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SEPARATION AND IDENTIFICATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY AND PROTEIN ASSAY USING THE BRADFORD METHOD

Chrisel Lubi, Wynzi Mallen, Jason Montesa, Rizziel Nemes, Peter Nocon Group 6, 2E Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT Proteins are heavy molecularly weighted molecules comprised by amino acid chains; humans need proteins to be able to sustain body functions properly. Myoglobin is an iron and oxygenbinding protein that was isolated from beef and was used in this experiment. The objectives of this experiment are (1) to determine and separate each amino acid present in the protein by paper chromatography. (2) To determine quantitatively the total protein concentration of a given sample thru Bradford Assay. The separation of each amino acids was accomplished by plotting ten standard amino acids that were used as a control variable to be compared with the acid, basic, and enzymatic hydrolysates of myoglobin. Bradford assay was the method used to determine the protein concentration of one to nine labeled test tubes with incrementing amount of concentration of diluted Bovine Serum Albumin (BSA). Another set of test tubes were labeled ten to twelve and were filled with a constant volume of milk sample diluted with different volumes of water. The absorbance of each test tube with different concentration of the samples was read at an absorbance of 595nm. binding to protein, the unprotonated, blue form becomes the most stable (Walker & Wilson, 2005). The absorbance of each test tube were obtained thru the utilization of a spectrophotometer. A Spectrophotometer is an instrument used to measure the light absorbed by a substance. These are used in colorimetric assays (spectrophotometry) to measure the amount of light transmitted through a sample at a given wavelength.

INTRODUCTION Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material. Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. (University of Wisconsin System, 2013) The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase. The Bradford dye assay is based on the equilibrium between three forms of Coomassie Blue G dye. Under strongly acid conditions, it is most stable as a doubly-protonated red dye form. Upon

Figure 1: Structure of Coomassie Brilliant Blue G-250 Dye

Once the absorbance of each test tube were obtained, a calibration curve was constructed. The Bradford Method eliminates some of the limitations in quantitation and protein concentration

determination as provided by the other methods of determining protein concentration. This method required the utilization of the Coomassie Brilliant Blue dye to bind to the protein. The binding of Coomassie Brilliant Blue dye to protein in acidic solution causes a shift in wavelength of maximum absorption of the dye from 465nm to 595nm and the absorbance at 595nm are directly related to the concentration of protein. The dye has an absorption of 470 and 650nm at a low pH. Though the Bradford Method has a number of advantages, one of its disadvantages is that it is sensitive to about 20 to 200g of protein. (Walker & Wilson, 2005) MATERIALS AND PROCEDURE Materials A. Paper Chromatography: Filter paper, Chromatography chamber(1000mL beaker covered with a watch glass), capillary tubes, amino acid standards: 2% w/v tryptophan, arginine, proline, cysteine, serine, aspartic acid, tyrosine, histidine, glycine, and alanine, acid, basic, and enzymatic hydrolysates of myoglobin, 1% Ninhydrin solution spray, 1Butanol:acetic acid: water (4:1:5). B. Bradford Assay Bradford reagent (Coomassie Brilliant Blue dye), Bovine Serum Albumin (BSA) standard (100 g/mL), evaporated milk sample, UVVIS Spectrophotometer Methods Paper Chromatography A 20x12 cm filter paper or chromatogram was first prepared, then a straight line was drawn 1.5 cm margin from the bottom of the longer edge of the filter paper. Then the chromatogram was marked with 13 equidistant points on the line that will serve as the spot of the amino acid standards. The standards were applied using the capillary tubes 5 times and while the hydrolysate samples were applied 10 times. The chromatogram was shaped into a cylinder and its edges were stapled before it was placed in the pre-equilibrated chamber and

was covered. The chromatogram was removed when the solvent front was approximately 1 cm from the top edge. The solvent front was immediately marked with pencil. After marking the solvent from the chromatogram was air-dried and sprayed with 1% Ninhydrin solution under a fume hood and was dried over the hotplate for 1-3 minutes. The appearance of colored spots indicating the presence of amino acids were encircled and the chromatogram was analyzed. Bradford Assay Nine test tubes were prepared and labeled each from test tube one to nine. The nine test tubes were prepared as follows:

Test Tube # 1 2 3 4 5 6 7 8 9

mL standard

0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

mL water 1.50 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05

Table 1. Bovine Serum Albumin

Another set of test tubes labeled from test tube 10 to 12 contained the milk samples which were diluted with distilled water in ratios of 1:500, 1:1000, and 1;2000. Each test tube was added with 1.5mL of Bradford Reagent, then each test tube were mixed well and was let to stand for 5 minutes. The samples were transferred into individual cuvettes and were inserted into the spectrophotometer. The absorbance was read at 595 nm. The data was gathered and plotted to the graphing paper. RESULTS AND DISCUSSION Results Paper chromatography

The results obtained from the separation of amino acids by paper chromatography was processed by marking the spots that appeared on the chromatogram.

2.47 0.26 Enz. Hyd. 8.55 0.9 Table 2. Distance travelled by the compounds and their computed Rf values.

As seen on table 2 the distance travelled by the standard amino acids are relatively close hence the values of the retention factors are also relatively near each other. Among the hydrolysates observed the basic hydrolysate was observed to have produce two spots while both the acid and enzymatic hydrolysates have produced only one spot. Using the Rf values of the standard amino acids the Rf values of the hydrolysates were compared that of the standards to determine which amino acids were separated. Bradford Method The Protein assay by using the Bradford method read the following absorbance measurements after utilizing the spectrophotometer. Test tube # 1 2 3 4 5 6 7 8 9 10 Concentration 0 3.33 5.00 6.67 8.33 10.00 11.67 13.33 15.00 x A 0 0.041 0.006 0.005 0.001 0.005 0.069 0.079 0.113 1.28

Figure 2: The Chromatogram with some of the spots still visible.

The values of the retention factor were calculated after. The retention factor has the following formula:
=

Figure 3: The equation of the retention factor which also defines it.

Table 3. Concentration and Absorbance (A) at 595nm using the UV-VIS spectrophotometer.

The distance travelled by the solvent is measured to be 9.5cm. The measured distance travelled by the standards and hydrolysates and computed Rf values are shown in table 2.
Compounds
Tryptophan Arginine Proline Cysteine Serine Aspartic acid Tyrosine Histidine Glycine Alanine Acid Hyd. Basic Hyd. Distance Travelled (cm) 4.94 1.99 3.42 1.05 1.99 1.99 4.00 1.52 1.52 3.04 6.17 5.03 Rf value 0.53 0.21 0.36 0.11 0.21 0.21 0.42 0.16 0.16 0.32 0.65 0.53

As seen on table 3 the concentration were computed through the following formula:
1 1 = 2 2

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.

Absorbance, 595nm

0.12 0.1 0.08 0.06 0.04 0.02 0 0 5 10 15 20

Concentration g/mL
Figure 4: Graph of absorbance versus concentration with the Best fit line.

primarily to measure the absorbance of the standard Bovine Serum Albumin and an unknown concentration. The instrument uses light that is visible and adjacent (near-UV and near-infrared (NIR)) ranges as shown in Figure 5. The absorption in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. (Bradford, 2000)

The unknown (x) concentration was calculated with the slope-intercept form: = +
Where:

= = = = = =

Figure 5: Schematic Diagram of a spectrophotometer.

1.280 0.326 0.070

= 13.628/ The protein concentration of the unknown sample was determined by using the linear regression method. Where y stands for the absorbance, m for the slope, x for the concentration, and b for the y-intercept. The computation yielded 13.628 mg/ml as the value for the protein concentration of the unknown sample. Figure 3 shows an imperfect line because of errors made in this experiment, however, because of the use of the best-fit line, the figure can relatively show that the graph is close to linear. Discussion The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. The Ultraviolet-Visible spectrophotometer (UV-VIS) was used

Light absorbance is based on the capacity of a solution to absorb radiant energy. If the radiant energy of intensity Io is allowed to pass through a solution placed in a transparent tube, the solution absorbs a portion of this radiant energy. The unabsorbed radiant energy is transmitted. If I is the intensity of the transmitted energy, I/I0 is the transmittance T. Though it is the transmittance that is detected by a spectrophotometer, it is the absorbance that has a linear relationship with the concentration of the absorbing species in the analyte solution. Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength it was based on BeerLamberts Law which relate the absorbance of a solution to the concentration of a particular solute to in that solution (NIST, 2009).
= log = log 0 = log 0

Figure 6: Beer-Lamberts Law equation.

Where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity. In paper chromatography, the sample mixture is applied by spotting little amounts

of the sample with equidistance places to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action, in which the polar solvent is attracted to the polar paper, and moves upward as a result until the evaporation halts the movement. The paper is composed of cellulose, a strongly polar polymer to which polar molecules (such as water) are attracted, while the solvent is less polar, usually consisting of a mixture of water and another, lesspolar liquid. The paper is called the stationary phase while the solvent is referred to as the mobile phase. A three step process is involved in a chromatographic experiment, first is application of the sample, second is by allowing the mobile phase to move up the paper thus "developing" the chromatogram, and third obtaining the quantitative measures by calculating Rf (Retention factor) values for each separated component in the developed chromatogram and making conclusions. The Rf value for a substance is dependent on the polarity of the specific substance, so the Rf value can be used to roughly identify the substance. The Rf value is a number that is defined as seen on figure 3. (Shakashiri, 2012) CONCLUSION (1) It is therefore concluded thru the experiment that tryptophan, serine, and cysteine were the amino acids determined as some of the component of myoglobin by using the paper chromatography. (2) It is therefore concluded that by the using the Bradford Method the group was able to determine the concentration of a given unknown sample which was calculated to be 13.628g/mL.

References Bradford, M. M. (2000). Analytical Biochemistry. A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. In M. Bradford, Analytical Biochemistry (pp. 72:248-254). Lowry, O., Rosebrough, A., Farr, A., & Randall, R. (1951). Biological Chemistry. NIST. (2009, November 13). The National Institute of Standards and Technology. Retrieved from http://www.nist.gov/pml/div685/grp03 /spectrophotometry.cfm Shakashiri, B. (2012). Paper Chromatography. Retrieved from http://scifun.chem.wisc.edu/ Smith, P. e. (1985). Analytical Biochemistry. Stoscheck, C. (1990). Quantification of Protein. Methods in Enzymology, 182:50-68. University of Wisconsin System. (2013, December 30). Paper Chromatography. Retrieved from Department of Chemistry: http://chem.wisc.edu/deptfiles/gench em/lab/labdocs/modules/paprchrom/ paprchromdesc.htm Walker, J., & Wilson, K. (2005). Principles and Techniques of Biochemistry and Molecular Biology, 6th Edition. New York: Cambridge University Press, Cambridge.