
and 0.15, respectively. The enzymatic
hydrolysate did not produce any visible result

at 595 nm. Then, the data was gathered and because glutenin, an insoluble gluten complex,

plotted to the graphing paper. is resistant to enzymatic hydrolysis (Wang et al,

2007). The amino acids that have the closest Rf
values to these are cysteine which has an Rf
value of 0.10 and aspartic acid which has 0.15.
RESULTS AND DISCUSSION
These values mean that these amino acids are
In Paper Chromatography, the following present in the protein, gluten.
results were obtained as shown in Figure 2.
Table 1. Computed Rf values of the samples
Sample/Standar Distance Rf Value
d Traveled
Acid 1.0 cm 0.10
Alkaline 1.5 cm 0.15
Enzymatic - -
Tryptophan 5.0 cm 0.50
Arginine 2.0 cm 0.20
Proline 3.5 cm 0.35
Cysteine 1.0 cm 0.10
Serine 2.0 cm 0.20
Aspartic acid 1.5 cm 0.15
Tyrosine 4.3 cm 0.43
Figure 2. Corresponding spots of the amino acid Histidine 2.2 cm 0.22
Glycine 2.5 cm 0.25
The Rf values of the standard amino Alanine 3.0 cm 0.30
acids and hydrolysates were then computed
.
using the formula: Table 2 shows the data gathered in
Bradford assay after undergoing the UV-Vis
distance ( cm ) m oved by solute
Spectrophotometer.
distance ( cm) moved by solvent
Rf = origin origin
Table 2. Concentration and Absorbance using UV-
Vis Spectrophotometer
Test Tube Concentratio Absorbanc
# n e concentration and V2 is the total volume of the
(595nm)
solution.
blank 0 0
2 3.33 0.030
3 5.00 0.001 Bovine Serum Albumin(BSA) is a
4 6.67 0.012 globular protein of molecular mass 66,210
5 8.33 0.001
consisting of 581 amino acids in a single chain
6 10.0 0.012
7 11.67 0.024 and 17 intramolecular disulfide bridges
8 13.33 0.074 connecting 34 half-cysteines with the formation
9 15.0 0.027
unknown x 0.113 of nine double loops. The protein is relatively
acidic and soluble in water, with an isoelectric
The concentration seen in Table 2 was computed point of 4.7. At pH 7.0, the net charge is -18.
using the formula: The 17 disulfide bridges form 9 loops gouped
into 3 similar domains. it is cigar shaped with
C1 V 1=C 2 V 2
molecular dimension 141 x 42 A. BSA has the
capacity to bind many of the organic compounds
Figure 3. Graph showing the absorbance and
concentration of the samples containing a hydrophobic contribution of at least
five to six methylene groups. However, BSA has
Absorbance using the UV-Vis Spectrophotometer the ability to bind many inorganic anions which
0.08 are very hydrophobic in nature. Fatty acids
0.07 anions are also known to be very tightly bound
0.06
and to exert a stabilizing effect. Therefore, BSA
0.05
0.04 is stabilized with caprylic acid.
0.03
0.02 It seems that BSA is the only one
0.01 protein considered in Paper Chromatography. It
0
is used as an additive in the mobile phase
whereas it is used as immobilized on a solid
The protein concentrations of the
anion exchanger or as an additive in HPLC.
standards were computed using the equation:
BSA is highly soluble in water, which requires
C1 V 1=C 2 V 2 hydrophobic plates as stationary phase.

This absorbance is caused by the shift of

the Coomasie dye under acid conditions
C1 is the Bovine Serum Albumin concentration characterized by red coloration into its blue form
which was 100g/ml, V1 was the volume of the as it binds to the protein. This binding stabilizes
standard protein, C2 is the unknown protein
the blue Coomasie dye and the amount of also easy to determine the desired information,
complex present in the solution is the measure as a linear graph can be drawn from the test
for protein concentration. results.

Figure 3 shows an imperfect line REFERENCES

because of errors made in the experiment.
Aboul-Enein, H.Y, Ali, I. Chiral Separations By
CONCLUSION Liquid Chromatography And Related
Technologies. pp 211
It can be concluded, based on the results
obtained, that cysteine and aspartic acids are David, G.L. Analytical Chemistry. p 86
present in the protein, Gluten.
Kowalska, T., Sherma, J. Thin Layer
Also, by using the Bradford Method, it Chromatography in Chiral Separations
is able to determine the concentration of an and Analysis. p 189
unknown protein. Data collected from the tests,
Walker, J.M. Methods in Molecular biology, Vol.
can be tabulated and graphed, to make
32: Basic Protein and Peptide Protocols
calculating the concentration easier.
Manickam, S.A. Biochemical Methods. p 220
The Bradford Method is a cost and time
effective method that gives reliable results. It is