ISOLATION, CHARACTERIZATION AND MODE OF NEUTRALIZATION OF A POTENT ANTIHEMORRHAGIC FACTOR FROM THE SERUM OF THE SNAKE BOTHROPS-ASPER

A potent antihemorrhagic factor (BaSAH(1)) 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 mu g of the purified factor BaSAH were needed to neutralize the hemorrhagic dose of B. asper whole venom compared to 60 mu g of the clinically used horse polyvalent immunoglobulins. Moreover, 0.35 mu g 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 degrees C but lost activity completely after 30 min of heating at 70 degrees 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 I-125-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.