Crotamine conformation: Effect of pH and temperature

Abstract

The pH-isomers of crotamine were characterized by spectropolarimetric titration. The neutral isomer (I) found at pH range between 4 and 8·5 corresponds to the native conformation. After heating at 80°C for 30 min in 8 M urea, it retains full biological activity and the small spectral changes induced by heating are readily reversible. Compared to the neutral isomer, the acid isomer (II) occurs at pH values lower than 2·0 and has an unmasked group presumably a carboxyl, with a p K′ of 2·6. The basic isomer (III) has two groups with ap K′ of 9·7 exposed. Also, the exposure of the N-terminal tyrosyl in II and the tyrosyl and two tryptophanyl residues in III are revealed by difference spectrophotometry. Both II and III represent partially pH-denatured conformations which undergo further structural unfolding upon heating. The unfolding ▵H° for isomers II and III were determined to be 8·2 and 6·1 kcal/mole, respectively. In addition, the spectral ▵H° calculated from the spectrophotometric data gives information about the spatial displacement of residues (mainly tryptophanyl and tyrosyl) which are more pronounced in III than in II.