Action of snake venom hemorrhagic principles on isolated glomerular basement membrane

1. 1. The reaction of venom hemorrhagic principles (HR1, HR2a and HR2b) with isolated glomerular basement membrane was studied in vitro. For comparison, experiments were also done with a bacterial collagenase which is known to induce the hemorrhage. Upon incubation of the basement membrane preparation with either of the four agents, its supernatant fluid gave positive reactions for proteins (or peptides) and carbohydrates. The optimal pH of the protein-liberating activities of HR1, HR2a and HR2b was around 8.0. 2. 2. The approximate ratio of the proteins or peptides liberated by HR2a, HR2b, HR1 and collagenase, under standard conditions, was 1:1:2:4; and for the carbohydrates liberated 0.5:0.5:2:4, thus suggesting that HR2a or HR2b acted on the basement membrane in a different manner from HR1. This interpretation is supported by comparison between the split-products of the membrane by these principles in gel filtration experiments. 3. 3. Liberation of both proteins and carbohydrates from basement membrane by HR1, HR2a and HR2b was inhibited by EDTA, cysteine, antivenin and antihemorrhagic factor from snake serum, all of which inhibit the hemorrhagic activity of these principles. The results suggest that one and the same entity is responsible for both the hemorrhagic and liberating activities in each venom principle. 4. 4. Purified HR1 was resolved partially into four components by polyacrylamide gel electrophoresis. Both liberating and hemorrhagic potencies, as expressed by their specific activities, however, were constant throughout, which further supports the idea that the same factor is responsible for both activities. 5. 5. The hemorrhagic effect of HR1, HR2a, HR2b and collagenase is attributed to enzymatic destruction of the basement membrane with consequent lowering of the stability of the vessel wall.