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...
Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by a-bungarotoxin. Several of these, exemplified by ,-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of Crotalus durissus terrificus, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests.It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating.Phospholipase from Crotalus adamanteus, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37°. P-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.Binding studies with 125I4abeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for jP-bungarotoxin and do not bind Crotalus adamanteus phospholipase. The crotoxin complex is split in the course of inding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B.Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for (3 type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.When the neurotoxin of the Brazilian rattlesnake (Crotalus durissus terrificus) was isolated in pure crystalline form in 1938 and was found to also carry the indirect (i.e., lecithin-dependent) hemolytic activity of that venom (1), it was then suggested (and has since been reiterated) that the ability of crotoxin to attack phospholipids might represent the basis of its neurotoxic potency.When, many years later, after crotoxin had been shown to consist of two proteins (2), the surprising finding was made that high neurotoxicity required the presence of both components (3-5), while in regard to hemolytic activity the larger and basic compone...
The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.
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