In the Laboratory
Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC
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Janice S. Barton,* Chung-Fei Tang, and Steven S. Reed Department of Chemistry, Washburn University, Topeka, KS 66621; *
[email protected] Rationale Amino acid composition and sequence determination represent important biochemical techniques for characterizing peptides and proteins. Prediction of protein secondary and tertiary structure (1–3) relies on accurate sequence information. Database comparisons for sequence alignment reveal sequence homologies and are instrumental in establishing evolutionary and functional relationships and for identifying motifs or supersecondary structure (1–3). Although automated Edman degradation and DNA sequence determination are prominent techniques for establishing the sequence of proteins, manual methods can enhance students’ understanding of the principles and techniques of sequence analysis. Thus, biochemical laboratory textbooks often include experiments for determination of the composition (4 ), N-terminal residue (5), or sequence of a dipeptide (6–8). Historically, these textbook experiments employed combinations of paper and thin-layer chromatography detection procedures, which have limited resolution. A composition determination procedure that involves separating fluorenylmethyl chloroformate derivatives by HPLC (9) and an amino terminal analysis of PTH (phenylthiohydantoin) amino acids by HPLC (7 ) adapt newer technologies to instructional laboratories. PTC (phenylthiocarbamyl) derivatives of amino acids can be used to define the composition of amino acid mixtures, and their resolution by HPLC (10, 11) drastically reduces the time required for amino acid analysis. Apparently, however, these PTC methods have not yet been incorporated into the laboratory curriculum. Most PTC-amino acids are negatively charged owing to the free α-carboxyl group; basic amino acids are the possible exception. In contrast, any charge on the PTH–amino acid would arise from the side chain. Consequently, the experimental conditions for separation of PTC– and PTH–amino acids by HPLC differ. Use of two HPLC protocols to determine the sequence of a dipeptide poses a logistics problem for use in biochemistry instructional laboratories. Instead, it is desirable to have one HPLC protocol for both composition and sequence analysis. Since PTC derivatives are converted into the PTH form during the Edman degradation to identify the N-terminal amino acid, it seemed prudent to employ PTH derivatives for composition determination as well. The experimental techniques presented in this communication use PTH derivatives and HPLC to determine both the composition and sequence of dipeptides. The method can also be used solely for amino terminal analysis. From this instructional procedure, students learn some principles and practical application of HPLC as well as a major synthetic method for peptide and protein analysis. The protocol for formation of PTH derivatives is simplified and replaces aqueous pyridine with ethanol and triethylamine. In this conversion process, the more common concentrated hydrochloric acid (HCl) is
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substituted for trifluoroacetic acid (TFA). Serine and threonine may serve as components of the unknown dipeptide when concentrated HCl replaces the dehydrating TFA. Formation of PTH derivatives in both the N-terminal and composition steps of sequence determination allows use of a single solvent system for reverse phase HPLC analysis. Methods This experiment involves N-terminal analysis by the Edman method and hydrolysis of a dipeptide with subsequent determination of dipeptide composition with a modified Edman procedure. Determination of the N-terminal residue using the Edman degradation procedure may be characterized by the steps of coupling, washing, cleavage, extraction, and conversion (12, 13). Subsequent to hydrolysis, peptide composition determination requires coupling, washing, and conversion. Student pairs can accomplish both procedures (synthesis of the N-terminal and hydrolyzed dipeptide PTH derivatives) in one 3-hour period. This time compaction is best achieved if each partner performs one of the two tasks, N-terminal or composition analyses, and the students have previously performed hydrolysis of the dipeptide used for composition analysis. The PTH–amino acids are analyzed by reverse phase HPLC on a C18 column with a one-step gradient elution procedure of 20-minute duration. Inexpensive columns are sufficient for simultaneous resolution of derivatives of aspartate, serine, glycine, alanine, proline (or valine), leucine, and phenylalanine (or lysine) using a single-step gradient elution process. Isocratic methods, with which we have no experience, are available (14 ). This procedure is similar to other HPLC protocols for PTC– and PTH–amino acids in that it requires maintenance of the column temperature near 50 °C. The temperature is maintained by an immersion circulator with thermostat that pumps heated water through a column jacket. The column jacket is a simple, inexpensive device (insulated copper tubing wound to fit around the column) that works well, giving good retention time precision. Results Dipeptide samples for students were selected for a combination of readily resolvable PTH–amino acids. These dipeptide combinations were composed of amino acids with side chains (aliphatic, acidic, basic, polar uncharged, and aromatic) of differing polarity. For chromatographic analysis, students were given the individual known PTH–amino acids and were expected to obtain a chromatogram for each known, their amino-terminal derivative, and the mixture of PTH derivatives generated from the hydrolyzed dipeptide. Identification of the N-terminal amino acid and the composition of the dipeptide were made from observed retention times. Table 1
Journal of Chemical Education • Vol. 77 No. 2 February 2000 • JChemEd.chem.wisc.edu
In the Laboratory
contains retention times for a complex mixture of PTH– amino acids separated on an PTH–Amino Acid Time/min Alltech Absorbosphere C18 Alanine 5.76 HPLC column. The value of Aspartate 2.41 standard deviations for tripliGlycine 4.38 cate retention times is ±0.07 Leucine 12.29 minutes or less. Lysine (ε-PTC) 11.87 As described here, the Norleucine 12.62 HPLC experiment requires Phenylalanine 11.63 one laboratory period for each partnership. The time requireProline 9.89 ment can be reduced by givSerine 3.60 ing the students the mixture Valine 9.68 of known PTH–amino acids Note: An Absorbosphere C18, and identification of the elu250 × 4.6-mm column was used. Norleucine may be used as an tion order. Each partnership internal standard. can then obtain chromatograms for the standard mixture, the N-terminal derivative, and the hydrolyzed dipeptide in about an hour. Two to three groups can operate the HPLC in a 3- to 4-hour period. For laboratory sessions with large enrollments, the number of students in a group can be increased or use of the HPLC can be scheduled outside of the class period. This experiment, developed over a period of three to five years, has been highly successful. Dipeptide combinations of amino acids differing in retention times by about 1 minute were successfully identified by students enrolled in an upperdivision biochemistry laboratory course with a prerequisite of one semester of organic chemistry laboratory. Table 1. Retention Times for PTH–Amino Acids
Chemicals and Equipment Needed Phenylisothiocyanate suitable for protein sequencing was obtained from Aldrich. Dipeptides and PTH–amino acids were obtained from Sigma. The acetonitrile should be HPLC grade. Nylon 66 filters with 0.45-µm pore size should be used to filter samples (4-mm diam) and solvents (45-mm diam). A rotatory evaporator and a variable micropipet (100–1000 µL) are very useful performance-enhancing items. The HPLC, equipped with a UV detector set at 254 nm, must have two pumps and the ability to deliver a solvent gradient either internally or by computer. An inexpensive C18 Econosphere
5-µm particle column (250 × 4.6 mm) may be purchased from Alltech. An immersion circulator with thermostat and a column water jacket are required for HPLC. Acknowledgments Partial support for this work has been provided by the National Science Foundation’s College Science Instrumentation Program, grant #CSI-855 8650399, and a research grant from Washburn University. WSupplemental
Material
The complete description of this experiment and supplemental material are available in this issue of JCE Online. Literature Cited 1. Fasman, G. D. In Prediction of Protein Structure and the Principles of Protein Conformation; Fasman, G. D., Ed.; Plenum: New York, 1990; pp 193–301. 2. Ghelis, C.; Yon, J. Protein Folding; Academic: New York, 1982. 3. Pascarella, S.; Colosimo, A.; Bossa, F. In Laboratory Methodology in Biochemistry: Amino Acid Analysis and Protein Sequencing, Fini, C.; Floridi, A.; Finelli, V. N.; Wittman-Liebold, B., Eds.; CRC Press: Boca Raton, FL, 1990; pp 109–128. 4. Crandall, G. D. Biochemistry Laboratory; Oxford University Press: New York, 1983. 5. Rendina, G. Experimental Methods in Modern Biochemistry; Saunders: Philadelphia, PA, 1971. 6. Clark, J. M. Jr.; Switzer, R. L. Experimental Biochemistry, 2nd ed.; Freeman: New York, 1977. 7. Boyer, R. F. Modern Experimental Biochemistry, 2nd ed.; Benjamin/Cummings: Redwood City, CA, 1993. 8. Rogers, P. W. J. Chem. Educ. 1996, 73, 189. 9. Clapp, C. H.; Swan, J. S. J. Chem. Educ. 1992, 69, A122– A126. 10. Bidlingmeyer, B. A.; Cohen, S. A.; Tarvin, T. L. J. Chromatogr. 1984, 336, 93–104. 11. Ebert, R. F. Anal. Biochem. 1986, 154, 431–435. 12. Schroeder, W. A. Methods Enzymol. 1967, 11, 445–461. 13. Robyt, J. F.; White, B. J. Biochemical Techniques, Theory and Practice; Waveland: Prospect Heights, IL, 1990; pp 347–349. 14. Hayakawa, K.; Oisumi, J. J. Chromatogr. 1989, 487, 161–166.
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