Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography.
A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.
D000596:Amino Acids / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006868:Hydrolysis; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D013438:Sulfhydryl Compounds; D014364:Tryptophan / Q000031:analogs & derivatives / Q000032:analysis*
D000596:Amino Acids; D011506:Proteins; D013438:Sulfhydryl Compounds; C044338:2,3-dihydrotryptophan; D014364:Tryptophan