Determination of the Amino Acid Sequence of an Unknown Dipeptide

Transcription

1 Wilson 1 Determination of the Amino Acid Sequence of an Unknown Dipeptide Martin C. Wilson Department of Biology, University of North Carolina - Asheville, Asheville, North Carolina 28804, United States Abstract: mcwilson@unca.edu A dipeptide of unknown amino acid composition was subjected to acid hydrolysis and heat to permit breakage of peptide bonds in the sample resulting in each individual amino acid constituent. Chromatography was then employed with a proper solvent system to compare a set of known amino acid standards against the unknown hydrolyzed sample to determine the two amino acids present in the unknown dipeptide. Ninhydrin was used to visualize the amino acid standards and unknown sample. After determination of amino acid composition in the dipeptide sample, the sample was subjected to N-group analysis to determine the aminoterminal end of the digested dipeptide sample. The dansylation technique was chosen to fluorescently label the amino acid that was present at the amino terminus of the dipeptide. Thin-layer chromatography was then selected using a proper solvent system to compare amino acid standards to the fluorescently-labeled dansylated amino acid derivative present at the amino terminus. UV-transillumination revealed the amino acid present at the N-terminus, and the sequence of the dipeptide was deduced from this information. Introduction: To understand the chemical, structural and functional properties of a certain protein, it is essential to determine the monomeric units of the protein (amino acids) and the order in which these units are assembled (the sequence of amino acids). Although the building blocks of all proteins are the same (20 amino acids), the number of peptides and proteins present in our cells is astonishing. The differences in the composition and sequence of amino acids code for differences in structure and function of the proteins and peptides. The general formula of all amino acids is shown in Figure 1. All amino acids consist of an amino group, a carboxylate group, and an R group. Amino acids vary from one another in the identity of the R group.

2 Wilson 2 H H + N H C C O H R O - Figure 1: General structure of an amino acid with the N-terminus displayed at left, while the C-terminus is depicted at right. When two amino acids come together to make a dipeptide, the carboxylate group of one and the amino group of the other are joined in an amide bond (peptide bond) with release of a molecule of water in a dehydrogenation reaction (Figure 2). Figure 2: Reaction scheme depicting the joining of 2 amino acid constituent molecules resulting if the formation of a peptide bond. A molecule of H 2 0 is released in the process of the reaction from the deprotonation of the N- terminus of one amino acid and the loss of a molecule of O from the other amino acid. The peptide (amide) bond in peptides can be hydrolyzed by strong acids and bases. Since acid hydrolysis is less destructive, it is often used along with a period of heating at high temperatures to completely hydrolyze a protein into the constituent amino acids. The separation and identification of these amino acids are based on chromatographic methods e.g. paper chromatography (PC) and thin-layer chromatography (TLC). Both PC and TLC are used in this experiment. The paper acts as a stationary phase, while the chromatography solvent is the mobile phase. Different amino acids will have different affinities for each phase. Therefore, each amino acid is carried a different distance along the paper. To visualize the amino acids, ninhydrin is sprayed onto the plate and produces pink-purple spots (except for proline which gives yellow spots) each referring to a specific amino acid thus giving insight into the composition of the peptide. Ninhydrin only reacts with free amino groups, so any compounds

3 Wilson 3 that do not contain a free amino group will not be detected by this method. Figure 3 depicts the reaction of ninhydrin with the amino group of amino acids. Figure 3: The reaction of ninhydrin with the free amino group of the amino acid. Two H 2 0 molecules are lost in the formation of a bond with the ninhydrin molecule, and a loss of a proton on the free amino group of the amino acid. Resonance stabilization and loss of C0 2 then result, followed by the loss of another H 2 0 molecule and an aldehyde group. Finally, another molecule of ninhydrin reacts with the originally modified ninhydrin molecule to give the purple-colored product. Dansyl chloride reacts with amino groups according to the following reaction: Figure 4: Reaction of dansyl chloride with the free amino group of the amino acid. The free amino group is deprotonated by reaction with base. Reaction of the peptide with acid then hydrolyzes the dipeptide (in our case) and allows the dansyl chloride to bond to the N-terminal amino acid comprising the dipeptide sequence. The dansylated product is highly fluorescent and is stable to the conditions of acid hydrolysis. The procedure involves labeling the terminal amino groups of the peptide with dansyl chloride, followed by complete hydrolysis of the peptide to release the dansylated terminal amino acid. (All other positions in the peptide simply yield the corresponding free amino acid.) The dansylated amino acid can then be identified by TLC chromatography and a proper solvent

4 Wilson 4 system. UV-transillumination is then employed to visualize the fluorescent dansylated derivates, and the amino acid located at the N-terminus of the dipeptide can be determined by comparison to dansylated amino acid standards that fluoresce under UV light as well. Methods: ~3 mg of the unknown dipeptide was measured on a weight boat. The sample was then transferred to a 1.5 ml centrifuge tube. Hydrolysis was accomplished in 200 µl of 6 N HCl and allowed to hydrolyze for a week. This step of the experiment was performed by Dr. Greg Kormanik, and all experimental procedures were performed at the University of North Carolina at Asheville in Buncombe Co., NC. All other procedures were performed by biochemistry students, with the exception of Dr. Kormanik s preparation and developing chromatograms in certain portions of this experiment. A 20 x 20 cm paper chromatogram was marked 2 cm from the bottom (to mark the line at which all samples would be applied), and the digested dipeptide sample was spotted as a 2-3 mm spot, with a second application spot of the digested sample using ¼ as much liquid. Amino acid standards were spotted on the origin line as well (13 amino acid standards). The chromatogram was stapled into a cylinder, and placed in ~1cm of mobile phase mixture in a developing chamber. A 75:30 mixture of 2-butanol/3.3% NH 4 OH was used as the mobile phase for developing this chromatogram. The paper chromatogram was allowed to develop until the solvent front was ~¾ from the top of the paper. The chromatogram was then removed from the chamber, and the solvent front line was marked lightly with a pencil so that R f values could be calculated for the amino acid standards and unknown spots. The chromatogram was then allowed to air-dry in the hood until no solvent remained, and it was sprayed with a ninhydrincollidine reagent. The paper was then placed in a 90 C oven and heated for ~3 minutes. Amino acids were stained either purple, blue, or yellow (proline) by this procedure. Amino acid composition was then determined for the hydrolyzed peptide by comparison to the stained amino acid standards. It was determined that phenylalanine and alanine were the amino acids present in the unknown dipeptide sample. 2 mg of unhydrolyzed dipeptide was then dissolved in 0.5 ml of 0.2 M NaHCO 3 in a 1.5 ml centrifuge tube. 200 µl of dansyl chloride in acetone solution was added and mixed. The reaction mix was incubated in a 40 C water bath for 1 hour. Dr. Kormanik then evaporated the acetone to dryness under nitrogen gas in an air hood. He then dissolved the formed residue in 0.5 ml of 6 M HCl, and allowed the tube to sit for ~1 week to permit optimum hydrolysis. The following week, the hydrolyzed dansylated dipeptide was evaporated on a watch glass with gentle heat in the flow hood. When only solid remained, the residue was dissolved in 0.5 ml CH 3 OH, and the liquid was again evaporated. The residue was then dissolved in ~0.5 ml CH 3 OH

5 Wilson 5 again, and the liquid was transferred to a micro centrifuge tube, and it was briefly spun (~1 min) to collect a clear supernatant. A silica TLC plate was prepared by marking an origin line ~2 cm from the bottom. A micropipette was then used to spot ~3 µl of dansylated derivative hydrolyzed peptide and ~3 µl each of chosen dansylated amino acids (amino acids chosen that were likely candidates for the N-terminal location of the dipeptide). The plate was developed in a chamber containing 10:1 toluene-glacial acetic acid until the solvent front line was ~¾ from the top of the plate. The solvent front line was then marked, and the plate was allowed to dry in the flow hood in preparation for UV-transillumination. The plate revealed no significant migration of any of the dansylated amino acid standards or the dansylated hydrolyzed peptide derivative. Therefore, Dr. Kormanik developed the TLC plate in a mobile phase of 1.5% formic acid/h 2 0. The newly developed TLC plate was then subjected to UV-transillumination again, and the standards and dansylated peptide derivative exhibited more favorable migration so that an amino acid could be chosen as the candidate for occupying the N-terminus. Results: Figure 5: Paper chromatogram depicting 2 applications of unknown hydrolyzed dipeptide at left, with 13 amino acid standards spotted as well, with the proline standard appearing yellow after ninhydrin staining. The chromatogram was developed in a 75:30 mixture of 2-butanol/3.3% NH 4 OH. The unknown dipeptide was determined to contain phenylalanine and alanine as the amino acid constituents from comparison with the amino acid standards. R f values were calculated for the unknown dipeptide sample and amino acid standards as well.

6 Wilson 6 Amino Acid Standards/Unknown Dipeptide Sample R f Values Color Unknown , Dark Purple Unknown , Gray Ala (A) Dark Purple Arg (R) 0.12 Light Purple Asn (N) Dark Purple Glu (E) Light Purple Gly (G) Light Purple Ile (I) 0.36 Dark Purple Leu (L) 0.36 Dark Purple Phe (F) 0.39 Gray Ser (S) Dark Purple Trp (W) Dark Brown Purple Tyr (Y) Dark Purple Val (V) Purple Pro (P) Yellow Table 1: Depiction of calculated R f values along with colors of the amino acid standards observed after application of the ninhydrin-collidine reagent. The 13 amino acids chosen for comparison to the unknown dipeptide sample are shown in three-letter code, but in the photograph, the one-letter code is chosen to display the identity of the amino acid. R f values most similar to the amino acids contained in the dipeptide are Ala (0.107 as compared to 0.093) and Phe (0.390 as compared to 0.253). Note the high difference in Rf values for Phe and the unknown dipeptide. This is most likely due to the solvent front line being inconsistent. Figure 6: TLC plate depicting dansylated derivatives of the hydrolyzed dipeptide and amino acid standards. The plate was developed in a chamber containing 10:1 toluene-glacial acetic acid mobile phase. Migration of the dansylated derivatives was minimal using this solvent system (except for isoleucine at far right), and no information could be ascertained about which amino acid was present at the N-terminus of the dipeptide.

7 Phenylalanine Alanine Wilson 7 Isoleucine Unknown dipeptide sample Figure 7: TLC plate depicting dansylated derivatives of the hydrolyzed dipeptide and amino acid standards in a 1.5% formic acid/h 2 0 mobile phase. Resolution was increased with a more polar solvent system, yet the amino acid that was present at the N-terminus of the dipeptide was difficult to identify, as the phenylalanine and alanine dansylated derivatives migrated much farther than the danyslated hydrolyzed dipeptide sample. (Note: the TLC plate was photographed with the glass plate side up, so the positions of the dansylated derivatives are inverted, so isoleucine is located at far left, and the dansylated dipeptide is located at far right). Conclusion: Chromatography and analysis of the N-terminus of the unknown dipeptide sample using dansylation suggest that phenylalanine and alanine are the amino acids composing the dipeptide sample. The paper chromatogram spotted with the hydrolyzed dipeptide sample and 13 amino acid standards revealed that phenylalanine and alanine were the most likely candidates that were consistent with the migration distance and color of the amino acid standards after being treated with a ninhydrin-collidine reagent. However, errors could have existed at this stage of the experiment. When developing the paper chromatogram in a developing chamber, a portion of the mobile phase was elevated with respect to the rest of the chromatogram. Capillary action resulted in the mobile phase being higher than at the remainder of the chromatogram where the amino acid standard glycine was located, and this is evident in the depicted photograph of the chromatogram, as Gly is much more smeared as compared to the other amino acid standards. Although it does not appear that the development of the Ala and Trp standards was not affected, it is still important to note that this incident occurred. Also, the chromatogram should have ideally been developed for ~5-7 hours, while our chromatogram was only developed for ~3 hours. A longer development would have meant more accurate R f values and assumptions for which amino acids comprised the unknown dipeptide sample.

8 Wilson 8 With respect to N-terminal analysis of the unknown dipeptide sample, when the last evaporation step with CH 3 OH was being conducted, only 0.2 ml of CH 3 OH should have been added so that the dipeptide would not become too diluted in solution. However, it was not possible for us to transfer the dissolved dansylated dipeptide derivative quickly enough without evaporation of CH 3 OH occurring, so an excess of CH 3 OH (~4 ml) was added to the watch glass, and this aliquot was collected in a microcentrifuge tube and was allowed to evaporate readily with the cap off until the desired volume of 0.2 ml was reached. This may have resulted in a sample that was too dilute, and this also explains why the unknown dansylated derivative was a much fainter band when performing TLC as compared to the dansylated amino acid standards. Ideally, when the first TLC plate was developed in a 10:1 toluene-glacial acetic acid mobile phase, development should have taken a much longer amount of time to accomplish. The solvent front line was a little under ¾ from the top of the chromatogram, and due to time constraints, the TLC was prevented from developing any further and photographed under UVtransillumination. Furthermore, this solvent system was not an ideal mobile phase to elucidate whether phenylalanine and alanine were present in the dipeptide sample, as a 10:1 tolueneglacial acetic acid mobile phase is fairly polar, with phenylalanine and alanine being readily nonpolar amino acids. A 2 nd TLC development was conducted by Dr. Kormanik in a 1.5% formic acid/h 2 0 mobile phase. Although better resolution and separation of the dansylated derivatives of both the standards and unknown sample were achieved, no conclusive evidence was found as to whether phenylalanine or alanine occupied the N-terminus of the dipeptide. However, based upon comparisons of ninhydrin staining on the paper chromatogram and from another group s data based upon evidence from their dansylated derivative TLC photograph, we feel confident that phenylalanine and alanine are the two amino acids present in this dipeptide, but there is inconclusive evidence as to which amino acid resides at the amino terminus. Nonetheless, this experiment has proven that analysis of protein sequence is a delicate science, and the utmost care, time, and devotion must be given to accurately and faithfully devise solvent systems and conditions that are most favorable to analyze the protein of interest. These techniques will help guide the elucidation of structural relatedness of the serum albumin protein from different species in the next laboratory report.

9 Wilson 9 References: "Concept 5 Review: The Peptide Bond." Pearson Education, Inc.Web. 11 Feb < "Dipeptide Lab Protocol." George Mason University, 17 May Web. 11 Feb <mason.gmu.edu/~jbougie1/teaching/.../dipeptide_lab_protocol.doc>. Kormanik, G Biochemistry Laboratory Handout. "Lecture 5: Peptides." State University of New York College of Environmental Science and Forestry. Web. 11 Feb < Lecture 05.ppt>. Reusch, William. "Proteins, Peptides, and Amino Acids." Michigan State University. Web. 11 Feb <