Junaid Mahmood Alam

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.

Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1)

A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle fibers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular infiltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The purified PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty infiltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked infiltration of inflammatory cells within alveoli. Our results suggest that the purified PLA2-H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria.