Isolation, characterization and mode of neutralization of a potent antihemorrhagic factor from the serum of the snake Bothrops asper

A potent antihemorrhagic factor (BaSAH¹) was isolated from the serum of the snake Bothrops asper by ammonium sulfate precipitation at 40-60%, Sephacryl S-200 and Sephadex G-50 gel filtration, DEAE-Sepharose, and hydrophobic Phenyl-Sepharose chromatography. The purified protein showed one band with an isoelectric point of 5.2 and a molecular weight of 66 kDa. 4 μg of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 μg of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 μg of BaSAH were sufficient to achieve complete neutralization of the main hemorrhagic toxin (BaH1), with a molar ratio of 2:1. The antihemorrhagic activity was stable between pH 1.5-9 and up to 60°C but lost activity completely after 30 min of heating at 70°C. BaSAH did not digest the hemorrhagic toxin BaH1 or formed a precipitin line with it, nor with the whole venom. Both ELISA experiments and chromatography of BaSAH after incubation with the ¹²⁵I-labeled hemorrhagic toxin BaH1 demonstrated that the mechanism of the neutralization involves a formation of an inactive soluble complex between the natural antihemorrhagin and the main hemorrhagin of B. asper venom.