Purification of an L-amino acid oxidase from Bungarus caeruleus (Indian krait) venom

Snake venoms are rich in enzymes such as phospholipase A2, proteolytic enzymes, hyaluronidases and phosphodiesterases, which are well characterized. However, L-amino acid oxidase (LAO EC.1.4.3.2) from snake venoms has not been extensively studied. A novel L-amino acid oxidase from Bungarus caeruleus venom was purified to homogeneity using a combination of ion-exchange by DEAE-cellulose chromatography and gel filtration on Sephadex® G-100 column. The purified monomer of LAO showed a molecular mass of 55 ±1 kDa estimated by SDS-PAGE. The specific activity of purified LAO was 6,230 ± 178 U/min/mg, versus 230 ± 3.0 U/min/mg for the whole desiccated venom, suggesting a 27-fold purification with a 25% yield. Optimal pH and temperature for maximum purified enzyme activity were 6.5 and 37ºC, respectively. Platelet aggregation studies show that purified LAO inhibited ADP-induced platelet aggregation dose-dependently at 0.01 to 0.1 µM with 50% inhibitory concentration (IC50) of 0.04 µM, whereas at a 0.08 µM concentration it did not induce appreciable aggregation on normal platelet-rich plasma (PRP). The purified protein catalyzed oxidative deamination of L-amino acids while the most specific substrate was L-leucine. The purified LAO oxidizes only L-forms, but not D-forms of amino acids, to produce H2O2. The enzyme is important for the purification and determination of certain amino acids and for the preparation of α-keto acids.(AU)

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.

Preliminary studies with a neurotoxin obtained from Bungarus caeruleus venom

The neurotoxin purified from the venom of Bungarus caeruleus causes a neuromuscular blockade on acetylcholine-induced muscle twitch response in isolated frog rectus abdominis muscle preparation. Neuromuscular blockade produced by d-tubocurarine on acetylcholine-induced muscle twitch response in an isolated frog rectus abdominis muscle preparation was reversed to normal muscle twitch response in presence of neostigmine. Whereas the purified neurotoxin produced an irreversible neuromuscular blockade in presence of the same strength of neostigmine. As it is already known, botulinum toxin, which also brings about neuromuscular blockade, is effectively used as a drug in the treatment of painful movement disorders. Since the purified toxin also causes paralysis of the muscle, we propose its possible efficacy in the treatment of neuromuscular disorders.(AU)

Isolation and purification of a neurotoxin from Bungarus caeruleus (common Indian krait) venom: biochemical changes induced by the toxin in rats.

Bungarus caeruleus (Indian common krait) is a venomous snake that is responsible for most of the snakebites in India. In the present study, we report the isolation and purification of neurotoxin and the biochemical changes and pathological effects induced by injection of purified neurotoxin into rats. The purpose of this study was to compare the effects of crude krait venom and the purified toxin. Both the crude venom and a sublethal dose of 60 microg/kg B. caeruleus purified toxin significantly increased the serum levels of alkaline phosphatase, alanine aminotransferase, and urea (p < 0.05). The crude venom but not the purified toxin increased the levels of lactate dehydrogenase, aspartate aminotranseferase, creatine kinase, and glucose. The kidneys showed congestion of the vessels, hemorrhage, and necrosis in venom-injected but not in toxin treated animals. The results of this study indicate that although crude krait venom has severe lethal, hemorrhagic, nephrotoxic, and proteolytic activities, the purified neurotoxin shows only moderate toxic activity, manifested as prominent local and systemic effects.

Dose-dependent serum biochemical alterations in Wistar albino rats after Palamneus gravimanus (Indian black scorpion) envenomation.

Palamneus gravimanus envenomated rats showed dose-dependent alterations in serum biochemical parameters. Sub lethal doses of 100, 200, and 400 microg/kg of P. gravimanus venom were injected intramuscularly into rats. Blood samples were collected by heart puncture before and 4 h after crude venom administration. Serum was analyzed for glucose, blood urea nitrogen (BUN), uric acid, total protein, cholesterol, sodium, potassium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase (AST-SGOT), alanine amino-transferase (ALT-SGPT), lactate dehydrogenase (LDH), and creatinine phosphokinase (CPK). Statistically significant increases in serum levels of glucose, creatinine, AST, ALT, BUN, CPK, and LDH and significant decreases in serum levels of total protein, uric acid, cholesterol, calcium, and potassium 4 h after venom administration could be due to the toxic action of P. gravimanus venom on certain organs in rats.

Biochemical and clinicopathological changes induced by Bungarus coeruleus venom in a rat model.

Bungarus coeruleus, a common venomous snake allied to the cobra, is responsible for most envenomations in India. This study examines the pathological effects of B. caeruleus venom and the associated biochemical changes in a rat model. Increased serum aspartate aminotransferase (48%), creatine kinase (30%), and lactate dehydrogenase (6%) were detected after a sublethal dose of 25 microg/kg of B. caeruleus venom was injected intramuscularly into rats observed for 180 min. The venom induced hyperglycemia and increased serum alkaline phosphatase (55%) and urea (90%) concentrations, whereas cholesterol and triglycerides remained normal. Histopathological changes in the heart-hemorrhage, multifocal areas of myocardial fiber necrosis-and constriction of blood vessels in the kidney, with congested vessels, hemorrhage and necrosis of proximal tubules, liver congestion, and hemorrhage were found.