SEPARATION AND IDENTIFICATION OF MYOGLOBIN IN ENZYMATIC HYDROLYSATE BY THIN-LAYER CHROMATOGRAPHY

Mariah Sharmane Santos, Denisa Louise Serranilla, Anna Patricia B. Tan, Gabriella Yna V. Villanueva, Camille Abigail Q. Vivo and Jarena Ria Z. Zolina Group 9 2A Pharmacy Biochemistry Laboratory

ABSTRACT
Thin-layer chromatography was conducted to be able to analyze the amino acid constituents of the enzymatic hydrolysate of Myoglobin based on the polarities of Proline, Alanine, Cysteine, Histidine, and Aspartic Acid. A 1-cm margin had been drawn across the longer bottom edge of the TLC plate. Eight equidistant points were plotted along the line where the given amino acids and protein hydrolysate samples had been applied. The TLC plate was then introduced in the solvent system where water served as the stationary phase while 1-Butanol served as the mobile phase for non-polar constituents and acetic acid as the mobile phase for polar constituents. Because of the strong polarity of the stationary phase, polar components of the samples had been attracted and had a greater affinity with the stationary phase while non-polar components had been carried through the mobile phase and had a greater affinity with it. The Rf values for each sample was computed and the amino acid constituents of the enzymatic hyrolysate of Myoglobin had been identified and these are Histidine and Aspartic Acid.

INTRODUCTION
Protein is a macromolecule formed by polymerization of amino acids. It is a primary constituent of living things where it provides most of the molecular machinery of cells. Amino acids are the subunits of protein where they are linked by peptide bonds to form a polypeptide chain. Each amino acid contains an amino group and a carboxyl group, both of which are bonded to the carbon. The -carbon is also bonded to a hydrogen and a side chain group (R-group). The R-group determines the identity of the particular amino acid. Amino acids are designated into two stereoisomers and these are the L- and D-amino acids. The amino acids on proteins are all of the L form. Myoglobin is an ironand oxygenbinding protein present in large concentrations in the muscle tissues. It forms pigments which is responsible for the red color of the organs. It is considered to be a monomeric hemeprotein and does not exhibit cooperative binding of oxygen, since positive cooperativity is a property of oligomeric proteins only. Instead, the binding of oxygen by myoglobin is unaffected by the oxygen pressure in the surrounding tissue. Myoglobin is often cited as having an "instant binding tenacity" to oxygen given its hyperbolic oxygen dissociation curve. High concentrations of Myoglobin in muscle cells allow organisms to hold their breaths longer. Amino acid components of Myoglobin had been characterized qualitatively by thin-layer chromatography. Thin-Layer Chromatography (TLC) is a simple and inexpensive technique that is often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction. As the solvent migrates along the TLC plate, each amino acid present is then carried out and are separated based on their polarities.

EXPERIMENTAL
A. Compounds Used y Amino Acid Standards: Proline, Alanine, Cysteine, Histidine, and Aspartic Acid y Acid, Basic, and Enzymatic Myoglobin hydrolysates y 1-Butanol:acetic acid: water (4:1:5) y 1% Ninhydrin solution in spray bottle y TLC Plate y Chromatography Chamber (1-L beaker covered with a watch glass) y Capillary tubes B. Procedure To begin the thin-layer chromatography set-up, a 12 x 15 cm TLC plate had been prepared where the origin had been drawn across the plate with a 1-cm margin from the bottom of the longer edge of the plate. Eight equidistant points had been marked on the line for the spotting of the given amino acids and hydrolysate samples. Using a capillary tube, the hydrolysate samples had been applied ten times while the amino acid standards were applied five times and had been air-dried in between applications. The TLC plate was then rolled into a cylinder and then stapled it where it would stay put inside the pre-equilibrated chamber containing 1Butanol, acetic acid, and water in a 4:1:5 ratio solvent system. The chamber had been covered and allowed the solvent to ascend undisturbed. The plate was then removed when the solvent front is approximately 0.5-cm from the top edge of the plate. The solvent front had been marked with a pencil line to be able to calculate the Rf values of The Rf values of the amino acid standards and the

the samples. The chromatogram had been air-dried and had been lightly sprayed with 1% Ninhydrin reagent. The chromatogram was then heated a few inches above a hot plate until colored spots was seen. After heating, all spots were encircled with a pencil and the Rf values of each sample was then computed.

enzymatic hydrolysate of Myoglobin listed in Table 1 indicated what type of amino acid is present in the sample. Table 1. Rf Values of the Amino Acid Standards and Enzymatic Hydrolysate of Myoglobin
Amino Acid Standards Pro Ala Cys His Asp Rf Values of the Spots Enzymatic Standards Hydrolysate 0.40 0.34 0.34 0.18 0.23 0.22 0.23

RESULTS AND DISCUSSION

Determination of the amino acid components of Myoglobin had been made possible with the use of thin-layer chromatography where the results are based on the polarities of the given amino acids. Amino acids contain an amino group and a carboxyl group, both of which are bonded to the carbon. The -carbon is also bonded to a hydrogen and a side chain group (R-group). The R-group determines the identity of the particular amino acid whether it is non-polar, neutral polar, acidic, and basic. The interaction of Proline, Alanine, Cysteine, Histidine, and Aspartic Acid with the solvent system is due to their R-group. To evaluate the samples, the Rf value is calculated with this equation:        

The identity of the amino acids present in the enzymatic hydrolysate of Myoglobin was determined by comparing the Rf value of the unknown component with that of the standard amino acids. Histidine and Aspartic Acid were the only separated and identified amino acid components of the enzymatic hydrolysate of Myoglobin since it has the closest Rf value to the both of it.

REFERENCES
Campbell, M.K., Farrell, S.O. (2009). Biochemistry (International Edition). 7th ed. Canada: Brooks/Cole, Cengage Learning. Clark, J. Understanding Chemistry: Amino Acids and Other Biochemistry Menu. http://www.chemguide.co.uk/organicprops/aminoac idmenu.html 1/15/2012 King, M.W. The Medical Biochemistry Page: Hemoglobin. http://themedicalbiochemistrypage.org/hemoglobinmyoglobin.html 1/15/2012 Kyrk, J. Amino Acids. http://www.johnkyrk.com/aminoacid.swf 1/15/2012

Based on observation, amino acids with a nonpolar R-group like Proline and Alanine would basically yield a higher Rf value which interprets that it has a greater affinity with the mobile phase that is why it would appear higher in the chromatogram. On the other hand, neutral polar amino acids including Cysteine would have a lesser Rf value and would then have a lesser affinity with the mobile phase. Lastly, acidic and basic amino acids like Histidine and Aspartic Acid would have the least Rf value and would strongly interact with the strongly polar stationary phase that is why it would appear close to it. Because amino acids would normally appear colorless, a 1% Ninhydrin reagent was lightly sprayed on to the chromatogram and was heated to be able to detect it. Although the Ninhydrin reagent resulted to purple colored spots because of its reaction with -amino acids, the only imino acid Proline gave a yellow colored spot because it is a secondary amine.

Thin Layer Chromatography.

http://www.wpi.edu/Academics/Depts/Chemistry/C ourses/General/tlc.html 1/15/2012

Pro

Ala

Cys

His

Asp

Figure 1. Chromatogram