Studies on the Tryptophan Residues in Porcine Pepsin

Abstract

Abstract The selectivity of 2-hydroxy-5-nitrobenzyl bromide for tryptophan, as demonstrated by Koshland and his coworkers, has been used to study some of the properties of the residues of this amino acid in pepsin. The aromaticity and absorbance of the alkylated tryptophan facilitate the isolation of peptides containing the modified residues. Pepsin freshly prepared from pepsinogen was reduced, carboxymethylated, and treated with 2-hydroxy-5-nitro-benzyl bromide. The total incorporation of reagent corresponded to 4 residues of tryptophan per molecule. Hydrolysis of pepsin with alkali or with enzymes, followed by ion exchange chromatography, gave the same result. Reduced and carboxymethylated pepsinogen, when treated with 2-hydroxy-5-nitrobenzyl bromide, also was found to contain 4 tryptophan residues per molecule. The zymogen derivative was hydrolyzed by chymotrypsin and a group separation of peptides was made by gel filtration on Sephadex G-50. Each of four groups containing alkylated tryptophan residues was further hydrolyzed by thermolysin. The mixtures of small peptides were fractionated on columns of Sephadex G-25 and G-15. The peptides containing alkylated tryptophan were retarded by adsorption on the gel and their elution could be followed by measurement of the color of the hydroxynitrobenzyl group. The amino acid compositions and sequences of all of the peptides isolated were consistent with the presence of the following four sequences in pepsin and pepsinogen: I, Val-Phe-Asp-Asn-Leu-Trp-Asp-Gln-Gly; II, Leu-Trp-Val-Pro-Ser; III, Val-Glu-Gly(Trp,Gln); IV, Leu-Asn-Trp-Val-Pro. Each of the above sequences in the protein gave rise to several peptides which were separable on the gel columns but showed identical amino acid compositions. The multiplicity of peaks is probably related to the chemistry of the alkylation, which Koshland and associates have shown may yield several isomeric, monosubstituted derivatives of the indole ring of tryptophan. When active pepsin (with the disulfide bonds intact) was treated with 2-hydroxy-5-nitrobenzyl bromide at pH 3.5, only 2 residues of the reagent were incorporated; one has been shown to be in Sequence I, above, the second in Sequence IV. The loss in specific activity against both hemoglobin and acetyl-l-phenylalanyl-l-diiodotyrosine resulting from this incorporation is only 25 to 30%, indicating that the reagent is not reacting with groups that are essential for catalytic activity. In alkali-denatured pepsin, 3 residues of tryptophan were accessible, whereas the 4th became reactive only after cleavage of the disulfide bonds of the protein by reduction and carboxymethylation. In the course of these experiments it was also observed that all 4 of the methionine residues of pepsin are unavailable to alkylation by iodoacetate or iodoacetamide at pH 2.2.