Biophysical, histopathological and pharmacological characterization of crotamine isoforms F22 and F32

Abstract

Two major crotamine isoforms (F22 and F32) were obtained after three chromatographic steps and were assayed in mouse phrenic nerve-diaphragm preparations. F32 and F22 (0.5 mug/ml, n = 4) produced a facilitatory effect, which increased isometric twitch-tension by 300 and 230%, respectively, after a 120 min incubation. At a concentration of 0.1 mug/ml, both isoforms increased the twitch-tension by about 160%. However, when the isoforms were co-incubated (final concentration, 0.5 mug/ml) for 30 min prior to testing, they did not cause the facilitation seen with greater than or equal to0.1 mug/ml of each isoform alone. Histologically, F32 and F22 at 0.5 and 1 mug/ml were quantitatively alike in inducing tissue myonecrosis. However, a mixture of the two isoforms (final concentration, 0.5 mug/ml) significantly attenuated the damage seen with either toxin alone. Mass spectrometry analysis showed that the isoforms had the same molecular mass (4.8 kDa) and that they existed as monomers with a highly stable structure. These results indicate that F22 and F32 acted on muscle cells of the mouse phrenic-nerve diaphragm preparation through similar mechanisms. Since the isoforms did not produce the expected summation in the increase in muscle twitch-tension, it is possible that they may have different affinities for the sodium channel subunits. (C) 2003 Elsevier Science Ltd. All rights reserved.