Bibhuti R. Dasgupta

Method for quantitative determination of free and peptide-linked tryptophan after reaction with 2-hydroxy-5-nitrobenzyl bromide

Abstract A method for quantitative determination of tryptophan in the presence of 20hydroxy-5-nitrobenzyl bromide substituted tryptophan is described. The procedure does not require removal of the excess reagent and can be applied to intact proteins substituted with the reagent. The procedure involves applying mathematically derived correction factors to the analysis data obtained from the Spies and Chambers method for tryptophan determination.

Study of the toxin of Clostridium botulinum. Effects of 2-Hydroxy-5-nitrobenzyl bromide on the biological activity of botulinum toxin

Abstract 2-Hydroxy-5-nitrobenzyl bromide, when reacted with the toxin of Clostridium botulinum Type A, reduced toxicity by 99% and modified 17.8 moles out of 77.2 moles of tryptophan reduced in the toxin. This treatment resulted also in the loss of the ability of the toxin to stimulate protective antibidy formation in rabbits by destroying an antigenic determinant responsible for the formation of neutralizing anti-body in the native toxin. This evidence, in addition to the results obtained by photo-oxidation of the toxin 5 , further supports the thesis previously advanced as to the critical role of tryptophan residues in the toxins reactive sites. 2-Hydroxy-5-nitro-benzyl substitution method yielded data consistent with the estimated number of active tryptophan residues obtained from photooxidation reaction kinetics.

Cation-exchange chromatography of Clostridium botulinum type A toxin on amberlite IRC-50 resin at pH 5.55.

The hemagglutinin-free toxin (the α fraction) isolated from crystalline toxin of Clostridium botulinum type A, homogeneous by several criteria, was examined on carboxymethyl-Sephadex, sulfoethyl-Sephadex and Amberlite IRC-50 (XE-64) columns to attempt further resolution and to provide additional tests of its homogeneity. Satisfactory conditions for chromatography were obtained only with Amberlite IRC-50 (XE-64) resin. As an analytical tool this resin proved more sensitive than a DEAE-cellulose column since it was able to separate the hemagglutinin when present as trace contaminant in the α fraction, which could not be achieved by anion-exchange chromatography. Although the IRC-50 column separated the 150 000 mol. wt. specie of the α fraction from its aggregated forms, the toxin could not be fractionated into subunits. These observations were consistent with the result of gel filtration of the α fraction and its aggregates on Sephadex columns. The present report also demonstrates for the first time that a protein as large as 150 000 mol. wt. can be successfully chromatographed on IRC-50 (XE-64) resin at pH 5.55.