Partial purification and some physicochemical properties of phospholipases A2 from the venom of the bushmaster snake (Lachesis muta) (original) (raw)
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Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1995
A neutral phospholipase A 2 (PLA 2) was separated from Pseudechis papuanus venom by a two-stage FPLC procedure of cation exchange and phenyl-Superose chromatography. It had a molecular mass of 15 kDa and a lower LDso value than a co-separated haemorrhagic fraction, indicating a higher lethal potency. In vitro tests confirmed the powerful inhibition of platelet aggregation by the PLA 2 and strong anticoagulant activity initially observed with whole venom. Ultrastructural studies showed that platelets lost their discoid shape and developed membranous projections with a general decrease in electron-density of the cytosol and disruption of the microfilaments following incubation with the enzyme. Amino acid sequence analysis of the N-terminus and some internal peptides demonstrated a high degree of homology with PLAzs from other Pseudechis venoms. Our results indicate that this fraction is the main agent responsible for the haemostatic disorders in envenomed patients.
Toxicon, 1988
Venom from southern copperhead snake (Agkistrodon contortrix rnntortrix). II. A unique phospholipase A, that induces platelet aggregation. Toxirnn 26, 199-206, 1988 .-A platelet aggregation factor was purified from the venom of southern copperhead snake (Agkistrodon rnntortrlx rnntortrlx) by DEAE~ellulose ionexchange chromatography, precipitation with ammonium sulfate, affinity chromatography using bovine serum albumin as ligand, and gel filtration on Cellulofme GCL-2000. It had molecular weights of 11,000 and 14,000, as determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. It consists of a single polypeptide, and was identified as a phospholipase A,. It was quite resistant to heat and various denaturing reagents including urea and SDS. It lost both phospholipase A= activity and platelet aggregating activity upon modification of histidine residues) with p-bromophenacyl bromide. Its specificity towards the~-position of phospholipid in esterolytic reaction was confu~med by gas-liquid chromatography using a pure synthetic phosphatidylcho6ne. Platelet aggregation by this phospholipase A~wa~completely inhibited by prostacyclin, but was little inhibited by aspirin which indicates almost no direct participation of released arachidonic acid in the aggregation mechanism.
Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom
Bayero Journal of Pure and Applied Sciences, 2021
The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km of 1.47mg/ml and Vmax of 3.32μ mo...
Biochemical Journal, 1980
The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by ge...
Indian Journal of Clinical Biochemistry, 2015
This study is designed to isolate and purify a novel anti-clotting protein component from the venom of Enhydrina schistosa, and explore its biochemical and biological activities. The active protein was purified from the venom of E. schistosa by ion-exchange chromatography using DEAE-cellulose. The venom protein was tested by various parameters such as, proteolytic, haemolytic, phospholipase and anti-coagulant activities. 80 % purity was obtained in the final stage of purification and the purity level of venom was revealed as a single protein band of about 44 kDa in SDS-polyacrylamide electrophoresis under reducing conditions. The results showed that the Potent hemolytic activity was observed against cow, goat, chicken and human (A, B and O positive) erythrocytes. Furthermore, the clotting assays showed that the venom of E. schistosa significantly prolonged in activated partial thromboplastin time, thrombin time, and prothrombin time. Venomous enzymes which hydrolyzed casein and gelatin substrate were found in this venom protein. Gelatinolytic activity was optimal at pH 5-9 and 1 H NMR analysis of purified venom was the base line information for the structural determination. These results suggested that the E. schistosa venom holds good promise for the development of novel lead compounds for pharmacological applications in near future. Keywords Venom Á Hemolytic Á Proteolytic activity Á Activated partial thromboplastin time (APTT) Á Thrombin time (TT) Á Prothrombin time (PT) Abbreviations BSA Bovine serum albumin EDTA Ethylene diamine tetra acetic acid PBS Phosphate buffered saline CaCl 2 Calcium chloride HCl Hydrochloric acid E. schistosa Enhydrina schistosa SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis Tris Tris(hydroxymethyl) aminomethane HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) RT Recalcification time APTT Activated partial thromboplastin time PT Prothrombin time TT Thrombin time
Journal of Pharmaceutical and Biomedical Analysis, 2013
Phospholipases A 2 are important components of snake venoms, the basic isoforms have been more extensively studied than the acidic groups, maybe due to their higher toxicity. Trying to better understand the role of the acidic isoforms on the envenomation process, an acidic phospholipase A 2 was purified from Bothrops moojeni snake venom through two chromatographic steps (BmooPLA 2) . The enzyme showed a relative molecular mass of 13,601 Da, pI 5.2, high phospholipase activity, bactericidal effect, moderate cytotoxic activity and was able to inhibit platelet aggregation. Moreover, BmooPLA 2 induced moderate in vivo edema and hypotensive effect. The 414 bp cDNA encoding the BmooPLA 2 was cloned and expressed in Escherichia coli. The recombinant BmooPLA 2 showed phospholipase and inhibitory activities on platelet aggregation similar to those of the native protein. A comparative study between BmooPLA 2 , the acidic (BthA-I) and basic (BthTX-II) PLA 2 from B. jararacussu venom showed that the effects of BmooPLA 2 and BthA-I-PLA 2 are similar. BmooPLA 2 is the first isolated and characterized non-myotoxic PLA 2 from B. moojeni snake venom. The recombinant PLA 2 can substitute the native toxin in studies aiming its biotechnological application in order to help the preservation of this endangered species. These data along with the preliminary structural studies here reported will provide a better understanding of this important class of proteins.
1994
By means of gel filtration, ionic exchange ch~mato~aphy and DEAE-coh.uun HPLC, an acidic phospholipase A, (PLA,) was purified from beaded lizard (~e~~e~ ~~} venom. The purified PLA is a single-chain polypeptide, consisting of about 163 amino acid residues with a molecular mass of 19000 Da as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ammo acid analysis. HHYV-PLA showed a rather specific inhibitory effect on platelet aggregation induced by U46619 and epinephrine in human platelet-rich plasma in a dose-and time-dependent manner, whereas it had little effect on collagen-and ADP-induced aggregation. ATP-release reaction induced by various agonists were dose-and time-dependently inhibited by HHV-PLA, even though platelet aggregation was apparently not affected in human washed platelets. When HHV-PLA was chemically modified with ~-bromophena~l bromide, both of its enzymatic achvity and antiplatelet activity were lost. Furthermore, exogenous l~ophosphati~lcholine and HHV-PLA treated phosphatidylcholine inhibited platelet aggregation induced by U46619 in human washed platelets. In conclusion, PLA enxyme from H horridum venom inhibits exclusively U46619-or thromboxane-induced platelet aggregation of human platelet-rich plasma probably by virtue of their PLA enzymatic acitvity on plasma phospholipids, converting phospholipids (e.g., phosphatidylcholine) into lysophospholipids, which in turn interfere with the coupling of TXA, receptor and its slgnalling transduction system.
Toxicon, 2008
Phospholipase A 2 (PLA 2 , EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A 2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE-Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl-Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA 2 s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA 2 s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.
International Journal of Biochemistry, 1990
The two ma-jot phospholip~e A, enzymes (OHPLA-DE1 and OHPLA-DE21 of king cobra (O~hio~~~gu~ harm&) &nom have be& purified .to el&trophoretic homogeneity.-2. The isoelectric noints of OHPLA-DE1 and OHPLA-DE2 were 3.8 I and 3.89. resmctivelv and the M,s were 14,000 ad 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromaiography; and 14,000 as estimated by SDS-PAGE. 3. The enzymes were not lethal to mice at a dosage of 10 pg/g body wt by i.v. route. Both phospholipase A, enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes.