Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10⁻¹ M and the V_max 7.9 × 10⁻¹ nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom.TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.     

Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K_m 2.3 × 10⁻¹ M and the V_max 0.52 × 10⁻¹ nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.     

BthMP: a new weakly hemorrhagic metalloproteinase from Bothrops moojeni snake venom

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5 kDa, pI = 7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A₂ activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 °C. Assays with metal ions showed that Ca²⁺ is an activator, whereas Zn²⁺ and Hg²⁺ inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.     

Purification and characterization of alpha-mucrofibrase,a novel serine protease with alpha-fibrinogenase activity from the venom of Chinese Habu (Trimeresurus mucrosquamatus)

A novel fibrinogenolytic protease, named alpha-mucrofibrase, was purified from the venom of Chinese Habu (Trimeresurus mucrosquamatus) by DEAE-Sephadex A-50 ion-exchange chromatography and Sephadex G-100 (super fine) gel filtration alpha-Mucrofibrase is a single-chain polypeptide of approximately 29 kDa. It is stable even at 95 degrees C, and the most susceptible hydrolysis substrate is S-2302. It cleaved primarily the Aalpha chain of fibrinogen followed by the Bbeta chain, while the gamma chain was partially affected. N-terminal sequence of this fibrinogenolytic enzyme has great homology with those of other snake venom serine proteases. The esterase activity of alpha-mucrofibrase is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by metal chelator (EDTA), suggesting this fibrinogenase belongs to the venom serine protease family.     

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.     

cDNA cloning, expression and fibrin(ogen)olytic activity of two low-molecular weight snake venom metalloproteinases

Two cDNA clones, AplVMP1 and AplVMP2, were isolated from a snake (Agkistrodon piscivorus leucostoma) venom gland cDNA library. The full-length cDNA sequence of AplVMP1 with a calculated molecular mass of 46.61 kDa is 1233 bp in length. AplVMP1 encodes PI class metalloproteinase with an open reading frame of 411 amino acid residues that includes signal peptide, pro-domain and metalloproteinase domains. The full-length cDNA of the AplVMP2 (1371 bp) has a calculated molecular mass of 51.16 kDa and encodes PII class metalloproteinase. The open reading frame of AplVMP2 with a 457 amino acid residues is composed of signal peptide, pro-domain, metalloproteinase and disintegrin domains. AplVMP1 and AplVMP2 showed 85% and 93% amino acid identical to PI class enzyme Agkistrodon contortrix laticinctus ACLPREF and PII class enzyme Agkistrodon piscivorus piscivorus piscivostatin, respectively. When expressed in Escherichia coli, most of recombinant proteins of AplVMP1 and AplVMP2 were in insoluble inclusion bodies, with soluble yields of 0.7 mg/l and 0.4 mg/l bacterial culture, respectively. Both affinity purified recombinant proteins show proteolytic activity on fibrinogen, although having an activity lower than that of crude A. p. leucostoma venom. Proteolytic activities of AplVMP1 and AplVMP2 were completely abolished after incubation with a final concentration of 100 μM of EDTA or 1,10-phenanthroline. Both AplVMP1 and AplVMP2 were active in a fibrin-agarose plate but devoid of hemorrhagic activity when injected (up to 50 μg) subcutaneously into mice, and had no capacity to inhibit platelet aggregation.     

Amino acid sequence of a thrombin like enzyme, elegaxobin II, from the venom of Trimeresurus elegans (Sakishima-Habu).

The amino acid sequence of a thrombin like enzyme , named elegaxobin II, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, achromobacter protease I, trypsin, endoproteinase Asp-N, and chymotrypsin. Elegaxobin II consisted of 233 amino acids and showed conservation of the catalytic amino acid residues (His(57), Asp(102), and Ser(195)) of chymotrypsin family serine protease in its amino acid sequence. The carboxyterminal amino acid, Leu, was determined using carboxypeptidase Y. This enzyme contains glucosamine and an N-linked glycosylation site. Elegaxobin II was 91% homologous in sequence to elegaxobin and protease I from the same snake venom, and it was 67, 75, 31 and 26% homologous in sequences to flavoxobin, KN-BJ 2, human kallikrein and bovine thrombin, respectively. Elegaxobin II lacked thrombin's ETW (146-148) loop, as well as its functionally important YPPW (60-insertion loop).     

α-Fibrinogenase from Agkistrodon rhodostoma (Malayan pit viper) snake venom

By means of DEAE-Sephadex A-50 column chromatography, Agkistrodon rhodostoma (Malayan pit viper) snake venom was separated into eleven fractions. Fraction II had fibrinogenolytic activity, and when further purified by gel filtration was homogeneous, as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis. It had a single peptide chain with a molecular weight of 25,360 and an isoelectric point greater than 10. The fibrinogenolytic activity was completely destroyed after heating for 30 min at 60°C at pH 5.6, 7.4 or 8.8. This enzyme cleaved specifically the α(A) chain of monomeric fibrinogen, without cleaving the β(B) chain or γ chain. The specific fibrinogenolytic activity was 51 mg fibrinogen/min per mg protein. This enzyme showed proteolytic activities toward fibrinogen, fibrin and casein, but was devoid of phospholipase A and tosyl-l-arginine methylester esterase activities which are found in the crude venom. The fibrinogenolytic activity was inhibited by EDTA and cysteine, but not by ε-aminocaproic acid.     

Ammodytase, a metalloprotease from Vipera ammodytes ammodytes venom, possesses strong fibrinolytic activity

Ammodytase, a high molecular mass metalloproteinase with fibrinogenolytic and fibrinolytic activities, was purified from long-nosed viper (Vipera ammodytes ammodytes) venom by gel filtration, affinity and ion-exchange chromatographies. The enzyme is a single-chain glycoprotein with apparent molecular mass of 70 kDa and isoelectric point of 6.6. Ammodytase shows very weak hemorrhagic activity, and only at doses higher than 20 μg. Consistent with this, it partially degrades some components of the extracellular matrix in vitro. It cleaves the Aα-chain of fibrinogen preferentially at peptide bonds Glu⁴⁴¹-Leu⁴⁴² and Glu⁵³⁹-Phe⁵⁴⁰. Its preference for bulky and hydrophobic amino acids at the P1′ position in substrates is demonstrated by its hydrolysis of only the Gln⁴-His⁵ and Tyr¹⁶-Leu¹⁷ bonds in the B-chain of insulin. Ammodytase is able to dissolve fibrin clots. It neither activates nor degrades plasminogen and prothrombin, and has no effect on collagen- or ADP-induced platelet aggregation in vitro. LC/MS and MS/MS analyses of its tryptic fragments demonstrated that ammodytase is a P-III class snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. Its similarity to hemorrhagins from V. a. ammodytes venom, accompanied by very low toxicity, makes ammodytase a promising candidate as an antigen to prepare antisera against these most dangerous components of the viper's venom. Moreover, its ability to degrade fibrin clots suggests its clinical use as an antithrombotic agent.     

Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis

A new PLA₂ (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE ∼14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed K_m 6.0 mM, V_max 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 °C, and it had a strict requirement of Ca²⁺ for its activity. The N-terminal sequence of PLA₂ was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA₂ from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA₂ from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD₅₀ than crotoxin complex from C. d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 μg/ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes.     

Molecular cloning of novel serine proteases and phospholipases A2 from green pit viper (Trimeresurus albolabris) venom gland cDNA library.

Green pit viper (Trimeresurus albolabris) is the most common venomous snake responsible for bites in Bangkok. It causes local edema and systemic hypofibrinogenemia resulted from the thrombin-like, as well as the fibrinolytic effects of the venom. However, the amino acid sequences of these venom proteins have never been reported. In this study, we have cloned five novel serine proteases from the Thai T. albolabris venom gland cDNA library. They were all closely homologous to the corresponding serine proteases from Chinese green viper (Trimeresurus stejnegeri), suggesting the evolutionary proximity of the two species. In addition, their functional activities could be deduced. There were predicted to be two thrombin-like enzymes (GPV-TL1 and GPV-TL-2), two isoforms of a fibrinogenolytic enzyme (albofibrase) and a plasminogen activator (GPV-PA), suggesting that defibrination syndrome in patients is a combination of these enzymatic effects. By multiple sequence alignment, no conserved residue or motif responsible for distinct functions of snake venom serine proteases could be observed. Moreover, one Lys 49 and one Asn 49 phospholipase A2 (PLA2) genes were cloned. Lys 49 PLA2 was predicted to devoid of catalytic activity, but showed a carboxy terminal cytotoxic region. No Asp 49 PLA2 was found in 150 clones screened. This explains the marked limb edema but no hemolysis in patients. These novel serine proteases have potentials to be therapeutic anti-thrombotic and thrombolytic agents in the future.     

Molecular cloning of serine proteases from elapid snake venoms

Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine protease, but there was no structural evidence previously. In the present study, the primary structure of OhS1 was determined by protein sequencing, in combination with RT-PCR and 5′-RACE methods. OhS1 precursor is composed of an 18-amino acid signal peptide, a 6-amino acid putative activation peptide and 236-amino acid mature protein. OhS1 homologues from Naja atra and Bungarus multicinctus were also cloned and reported. These elapid venom serine proteases exhibited 60% sequence identity with serine proteases from the snake venoms of the Viperidae and Colubridae family. Phylogenetic analysis indicated that snake venom serine protease might have a common ancestor.     

Anticoagulant activity of Moon jellyfish (Aurelia aurita) tentacle extract

Moon jellyfish (Aurelia aurita) tentacle extract was studied for its anticoagulant activity in vitro. The Jellyfish Tentacle Extract (JFTE) showed very strong fibrinogenolytic activity by cleaving Aα and Bβ chain of fibrinogen molecule. The fibrinogenolytic activity was found to be stronger than some snake venom derived anticoagulants. JFTE also completely liquefied fibrin clots in 24 h. JFTE was found to contain both high and low molecular weight proteins/peptides. The fibrinogenolysis appears to be caused by high molecular weight fractions of the extract. It has been also noted that PMSF significantly reduced fibrinogenolytic activity and heating totally abolished it. Autolytic degradation of the high molecular weight protein was also noted. Autolysis slowed down, but did not abolish the fibrinogenolytic activity of the extract.     

Amino acid sequence of a kinin-releasing enzyme, KR-E-1, from the venom of Agkistrodon caliginosus (Kankoku-mamushi)

The amino acid sequence of a bradykinin-releasing enzyme, named KR-E-1, isolated from the venom of Agkistrodon caliginosus (Kankoku-mamushi) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, hydroxylamine, achromobacter protease I, trypsin, V8 protease, arginine endopeptidase, and endoproteinase Asp-N. KR-E-1 consisted of 235 amino acids and showed conservation of the catalytic amino acid residues (His⁵⁷, Asp¹⁰², and Ser¹⁹⁵) of the chymotrypsin family of serine protease in its amino acid sequence. The carboxy-terminal amino acid, Phe, was determined using carboxypeptidase Y. This enzyme contains glucosamine and an N-linked glycosylation site. KR-E-1 showed 32, 31, 65, 65, and 67% sequence homology to human kallikrein, bovine thrombin, KN-BJ 2, elegaxobin, and elegaxobin II, respectively. The characteristic of structure of KR-E-1 was found to involve hydrophobic amino acid residues abundantly localizing in positions 1-50, with lysine residues abundantly localizing in positions 73-101.     

Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu).

A thrombin like enzyme, named elegaxobin II, with Lys-bradykinin releasing activity was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel-filtration on Sephadex G-100, and ion-exchange chromatography on the Q-Sepharose Fast Flow. By this procedure, about 9mg of purified enzyme was obtained from 1.1g of the venom. The purified enzyme showed a single protein band, the molecular weight of which was estimated to be about 35,000Da by sodium dodecyl sulfate-PAGE) under reducing condition, and this enzyme was found to contain a carbohydrate moiety. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 250 TAME units/mg of protein. This enzyme clotted only rabbit fibrinogen, whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin conversion, this enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas it did not release fibrinopeptide B. Furthermore, elegaxobin II released Lys-bradykinin when the enzyme was incubated with bovine plasma. The esterase activity was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), suggesting that this enzyme is a serine protease. The N-terminal sequence (Val-Ile-Gly-Gly) of this enzyme was identical to the typical sequence of serine proteinases.     

Venom from the centipede Scolopendra viridis Say: Purification, gene cloning and phylogenetic analysis of a phospholipase A2

Venom components from the centipede Scolopendra viridis Say were studied, using both the soluble venom and a cDNA library prepared from mRNA of the venomous glands. Separation of the soluble venom by high performance liquid chromatography (HPLC) permitted to obtain at least 54 different fractions. The fraction eluting at 46.24 min showed phospholipase activity. The enzyme was purified to homogeneity and the first 25 amino acid residues were identified by Edman degradation. From the cDNA library several genes were cloned, one of which codes for a protein with identical amino acid sequence as the one experimentally determined. The cloned gene codes for a signal peptide of 28 amino acids and a mature peptide of 119 residues. The molecular weight of the enzyme was estimated by mass spectrometry and shown to be 13,752 Da, which matches exactly with the molecular mass expected from the deduced amino acid sequence of the gene. Phylogenetic analysis of this sequence, in comparison with other known from venomous animals, showed that it is more similar to snake phospholipases than to insect or arachnid sequences, suggesting that it has been submitted to convergent evolution. To the best of our knowledge this is the first time that a phospholipase from this species of animal is fully characterized. We have named it Scol/Pla.     

Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom

A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC₅₀ of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca²⁺]_i measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca²⁺ releasing from Ca²⁺ store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.     

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C₁₆₄I₁₆₅M₁₆₆ motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity.     

Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice

Systemic alterations induced by a Bothrops alternatus hemorrhagin, named baltergin, a 55 kDa fibrinogenolytic metalloproteinase isolated from venom of north-eastern Argentina specimens, were studied in mice. It caused macroscopic hemorrhagic spots in lungs which was injected intravenously with a minimum pulmonary hemorrhagic dose of 10 μg. Histological observations of lungs showed mainly hemorrhagic areas, evidenced by the presence of erythrocytes in the alveolar spaces, congestion and increase of thickness of alveolar septum due to polymorphonuclear infiltrate and mononuclear cells. Neither macroscopic hemorrhage in other organs nor histological alterations in heart and cerebrum/cerebellum were observed at doses assayed. However, kidney and liver were mildly affected. Kidney examination revealed congestion, subcapsular hemorrhage with local capsule detachment, inflammatory infiltrate and degeneration of tubular cells. Congestion of blood vessels and hydropic degeneration of hepatocytes were observed in liver. Besides, baltergin was able to further hydrolyze type IV collagen. Although the enzyme showed to be less lethal than whole venom, it induced severe pulmonary bleeding and affected kinder and liver in minor grade. In conclusion, baltergin is able to alter the integrity of capillary vessels and simultaneously, to interfere on the hemostatic system. Thus, this metalloproteinase contribute markedly to systemic alterations characteristic of B. alternatus envenomations.     

Biochemical characterization of a thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon saxatilis) venom.

A thrombin-like enzyme and a fibrinolytic serine protease were purified to homogeneity from the venom of a Korean snake Agkistrodon saxatilis emelianov. Both the purified enzymes migrated as a single protein band corresponding to 39 kDa in SDS-PAGE. However, the molecular mass was reduced to 28 kDa by enzymatic removal of the N-linked carbohydrates in those two different enzyme species. Although the thrombin-like enzyme and the fibrinolytic protease show homologous features in their molecular sizes and N-terminal amino acid sequences, yet they can be clearly distinguished from each other in terms of substrate specificity, susceptibility to inhibitors and fibrinogen degradation. It is postulated that these two enzymes are capable of functioning in a cooperative manner to effectively remove fibrinogen and consequently to reduce the blood viscosity.