HEMORRHAGIC PRINCIPLES IN THE VENOM OF BITIS ARIETANS, A VIPEROUS SNAKE .1. PURIFICATION AND CHARACTERIZATION

Two hemorrhagic principles (Bitis arietans hemorrhagin a and b: abbrev iated as BHRa and BHRb) were purified from the venom of the viperous s nake Bitis arietans (puff adder) by gel filtration, ion-exchange and a bsorption chromatography. A 10-fold purification was achieved for BHRa and 7-fold for BHRb with an overall yield of 6.4% of hemorrhagic acti vity. The hemorrhagins were homogeneous according to disc- and SDS-pol yacrylamide gel dectrophoresis and immunodiffusion. BHRa and BHRb, con sist of 623 and 685 amino-acid residues and their apparent molecular w eights were 68000 and 75000, respectively. They were also immunologica lly distinct. The purified hemorrhagins express proteolytic activity w ith heat-denatured casein and hide powder azure. The proteolytic activ ity with heat-denatured casein was almost the same as that of the crud e venom, but that with hide powder azure was less than one-tenth of th at of the crude venom. The purified hemorrhagins were free of arginine esterase and phospholipase A, activities and they are acid labile hem orrhagic toxins. Their hemorrhagic activity was inhibited by EDTA, cys teine and by polyvalent anti-snake serum, but not by phenylmethanesulf onyl fluoride or soybean trypsin inhibitor.