Arinos Magalhães

Action of metalloproteinases mutalysin I and II on several components of the hemostatic and fibrinolytic systems.

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) have been previously isolated from bushmaster (Lachesis muta muta) snake venom. Hemorrhagic activity was observed with as little as 0.5 microg (2000 units/mg) and 17.8 microg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aalpha>Bbeta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5. 25 and 16.3 micromol fibrinogen/min/micromol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not inhibited by serine proteinase inhibitors (SERPINS). Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 microM) completely digested the alpha- and gamma-gamma chains and partially the beta-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the alpha-chain of fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g., streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently blocked aggregation of human PRP elicited by 10 microg/mL of collagen with an IC(50) of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished by alpha2-macroglobulin (alpha2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by alpha2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom.

Characterization of the local tissue damage induced by LHF-II, a metalloproteinase with weak hemorrhagic activity isolated from Lachesis muta muta snake venom

Local tissue damage induced by LHF-II, a 22-kDa hemorrhagic metalloproteinase from Lachesis muta venom was studied. Intravital microscopy experiments evidenced hemorrhagic events 2 min after LHF-II application onto cremaster muscle, characterized by microhemorrhages in capillary vessels and venules. However, histological analysis showed only mild hemorrhage in the gastrocnemius muscle. LHF-II degraded laminin, fibronectin and type IV collagen upon incubation in vitro, but was not cytotoxic to capillary endothelial cells in culture. Intramuscular injection of LHF-II induced a mild myonecrosis, with early small increments in plasma creatine kinase activity. It also induced edema in the mouse footpad at doses where hemorrhage is absent. Injection of LHF-II induced the synthesis of matrix metalloproteinases evidenced in muscle homogenates and in exudate samples. It is concluded that LHF-II has weak hemorrhagic and myotoxic activities, and that its role in the pathogenesis of L. muta-induced local tissue damage is associated with edema formation and degradation of extracellular matrix components, either directly or by activation of endogenous matrix metalloproteinases.

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.

Purification of a hemorrhagic factor (LHF-I) from the venom of the bushmaster snake, lachesis muta muta

Hemorrhagic factor (LHF-I), was isolated from Lachesis muta muta venom by a five-step procedure. Homogeneity was demonstrated by the presence of a single band on polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion. LHF-I is a glycoprotein with molecular weight of approximately 100,000 as determined by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. Caseinolytic activity was associated with the hemorrhagic activity throughout the purification procedure.

Purification and properties of the thrombin-like enzyme from the venom of Lachesis muta mvta

Abstract 1. 1. The coagulating enzyme of the Lachesis muta muta venom was purified to homogeneity by a combination of a gel filtration in Sephadex G-100 and affinity chromatography on agarose-agmatine resin. 2. 2. Several forms of the enzyme were prepared by isoelectric focusing with pIs ranging from 3.1 to 5.0; the asialoenzyme focused as a narrow band at pH 8.7. SDS-PAGE analysis of the purified enzyme revealed a single broad band with apparent M r of 41–47 kDa. 3. 3. The enzyme cleaves only fibrinopeptide A from fibrinogen; it does not activate factor XIII and is devoid of kallikrein-like activity. 4. 4. Kinetic properties of the enzyme were determined for representative synthetic chromogenic substrates and inhibitors.

Purification and partial characterization of a thrombin-like enzyme from the venom of the bushmaster snake, Lachesis muta noctiv aga

Abstract A thrombin-like enzyme has been purified from the venom of Lachesis muta noctivaga (41-fold purification and 43% yield). The steps included: gel filtration with Sephadex G-100, hydroxylapatite and DEAE-cellulose chromatography, and finally Sephadex G-150 filtration (twice). The material was homogeneous in polyacrylamide electrophoresis and gel filtration on Sephadex G-150. The enzyme is a glycoprotein of molecular weight 36,300 as determined by gel filtration. the thrombin-like enzyme hydrolyses tosyl- l -arginine methyl ester ( Km = 1·45 × 10 −4 M, Vm = 353 μ mole/min·mg, K cat = 212sec −1 ) with an optimum pH of 8·30. The enzyme also hydrolyses α- N -benzoyl- dl -arginine p -nitroanilide and is competitively inhibited by benzamidine, β-naphtamidine and phenylguanidine. Clotting and amidase activities are inhibited by diisopropylfluorophosphate. The enzyme molarity was determined by active site titration with p -nitrophenyl- p ′-guanidino benzoate (92% pure). Injected in dogs 2 μg of the purified enzyme reduce, in 30 min, the plasma fibrinogen concentration to values less than 15% of the original level. In vitro the activity for human fibrinogen-fibrin conversion was equivalent to 1650 ± 12 NIH thrombin units/mg protein enzyme.

Purification and characterization of a fibrinogen-clotting enzyme from the venom of jararacuçu (Bothrops jararacussu)

A clotting enzyme of the venom of Bothrops jararacussu, denoted FC-Bj, was purified by gel chromatography on Sephadex G-100 followed by HPLC on DEAE-5PW-PAK and gel filtration on Sephacryl S-200HR. The enzyme was identified as an acidic glycoprotein which probably consists of a single polypeptide chain with isoelectric point values in the range 3.3-4.4 and containing approx. 19% carbohydrates. On polyacrylamide gel electrophoresis (PAGE) at pH 8.3, the enzyme presented a diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the enzyme showed two protein bands corresponding to mol. wts of 50,600 and 60,000. After treatment of the enzyme with neuraminidase, a strongly stained band and a band weaker in staining intensity were observed on SDS-PAGE, thereby reducing the mol. wts to 44,500 and 56,300, respectively. The clotting factor possessed N-alpha-benzoyl-DL-arginine p-nitroanilide hydrolysing activity and coagulated fibrinogen to fibrin. These activities were 0.548 units/mg and 50.55 NIH thrombin units/mg, respectively. The proteinase was of the serine type, as indicated by sensitivity to phenylmethanesulfonyl fluoride and benzamidine. However, the amidolytic activity of this enzyme was resistant to inhibitors such as heparin, aprotinin, agmatine, EDTA, I-2581 and TLCK. The importance of disulfide bridges for the structural integrity of the purified enzyme was indicated by the loss of amidolytic activity in the presence of beta-mercaptoethanol and dithiothreitol. SDS-PAGE of fibrinogen degraded with this enzyme revealed the disappearance of the A alpha and B beta chains and the appearance of lower mol. wt fragments. The enzyme was able to hydrolyse synthetic chromogenic substrates with arginine as the C-terminal residue, and the kinetic parameters were determined. It hydrolysed the plasma kallikrein substrate H-D-Pro-Phe-Arg-pNA (S-2302) and produced kinin-releasing activity causing ileum contraction. In addition, hypotension and bradycardia were observed in urethane-anesthetized rats upon i.v. injection of the enzyme.

Coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes.

Two isoforms of a thrombin-like enzyme designated TLE-B and TLE-P were purified from the venoms of Lachesis muta muta (bushmaster) snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 44000 and 43000 under reducing conditions on SDS-PAGE, which decreased to 27000 after deglycosylation with N-glycosidase F (PNGase F). The purified proteinases split off fibrinopeptide A rapidly from human fibrinogen and fibrinopeptide B more slowly. In addition, both enzymes released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. Kinetic properties of these enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, Western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as the venoms of L. muta muta and Bothrops snakes. Incubation of human alpha2-macroglobulin (alpha2-M) with each enzyme at molar ratios of 1:1, 1:2 and 1:4 enzyme:inhibitor resulted in retarding their clotting activities by approximately 12 times, whereas their amidolytic activities were not affected. However, the Mr 180000 subunits of alpha2-M were not cleaved by these enzymes, suggesting that alpha2-M inhibits TLEs by steric hindrance. Similarly, inhibitions of their clotting activities were obtained using high concentrations of rabbit IgG (40 microg, corresponding to molar ratio enzyme:inhibitor of 1:2) against TLE-B.

Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human α2-macroglobulin and rabbit immunoglobulin

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

Screening of expression libraries using ELISA: identification of immunogenic proteins from Tityus bahiensis and Tityus serrulatus venom

The present report describes the use of ELISA with cDNA expression libraries in the identification of immunogenic proteins. The methodology described was applied using libraries constructed with mRNA isolated from Tityus serrulatus and Tityus bahiensis venom glands. In addition we describe for the first time the sequence of a neurotoxin from Tityus bahiensis venom gland named TbTx5 whose amino acid sequencing showed 93% similarity with the Tityus bahiensis TbTx IV-5 neurotoxin. The methodology described can be used for the generation of an immunogenic bank in order to contribute to genome and proteome projects.