PERTURBATION OF THE STRUCTURE OF CLOSTRIDIUM PERFRINGENS ENTEROTOXIN BY SODIUM DODECYL SULFATE, GUANIDINE HYDROCHLORIDE, pH AND TEMPERATURE

Abstract

The effects of sodium dodecyl sulfate, guanidine · HCl, heat and pH on the tertiary structure of Clostridium perfringens type A enterotoxin were determined by UV difference spectroscopy and by accessibility of the amino groups to 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) and the one sulfhydryl group to 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB). Biological stability of enterotoxin at several pH's at 55°C in the presence and absence of borate and dipicolinate ions was determined by the guinea pig skin test. Sodium dodecyl sulfate and heat did not unfold the molecule while treatment with >4.5 M guanidine · HCl at ≥25°C completely unfolded the molecule based on UV difference spectral changes and the accessibility of the amino groups to reaction with TNBS. Sodium dodecyl sulfate, at 0.2%, made the sulfhydryl group accessible to DTNB but did not affect the accessibility of the amino groups (7.89 ± 0.15 groups in the native molecule). Guanidine · HCl, at 6.5 M for 30 min and ≥25°C, made all 17 e-amino groups and the one α-amino group accessible to TNBS. At pH's above 8 spectral differences, in relation to the spectrum at pH 6.8, were associated primarily with ionization of the tyrosine residues while at pH's below 5.5 the spectral changes were probably a result of protonation of carboxyl groups in the vicinity of tyrosine and tryptophan residues, and of aggregation.