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
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 separation of the solutes (amino acids) is based on the liquid-liquid partitioning of amino acids in paper chromatography. The partitioning takes place between the water molecule (static phase) absorbed to the cellulosic matter of the paper and the organic (mobile) phase.

The Bradford method, on the other hand, is a rapid and accurate method for the estimation of protein concentration. It is essential in many fields of protein study. This technique is simpler, faster, and more sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to less interference by common reagents and nonprotein components of biological samples. The Bradford assay relies on the binding 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.

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

Figure 1. Structure of Coomassie Brilliant Blue G-250

solvent front was immediately marked with pencil. The paper was air-dried and sprayed with 1% ninhydrin reagent and was allowed to get

MATERIALS AND METHODS Materials

Paper Chromatography:

dry. All the observed spots were encircled and the Rf value of each molecule was computed. Bradford Assay: A series of test tube were prepared as

Filter paper, 1000 mL-beaker, capillary tubes, amino acid standards: 2% w/v tryptophan, arginine, proline, cysteine, serine, aspartic acid, tyrosine, histidine, glycine, and alanine, protein samples: enzymatic, acid, alkaline hydrolysates, 1% Ninhydrin solution spray, 1-Butanol:acetic acid:water (4:1:5)

follows:
Tube #
mL standard mL H2O

1
0

2
0.10

3
0.15

4
0.20

5
0.25

6
0.30

7
0.35

8
0.40

9
0.45

1.50

1.40

1.35

1.30

1.25

1.20

1.15

1.10

1.05

Bradford Assay:

The milk sample was diluted to 1:500, 1:1000 and 1:2000 with distilled water. 1.5

mL of the diluted sample was taken and labeled as 10, 11 and 12, respectively. Then, 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 at 595 nm. Then, the data was gathered and plotted to the graphing paper.

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 because glutenin, an insoluble gluten complex, 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. These values mean that these amino acids are present in the protein, gluten.

RESULTS AND DISCUSSION

In Paper Chromatography, the following results were obtained as shown in Figure 2. Table 1. Computed Rf values of the samples Sample/Standard Distance Rf Value 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 Histidine 2.2 cm 0.22 Glycine 2.5 cm 0.25 Alanine 3.0 cm 0.30

Figure 2. Corresponding spots of the amino acid

.
Table 2 shows the data gathered in Bradford assay after undergoing the UV-Vis Spectrophotometer.

The Rf values of the standard amino acids and hydrolysates were then computed using the formula:

( (

) )

Table 2. Concentration and Absorbance using UVVis Spectrophotometer

Where the distance traveled by the solvent is measured as 10 cm.

Test Tube # blank 2 3

Concentration Absorbance (595nm) 0 0 3.33 0.030 5.00 0.001

4 5 6 7 8 9 unknown

6.67 8.33 10.0 11.67 13.33 15.0 x

0.012 0.001 0.012 0.024 0.074 0.027 0.113

consisting of 581 amino acids in a single chain and 17 intramolecular disulfide bridges connecting 34 half-cysteines with the formation of nine double loops. The protein is relatively acidic and soluble in water, with an isoelectric point of 4.7. At pH 7.0, the net charge is -18.

The concentration seen in Table 2 was computed using the formula:

The 17 disulfide bridges form 9 loops gouped into 3 similar domains. it is cigar shaped with 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 the ability to bind many inorganic anions which are very hydrophobic in nature. Fatty acids anions are also known to be very tightly bound and to exert a stabilizing effect. Therefore, BSA is stabilized with caprylic acid. It seems that BSA is the only one protein considered in Paper Chromatography. It is used as an additive in the mobile phase

Absorbance using the UV-Vis Spectrophotometer

0.08 0.06 0.04 0.02 0

The protein concentrations of the standards were computed using the equation:

whereas it is used as immobilized on a solid anion exchanger or as an additive in HPLC. BSA is highly soluble in water, which requires hydrophobic plates as stationary phase. This absorbance is caused by the shift of

C1 is the Bovine Serum Albumin concentration which was 100g/ml, V1 was the volume of the standard protein, C2 is the unknown protein concentration and V2 is the total volume of the solution. Bovine Serum Albumin(BSA) is a globular protein of molecular mass 66,210

the Coomasie dye under acid conditions characterized by red coloration into its blue form as it binds to the protein. This binding stabilizes the blue Coomasie dye and the amount of complex present in the solution is the measure for protein concentration.

Figure 3 shows an imperfect line because of errors made in the experiment. CONCLUSION It can be concluded, based on the results obtained, that cysteine and aspartic acids are present in the protein, Gluten. Also, by using the Bradford Method, it is able to determine the concentration of an unknown protein. Data collected from the tests, can be tabulated and graphed, to make calculating the concentration easier. The Bradford Method is a cost and time effective method that gives reliable results. It is also easy to determine the desired information, as a linear graph can be drawn from the test results.

REFERENCES
Aboul-Enein, H.Y, Ali, I. Chiral Separations By Liquid Chromatography And Related Technologies. pp 211 David, G.L. Analytical Chemistry. p 86 Kowalska, T., Sherma, J. Thin Layer Chromatography in Chiral Separations and Analysis. p 189 Walker, J.M. Methods in Molecular biology, Vol. 32: Basic Protein and Peptide Protocols Manickam, S.A. Biochemical Methods. p 220