Snake venom hemorrhagins.

Viperine and crotaline snake venoms contain one or more hemorrhagic principles called hemorrhagins. These are zinc-containing metalloproteases characterized by the presence of a protease domain, with additional domains in some of them. They act essentially by degrading the component proteins of basement membrane underlying capillary endothelial cells. The toxins also act on these cells causing lysis or drifting apart, resulting in hemorrhage per rhexis or per diapedesis. Some of these toxins have been found to exert additional effects such as fibrinogenolysis and platelet aggregation that facilitate hemorrhage. The structural and functional features of this class of toxins have been discussed in this review in an attempt to get a better understanding of their toxicity. This can be of immense therapeutic value in the management of snake venom poisoning, as hemorrhagins are among the major lethal factors in snake venom.

Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity.

A membrane protease possessing thrombin-like activity was purified to homogeneity from mitochondria of rat submaxillary gland. The molecular mass of the enzyme was determined to be 45 kDa by SDS/PAGE under reducing conditions and by gel filtration on a Sephadex G-100 column. The enzyme is a glycoprotein and has an isoelectric point of 3.25. Maximum activity was observed at pH 10.5. Inhibition by di-isopropyl fluorophosphate, benzamidine, aprotinin and antipain suggested the enzyme to be a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH2Cl and chymostatin did not alter the activity. The enzyme showed affinity towards different synthetic substrates (p-nitroanilide derivatives) containing arginine at the P1 position. Kinetic studies revealed that Kcat./Km was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation initiated by the enzyme was not altered by concanavalin A, indicating that the carbohydrate moiety of the enzyme is not essential for this reaction. Further, this enzyme can catalyse the formation of fibrin clots from purified fibrinogen, which describes its thrombin-like activity. However, an antibody raised against the purified enzyme inhibited the plasma-clotting as well as fibrinogen-clotting activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of aprotinin, a protease inhibitor. Release of fibrinopeptides A and B from bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our studies reveal that the enzyme reported here differs from trypsin, chymotrypsin and other mitochondrial proteases reported so far.

A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity.

An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.

Neutralization of pathophysiological manifestations of Russell's viper envenoming by antivenom raised against gamma-irradiated toxoid.

Rabbits were immunized against gamma-irradiated (100 krads) Russell's viper venom toxoid adsorbed to aluminium phosphate gel. The antivenom (0.1 ml) neutralized 5 LD50, 8 minimum hemorrhagic doses (MHD) and 14 minimum necrotic doses (MND) of venom. The coagulant and protease activities of the viper venom were neutralized more effectively than phospholipase A activity, by the toxoid antivenom.

Pyroglutamate aminopeptidase in rat submaxillary gland.

A significant amount of pyroglutamate aminopeptidase (PGAP) activity was found to be present in 27,000 x g supernatant of rat submaxillary gland, maximum activity being at pH 6.5. EDTA stimulated the enzyme activity by 95% at pH 8.0 while at pH 6.5 it did not have any significant effect. On comparison of its properties submaxillary PGAP appears to be different from brain, pituitary and other reported PGAPs. Submaxillary PGAP could also catalyze efficiently the formation of cyclo (His-Pro) from TRH. Cyclo (His-Pro) formation by submaxillary enzyme was more pronounced than that by liver PGAP.

Neutralization potency of Russell's viper venom toxoid antivenom, as compared with standard antivenom.

Rabbits were immunized with gamma (gamma) irradiated Russell's viper venom toxoid, adsorbed to aluminium phosphate adjuvant. Antibody (raised against toxoid inoculation) titer was compared to a commercial antivenom on the basis of its ability to neutralize hemorrhagic, necrotic and lethal effects of viper venom. Toxoid immunization (on day 0, 15 and 30) produced antivenom which showed approximately one-third antilethal, half antihemorrhagic and antinecrotic titers as compared to those of the commercial hyperimmunized, concentrated horse antivenom.

Silanized silica bound trypsin as analytical probe.

Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.

Epidemiology of snake bite in the district of Burdwan, West Bengal.

An epidemiological field survey on snake bite was conducted on 26 randomly selected villages with a population of 18,892 in the district of Burdwan, West Bengal to assess the magnitude of the problem in a decade (1980-1989). Total number of snake bite, number of presumably poisonous snake bite and deaths due to snake bite poisoning were 307, 48 and 31 respectively. The death rate among snake bite victims was 10.09%. Males (54.72%) were bitten more than females (45.23%) and highest incidence of snake bite was found in the age group of 21-30 years and during the months of July and August. Majority of the snake bites (53%) were encountered in the lower extremities. Among the snake bite patients 201 (65.47% went to the traditional healers (ozhas) and 68 (22.14%) persons received hospital treatment, while 12 (3.09%) people neither went to the ozhas nor to hospital and 26 (8.46%) persons went to hospital after consulting the ozhas. If the present data are extrapolated for the total population of the district, average number of snake bite and death per year would be 7,857 (0.16%) and 793 (0.016%) respectively. Deaths due to snake bite per 100,000 population varied from 5.28 to 31.75 (average 16.4) over 10 years.

Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland.

A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A.

Active immunization of rabbit with gamma irradiated Russell's viper venom toxoid.

Russell's viper venom detoxified by gamma (gamma)-radiation (100 kR or 200 kR) was used as a toxoid for active immunization of rabbits following a short or long schedule of immunization without any adjuvant. Effective neutralization of venom toxin by immune sera of rabbits was observed with both schedules. Sera of rabbits immunized with 100 kR irradiated venom toxoid (100 kR toxoid antisera) were more potent than 200 kR toxoid antisera. The presence of antibody in the immune sera was detected by immunoelectrophoresis. The effect of gamma-radiation on some enzymes and venom protein profiles was studied. Phosphodiesterase, protease and hyaluronidase were inhibited by radiation though phospholipase A activity remained unaffected. Radiation did not produce any gross change in the protein profile of crude viper venom. Phosphodiesterase and protease activities of viper venom were neutralized more effectively by 100 kR toxoid antisera in the short schedule than in the long schedule of immunization.

Studies on the effect of reserpine on the peroxidase activity of submaxillary gland.

In an earlier report we have described how the administration of reserpine (0.5 mg/kg) stimulates the peroxidase activity of rat and mouse submaxillary gland [R. Chakraborty, B. Mukherjee and R. N. Hati, Life Sci. 35, 1913 (1984)]. Further studies have been carried out in this area. This stimulation of the enzyme activity started from 0.02 mg reserpine/kg body wt. The effect of reserpine was found in all subcellular fractions of submaxillary gland. Iodinating activity of the enzyme preparation was also increased by reserpine treatment. Kinetic studies indicate that reserpine increased the Vmax of the enzyme without altering Km. Protein synthesis inhibitors inhibited the increase of the enzyme activity by reserpine. Dibutyryl c-AMP, theophylline or both drugs together did not alter the peroxidase activity. The effect of reserpine was also found in experiments using slices and single cell preparations of submaxillary gland, suggesting that the drug is probably acting directly on the acinar cells.

Increase of the peroxidase activity of mouse submaxillary gland by reserpine.

The peroxidase activity of mouse submaxillary gland was found to be elevated by about 128% at 22 hr. after the administration of reserpine (0.5 mg/kg). This effect of reserpine was observed in rat also. Neither pretreatment with 6-hydroxy dopamine (6-OH dopamine) nor surgical sympathetic denervation could abolish the increase of the peroxidase activity elicited by reserpine. Also, treatment with propranolol, dibenamine or atropine sulfate failed to reverse the effect of reserpine. These results suggest that neither catecholamine nor acetyl choline is involved in this reserpine action.