Crotoxin, the Brazilian rattlesnake neurotoxin, generally behaves as a homogeneous protein; however, it is a molecular complex of an acidic and a basic protein. These can be separated after alkylation or acylation of the amino groups, or on carboxymethyl cellulose at pH 4, or on DEAE-cellulose in 6 M urea. The two proteins differ greatly in composition, but one or both may exist in the form of closely related variants. Their molecular weights appear to be about 8400 and 13,000.The acidic protein lacks the hemolytic and neurotoxic activity of crotoxin and the basic protein shows only the high, indirect hemolytic activity; a mixture of the two components shows the high neurotoxicity of crotoxin.In 1938, Slotta and Fraenkel-Conrat isolated and crystallized the toxic principle of the venom from Crotalus durissus terriflcus (neotropical rattlesnake) (1). Studies in the ultracentrifuge (2) and in the Tiselius electrophoretic apparatus (3) provided significant evidence for the homogeneity of this protein, crotoxin, and indicated a molecular weight of 30,000 and an isoelectric point of 4.7. This protein carried both the neurotoxic and the indirect, i.e. lecithin-requiring, hemolytic activity of that venom (1). Later, these activities were attributed to separate proteins (4), although no readily reproducible method, nor evidence, for their separation was described (4, 5). The first definitive evidence for the existence of two quite dissimilar proteins in crotoxin came from studies in which the protein was treated with fluorodinitrobenzene (6). Two dinitrophenylated proteins could then be separated, one being soluble and the other insoluble in water. These two proteins differed greatly in their amino acid compositions. No biological activities were detected in these DNP-protein fractions.The purpose of the present study was to purify the two components of crotoxin to a suitable form for biological evaluation. One approach was to replace the dinitrophenyl group by other substituents of amino groups that would favor dissociation of the two proteins, but that could be removed by hydrolysis under mild conditions, e.g., maleyl and methyl maleyl residues (7,8). The other approach was to attempt separation of the two proteins by column chromatography. The previously proposed procedure, DEAE-cellulose near pH 7.0 (4, 5), did not separate proteins of different aminoacid composition (9), but when 6 M urea was added to the solvent (at pH 6.0), or when the native protein was passed over carboxymethyl (CM)-cellulose, an acidic and a basic protein could be isolated. These corresponded in composition to the more soluble and insoluble fractions, respectively, obtained after alkylation or acylation of the amino groups of crotoxin.When the biological activities of these isolated components were tested, the basic protein retained the hemolytic activity, but neither protein showed the toxicity of crotoxin. The combination of the two components, however, was as neurotoxic as was the original complex.
MATERIALS AND METHODSFluorodinitro...
