GLUTEN FROM WHEAT FLOUR: BRADFORD ASSAY AND PAPER CHROMATOGRAPHY

Chryss Yancee G. Oliva, Joel C. Piansay, Magnolia Grace G. Quinto, Marian Angelu C. Ramos, Rose Ann M. Refuela Group 7, 2E Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Gluten that was isolated from wheat flour was used in this experiment for paper chromatography and Bradford Assay. 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. Paper chromatography separates small molecules such as amino acids which is one of the objectives of this experiment. Bradford assay however was used to determine the total protein concentration of the protein which is gluten. Under paper chromatography, 10 standard amino acids were used to compare and determine which amino acid is present in the said protein. Rf values were computed and it resulted to gluten having almost the same Rf values with arginine, cysteine, serine, aspartic acid, histidine, and glycine. Therefore gluten contains the same 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 13.628 g/ml.

INTRODUCTION
Paper chromatography, developed in 1941 by Archer Martin and Richard Synge, played an indispensable role in biochemical analysis due to its ability to efficiently separate small molecules such as amino acids, oligopeptides, nucleotides, and oligonucleotides and its requirement for only the simplest of equipment. The rates of migration of the various substances being separated are governed by their relative solubilities in the polar stationary phase and the nonpolar mobile phase. In a single step of the separation process, a given solute is distributed

between the mobile and stationary phases according to its partition coefficient, an equilibrium constant defined as:

Therefore, the molecules are separated based on their polarities, with the nonpolar molecules moving faster than polar ones. (Voet & Voet, 2011) The migration rate is expressed in ratio:

The Bradford assay is a common procedure for determining microgram quantities of protein. The Coomassie Blue G (Serva Blue G) dye is mixed with phosphoric acid and ethanol and under these conditions has an absorbance maximum of 465 nm. When the dye is mixed with protein, the absorbance maximum of the dye shifts to 595 nm. The protein stabilizes the anionic form of the dye by hydrophobic and ionic interactions by interacting principally with arginine residues, and to a lesser extent histidine, lysine, tyrosine, tryptophan and phenlyalanine residues. (Shand, 2003)

encircled and the Rf value of each molecule was computed. B. Bradford Assay A series of test tubes were prepared as follows:
Tube # mL stand ard mL H2O 1
0 1. 50

2
0. 10 1. 40

3
0. 15 1. 35

4
0. 20 1. 30

5
0. 25 1. 25

6
0. 30 1. 20

7
0. 35 1. 15

8
0. 40 1. 10

9
0. 45 1. 05

Table 1. Preparation of test tubes with the corresponding volumes of standard and water.

MATERIALS AND METHODS Materials

A. Paper Chromatography 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 proteins, 1% Ninhydrin solution spray, 1-Butanol:acetic acid:water (4:1:5). B. Bradford Assay Bradfrod Reagent (Coomasie dye), 100g/mL Bovine Serum Albumin (BSA), evaporated milk, UV-Vis Spectrophotometer

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. The data was gathered and plotted to the graphing paper.

RESULTS AND DISCUSSION Results

A. Paper Chromatography The Rf values of the following was computed using the following formula.

Methods
A. Paper Chromatography A 20x12 filter paper was prepared and drawn with a straight line 1.5 cm from the bottom edge. The 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 Ninhydrin solution and was allowed to get dry. All the observed spots were

Where the distance traveled by the solvent front is 9.5 cm.

Sample/Standard Acid Alkaline Enzymatic Tryptophan Arginine Proline Cysteine Distance Traveled 1.9 cm 1.3 cm 4.7 cm 1.6 cm 3.7 cm 1.5 cm Rf Value 0.2000 0.1368 0.4947 0.1684 0.3895 0.1579

Absorbance

Serine 2.1 cm 0.2211 Aspartic acid 2.0 cm 0.2105 Tyrosine 4.1 cm 0.4316 Histidine 1.7 cm 0.1789 Glycine 1.5 cm 0.1579 Alanine 2.7 cm 0.2842 Table 2. Computed Rf values of the samples and standards.

Absorbance at 595 nm using UV-Vis Spectrophotometer

1.5 1 y = 0.0706x + 0.3265 0.5 0 0 5 10 15 20 Concentration g/ml

Table 2 shows the computed Rf values of the samples namely Acid (0.2000), Alkaline (0.1368) and Enzymatic which did not produce any visible result. It also shows the Rf values of the amino acid standards: tryptophan (0.4947), arginine (0.1684), proline (0.3895), cysteine (0.1579), serine (0.2211), aspartic acid (0.2105), tyrosine (0.4316), histidine (0.1789), glycine (0.1579), alanine (0.2842). B. Bradford Assay The following data were gathered after computing for the concentration and undergoing the UV-Vis Spectrophotometer: Test Tube # 1 2 3 4 5 6 7 8 9 10 (Unknown) Concentration 0 3.33 5.00 6.67 8.33 10.0 11.67 13.3 15.0 x Absorbance (595nm) 0 0.482 0.959 1.039 1.073 1.08 1.13 1.138 1.21 1.28

Figure 1. Graph for the Absorbance and Concentration of the samples. The following procedure was done to compute for the protein concentration of the unknown:

Table 3. Computed concentration and absorbance at 595nm using UV-Vis Spectrophotometer

Table 3 shows the computed concentrations using the data found in Table 1 through following equation: Figure 2. The cuvettes were placed inside the spectrophotometer This table also shows the absorbance at 595 nm using UV-Vis Spectrophotometer.

Figure 3. UV-Vis Spectrophoto meter

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. The samples were transferred to individual cuvettes and were transferred to the UV-Vis Spectrophotometer. The machine was set to 595nm and automatically displays the absorbance values which can be seen on Table 3. This absorbance is caused by the shift of 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. To determine the protein concentration of the unknown sample, the linear regression method was used in the following equation, where y stands for the absorbance, m for the slope, x for the concentration, and b for the y-intercept. After the computation, 13.628 mg/ml was the computed value for the protein concentration of the unknown sample. Figure 1 shows an imperfect line because of errors made in this experiment, however, because of the use of the best-fit line, the figure can show that the graph is close to linear.

Discussion
A. Paper Chromatography As seen on Table 2, the acidic and alkaline hydrolysate samples have the Rf values of 0.2000 and 0.1368, respectively. The enzymatic hydrolysate did not produce any visible result because according to Wang and his colleagues (2007) glutenin, an insoluble gluten complex, is resistant to enzymatic hydrolysis. The amino acids that have the closest Rf values to these are arginine (0.1684), cysteine (0.1579), serine (0.2105), aspartic acid (0.2105), histidine (0.1789), and glycine (0.1579). These values mean that these amino acids are present in the protein gluten. According to Verbeeg and van den Berg (2009), gluten has the following percentage of amino acids: 2.40% arginine, 2.10% cysteine, 5.20% serine, 2.90% aspartic acid, 2.20% histidine, and 3.10% glycine. Glutamic acid having a percentage of 37.30% has the highest percentage composition but was unfortunately not included in the standards used in this experiment.

CONCLUSION
1. Using paper chromatography, the amino acids present in gluten are arginine, cysteine, serine, aspartic acid, histidine, and glycine. 2. The computed protein concentration of the unknown sample is 13.628 g/ml.

B. Bradford Assay Bradford assay was used to determine the protein concentration. The protein concentrations of the standards were computed using the equation:

REFERENCES
Shand, R.F. (2003). Molecular Techniques. Retrieved Jan 1, 2013 http://www4.nau.edu/biology/bio349l/Do cuments/bio349l/Protocols_for_week_4. pdf Verbeek, C. J. R. & van den Berg, L. E. (2009). Recent Developments in Thermo-Mechanical Processing of Proteinous Bioplastics. Recent Patents on Materials Science (2) 171-189 Voet D. & Voet J. (2011). Biochemistry 4th edition. USA: John Wiley & Sons Inc. 144-145 Wang, J. S., et. al. (2007). Characterization of hydrolysates derived from enzymatic hydrolysis of wheat gluten. J Food Sci. 72 (2) 103-7