Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

Abstract

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis

Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease fromBothrops pirajaiSnake Venom

“…Like the majority of serine proteases [12, 13, 20, 44–48], these differences indicate that BpirSP-39 seems to be a glycosylated protein. The difference detected during electrophoretic migration was probably caused by the carbohydrate microheterogeneity of the enzyme since this fraction can vary the weight of the serine protease up to 30%.…”

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

“…P-I SVMPs present variable molecular masses ranging from 20 to approximately 30 kDa, e.g. neuwiedase (20 kDa), BthMP (23 kDa), leucurolysin-a (23 kDa), atroxlysin-I (23 kDa), BpirMP (23 kDa), BaP1 (24 kDa), BnP1 (24 kDa), BH2 (26 kDa), Batroxase (27 kDa by SDS-PAGE and 22.9 kDa by MALDI-TOF MS) and BaltMP-I (29 kDa) [ 25 – 28 , 31 , 32 , 41 – 44 ]. Nevertheless, it should be taken into account that some of those differences in the molecular masses could be attributable to the different sensitivity of the methodologies used for their resolution, e.g.…”

“…Most P-I SVMPs are fibrinogenolytic enzymes that preferentially degrade the Aα chains of fibrinogen, while also degrading the Bβ chains at slower ratios [ 6 ]. Examples of fibrinogenolytic metalloproteases from Bothrops venoms include BthMP, BmooMP-α, atroxlysin-I, Batroxase, BaltMP-I, BaP1, neuwiedase and BpirMP [ 25 , 26 , 28 , 30 – 32 , 41 , 44 ]. Some SVMPs also showed fibrinolytic activity, including BnP1, bothrojaractivase, BthMP, BpirMP, atroxlysin-I and Batroxase [ 28 , 31 , 32 , 41 , 42 , 50 ].…”

An alternative method to isolate protease and phospholipase A2 toxins from snake venoms based on partitioning of aqueous two-phase systems

“…Based on literature findings, we are able to assume that B. alternatus venom exhibits a variety of phospholipase A 2 and protease enzymes in its composition (30)(31)(32)(33)(34)(35)(36)(37)(38). At initial extraction steps all protein fractions are present in the system and each enzyme isoform is distributed between the phases, according to its structural properties.…”