RESUMEN
In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bß but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152-341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.
Asunto(s)
Anticoagulantes/aislamiento & purificación , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/aislamiento & purificación , Serina Proteasas/aislamiento & purificación , Trimeresurus/metabolismo , Animales , Anticoagulantes/metabolismo , Anticoagulantes/toxicidad , Coagulación Sanguínea/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cromatografía de Fase Inversa , Creatina Quinasa/sangre , Creatina Quinasa/metabolismo , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/toxicidad , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/metabolismo , Hemorragia/inducido químicamente , Humanos , Ratones , Peso Molecular , Serina Proteasas/metabolismo , Serina Proteasas/toxicidad , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Tiempo de Coagulación de la Sangre TotalRESUMEN
A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.
Asunto(s)
Quelantes/farmacología , Venenos de Crotálidos/antagonistas & inhibidores , Ácido Edético/farmacología , Hemorragia/tratamiento farmacológico , Metaloproteasas/antagonistas & inhibidores , Necrosis/tratamiento farmacológico , Mordeduras de Serpientes , Trimeresurus/fisiología , Animales , Antivenenos/química , Antivenenos/farmacología , Coagulación Sanguínea , Quelantes/química , Cromatografía en Gel , Creatina Quinasa/análisis , Creatina Quinasa/metabolismo , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/toxicidad , Ácido Edético/química , Hemorragia/patología , Hemorragia/prevención & control , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/metabolismo , Masculino , Metaloproteasas/química , Metaloproteasas/aislamiento & purificación , Metaloproteasas/toxicidad , Ratones , Peso Molecular , Músculos/efectos de los fármacos , Músculos/patología , Necrosis/patología , Necrosis/prevención & control , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Zinc/metabolismoRESUMEN
In the present study, we describe the purification and characterization of a metalloprotease 'trimarin' from Trimeresurus malabaricus snake venom. Trimarin is a single-chain basic protein, with a molecular mass of 29.6kDa. Trimarin showed proteolytic activity towards casein and fibrinogen, which was irreversibly inhibited by EDTA and 1,10-phenanthroline. The metal ion associated with trimarin was found to be Zn(2+). Trimarin exhibited pharmacological activities including hemorrhage, myotoxicity, procoagulant and factor Xa-like activities. The hemorrhage and myotoxicity correlated with degradation of extracellular protein components type-IV collagen and fibronectin. Myotoxicity due to muscle tissue necrosis was substantiated with increased serum CK activity. Trimarin showed procoagulant activity with reduced re-calcification time of citrated human plasma. Trimarin shortened the activated partial thromboplastin time (aPTT) and prothrombin time (PT), suggesting its involvement in common pathway of blood coagulation. Trimarin coagulated the citrated human plasma in the absence of CaCl(2), but it was lacking thrombin like activity as it did not clot the purified fibrinogen. Remarkably, the enzyme clotted the factor X deficient human plasma, suggesting that trimarin has factor Xa-like activity. Thus, trimarin may play a key role in the pathophysiological conditions that occur during T. malabaricus envenomation, and may be used as a biological tool to explore many facets of hemostasis.
Asunto(s)
Venenos de Crotálidos/química , Metaloendopeptidasas/química , Trimeresurus , Animales , Coagulación Sanguínea/efectos de los fármacos , Factor Xa/química , Factor Xa/metabolismo , Fibrinógeno/metabolismo , Humanos , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/farmacología , RatonesRESUMEN
Vanillic acid has been investigated for its inhibitory effect on Naja naja, Daboia russellii, and Trimeresurus malabaricus venom 5'-nucleotidase activity. Trimeresurus malabaricus venom 5'-nucleotidase activity was 1.3- and 8.0-fold higher than that of N. naja and D. russellii venoms, respectively. Substrate specificity studies showed that for all the venoms tested, 5'-AMP was the preferred substrate for 5'-nucleotidase. This indicates the central role of adenosine in snake envenomation. Vanillic acid selectively and specifically inhibited 5'-nucleotidase activity among several enzymes present in the three venoms tested. The inhibitor was competitive, as the inhibition was relieved by increased substrate concentration. It appears that the COOH group in vanillic acid is the determining factor for inhibition as vanillin, a structurally similar compound with respect to vanillic acid, had no inhibitory activity. This study for the first time exemplifies vanillic acid as a pharmacological tool in evaluating the role of 5'-nucleotidase in snake envenomation.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Venenos de Serpiente/enzimología , Ácido Vanílico/farmacología , 5'-Nucleotidasa/metabolismo , Animales , Propuestas de Licitación , Elapidae/metabolismo , Inhibidores Enzimáticos/química , Especificidad por Sustrato , Trimeresurus/metabolismo , Ácido Vanílico/químicaRESUMEN
The snake venom proteins affect hemostasis by either advancing/delaying blood coagulation. Apart from proteases and phospholipase A(2)s (PLA(2)s), 5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation. In this study, the possible involvement of Naja naja venom 5'nucleotidase in mediating anticoagulant affect is evaluated. Vanillic acid selectively and specifically inhibited 5'nucleotidase activity among other enzymes present in N. naja venom. It is a competitive inhibitor as evident of inhibition relieving upon increased substrate concentration. Vanillic acid dose dependently inhibited the anticoagulant effect of N. naja venom up to 40%. This partial involvement of 5'nucleotidase in mediating anticoagulant effect is substantiated by concanavalin-A (Con-A) inhibition studies. Con-A, competitively inhibited in vitro protease and 5'nucleotidase activity up to 100%. However, it did not exhibit inhibitory activity on PLA(2). The complete inhibition of anticoagulant effect by Con-A upon recalcification time suggests the participation of both 5'nucleotidase and protease in mediating anticoagulant effect of N. naja venom. Vanillic acid and Con-A inhibition studies together suggest that probably 5'nucleotidase interacts with one or more factors of intrinsic pathway of blood coagulation to bring about anticoagulant effect. Thus, this study for the first time demonstrates the involvement of 5'nucleotidase in mediating N. naja venom anticoagulant effect.
Asunto(s)
5'-Nucleotidasa/fisiología , Anticoagulantes/farmacología , Venenos Elapídicos/farmacología , Ácido Vanílico/farmacología , 5'-Nucleotidasa/antagonistas & inhibidores , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hemostasis/efectos de los fármacos , HumanosRESUMEN
The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.
Asunto(s)
Venenos de Crotálidos/toxicidad , Venenos Elapídicos/toxicidad , Metaloproteasas/metabolismo , Venenos de Víboras/toxicidad , Animales , Coagulación Sanguínea/efectos de los fármacos , Hidrolasas de Éster Carboxílico/metabolismo , Caseínas/metabolismo , Elapidae , Fibrinógeno/metabolismo , Hemostasis/efectos de los fármacos , Humanos , India , Metaloproteasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Tiempo de Protrombina , Trombina/metabolismo , Trimeresurus , ViperidaeRESUMEN
Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.