J. Biochem, 1990, Vol. 107, No. 6 821-825
© 1990 Japanese Biochemical Society
research-article |
Interchain Disulfide Bonds in Crotamine Self-Association
*Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de S{small tilde}o Paulo 14.049 Ribeir{small tilde}ao Preto—SP, Brasil
**Departamento de Bioquímica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo 14.049 Ribeirão Preto—SP, Brasil
***Departamento de Bioquí;mica, Institute de Ciêencias Biológicas, Universidade Federal de Minos Gerais 30.000 Belo Horizonte—MG, Brasil
1 To whom correspondence should be addressed.
Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazillian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4, 500–5, 000 for RCC and 9, 000–10, 000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition, N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9, 760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4, 880 Da each.