Abstract
Precolumn derivatization of amino acids followed by their resolution by reverse-phase high-performance liquid chromatography (RP-HPLC) is now the preferred method for quantitative amino acid analysis. The derivatization step introduces covalently bound chromophores necessary not only for interactions with the apolar stationary phase for high resolution, but also for photometric or fluorometric detection.
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References
Marfey P. (1984) Determination of D-amino acids. II. Use of a bifunctional reagent, 1, 5-difluoro-2, 4-dinitrobenzene. Carlsberg Res. Commun. 49, 591–596.
Kochhar S. and Christen P. (1989) Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2, 4-dinitrophenyl-5-L-ala-nine amide. Anal. Biochem. 178, 17–21.
Kochhar S. and Christen P. (1988) The enantiomeric error frequency of aspartate aminotransferase. Eur. J. Biochem. 175, 433–438.
Szókán G., Mezö G., and Hudecz F. (1988) Application of Marfey’s reagent in racemization studies of amino acids andpeptides. J. Chromatog. 444, 115–122.
Martínez del Pozo A., Merola M., Ueno H., Manning J. M., Tanizawa K., Nishimura K., et al. (1989) Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase. J. Biol. Chem. 264, 17784–17789.
Szókán G., Mezö G., Hudecz F., Majer Z., Schön I., Nyéki O., et al. (1989) Racemization analyses of peptides and amino acid derivatives by chromatography with pre-column derivatization. J. Liq. Chromatogr. 12, 2855–2875.
Adamson J. G., Hoang T., Crivici A., and Lajoie G. A. (1992) Use of Marfey’s reagent to quantitate racemization upon anchoring of amino acids to solid supports for peptide synthesis. Anal. Biochem. 202, 210–214.
Goodlett D. R., Abuaf P. A., Savage P. A., Kowalski K. A., Mukherjee T. K., Tolan J. W., et al. (1995) Peptide chiral purity determination: hydrolysis in deuter-ated acid derivatization with Marfey’s reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry. J. Chromatog. A707, 233–244.
Graber R., Kasper P., Malashkevich V. N., Sandmeier E., Berger P., Gehring H., et al. (1995) Changing the reaction specificity of a pyridoxal-5′-phosphate-dependent enzyme. Eur. J. Biochem. 232, 686–690.
Vacca R. A., Giannattasio S., Graber R., Sandmeier E., Marra E., and Christen P. (1997) Active-site Arg→ Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase. J. Biol. Chem. 272, 21932–21937.
Wu G. and Furlanut M. (1997) Separation of DL-dopa by means of micellar elec-trokinetic capillary chromatography after derivatization with Marfey’s reagent. Pharmacol. Res. 35, 553–556.
Goodnough D. B., Lutz M. P., and Wood P. L. (1995) Separation and quantification of D-and L-phosphoserine in rat brain using N-alpha-(2, 4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey’s reagent) by high-performance liquid chromatography with ultraviolet detection. J. Chromatog. (B. Biomed. Appl.) 672, 290–294.
Yoshino K., Takao T., Suhara M., Kitai T., Hori H., Nomura K., et al. (1991) Identification of a novel amino acid, o-bromo-L-phenylalanine, in egg-associated peptides that activate spermatozoa. Biochemistry 30, 6203–6209.
Tran A. D., Blanc T., and Leopold E. J. (1990) Free solution capillary electro-phoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with ¢L-and ¢D-Marfey’s reagents. J. Chromatog. 516, 241–249.
Thompson J. S. and Edmonds O. P. (1980) Safety aspects of handling the potent allergen FDNB. Ann. Occup. Hyg. 23, 27–33.
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Kochhar, S., Mouratou, B., Christen, P. (2001). Amino Acid Analysis by High-Performance Liquid Chromatography after Derivatization with 1-Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey's Reagent). In: Cooper, C., Packer, N., Williams, K. (eds) Amino Acid Analysis Protocols. Methods in Molecular Biology™, vol 159. Humana Press. https://doi.org/10.1385/1-59259-047-0:049
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DOI: https://doi.org/10.1385/1-59259-047-0:049
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