Oxidative Cleavage of Tryptophanyl Peptide Bonds during Chemical- and Peroxidase-catalyzed Iodinations

Abstract

Abstract Tryptophanyl peptide bonds are oxidatively cleaved by active that is generated with H2O2, iodide, and a peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7). Complete oxidation of tryptophan derivatives and peptides to the oxindole occurs within 2 min at pH 3 to 5 with either lactoperoxidase or horseradish peroxidase, and a 2.5-fold molar excess of iodide plus H2O2. Under identical conditions, simple tryptophan peptides are cleaved 30 to 40% in 10 min at pH 5.0, and the rate of cleavage is parallel to, but less than, the rate of oxidation. All three constituents (iodide, H2O2, and peroxidase) are required for oxidation and fission to proceed. Iodinating reagents yield similar results, except that they oxidize tryptophan derivatives and peptides (Z-Trp-X, X-Trp-X) over the pH range of 2 to 11. Fission of the tryptophan peptide proceeds in acid media with a maximum at pH 5.0, and no cleavage occurs in alkaline media. A 2.5-fold molar excess of positive halogen reagents, ICl, iodide oxidized with chloramine-T, and N-iodosuccinimide completely oxidize tryptophan peptides at pH 5.0 within 1 min, whereas oxidation by I2 and I3- is 2 and 8 times slower, respectively. Chloramine-T per se oxidizes tryptophan peptides at about the same rate as I2 below pH 6.5, but is unreactive above this pH. All of the iodinating reagents induce complete oxidation within 1 min at pH 9.5. Thirty to forty percent of a tryptophan peptide (e.g. Z-Trp-Gly or Z-Trp-Leu) is cleaved in 10 min at pH 5 by a 2.5- to 10-fold molar excess of all the reagents, except for I3- which promotes half as much cleavage. With all of the iodinating reagents, the rate of cleavage is parallel to, but less than, the rate of oxidation. Iodimetric titrations and pH profiles suggest that the oxidation and oxidative cleavage of tryptophan peptides during iodination proceeds via the mechanism proposed for brominating agents (Patchornik, A., Lawson, W. B., Gross, E., and Witkop, B. (1960) J. Amer. Chem. Soc. 82, 5923). Two equivalents of I+ or I2 convert the indole nucleus to the oxindole, which undergoes spontaneous hydrolysis after cyclization to the iminolactone. Unlike brominating reagents, a third iodine equivalent does not form a stable iodooxindole derivative with several different tryptophan peptide models or oxindole itself. Two exceptions are Z-Trp-Gly and Z-Trp, which are probably iodinated by ICI on the benzyloxycarbonyl moiety. Free tryptophan is oxidized by 2.6 to 2.9 eq of iodine (ICl or I2), but it does not become iodinated in labeling experiments with 131I2 or 131ICl.