Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.     

Purification of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

During the isolation of a capillary permeability-increasing enzyme from the venom of A. caliginosus, a kininogenase was also purified from the venom by gel filtration on Sephadex G-100, ion-exchange chromatographies on CM-Sephadex C-50 and DEAE-Sephadex A-50, and gel filtration on Sephadex G-75. By this procedure, 11 mg of the purified enzyme were obtained from 4 g of the venom. The purified enzyme was homogeneous by polyacrylamide disc gel electrophoresis at pH 8.3 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and did not show any caseinolytic or clotting activity. The purified enzyme released bradykinin from purified bovine high mol.wt kininogen. The capillary permeability was increased by injection of the purified enzyme into the depilated skin of the back of a rabbit. It is supposed that the capillary permeability-increasing activity exerted by the enzyme is due to the release of bradykinin.     

Purification and biochemical characterization of F II(a), a fibrinolytic enzyme from Agkistrodon acutus venom.

A fibrinolytic enzyme, F II(a), was isolated from Agkistrodon acutus venom by ion-exchange chromatography and gel filtration. F II(a) consisted of a single polypeptide chain with a molecular weight of 26,000 and an isoelectric point of 4.6. F II(a) was shown to solubilize fibrin and fibrinogen. F II(a) cleaved, primarily, the alpha chain of fibrinogen and fibrin followed by the beta chain, while the gamma chain was minimally affected. Thus, the enzyme was an alpha,beta-fibrinogenase. The cleavage pattern of fibrinogen clearly varied from plasmin cleavage of the same molecule. In vivo, F II(a) had no influence on the rat's tissue-type plasminogen activator and plasminogen activator inhibitor-1 activities in plasma. At the dosage of 5mg/kg, histological examination of heart, liver and lung tissue showed no hemorrhage. F II(a) is an enzyme that hydrolyzed fibrin directly without hemorrhagic activity.     

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.     

Purification of a kininogenase (kininogenase-2) from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

From the partially purified capillary permeability-increasing enzyme obtained from A. caliginosus venom, another kininogenase (kininogenase-2) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and SDS-gel electrophoresis. The kininogenase-2 had arginine ester hydrolytic and capillary permeability-increasing activities, and did not show any caseinolytic or clotting activity in a similar manner to a previously purified kininogenase (kininogenase-1). The purified kininogenase-2 liberated bradykinin on incubation with purified bovine high mol. wt kininogen. The rate of bradykinin release from the kininogen by kininogenase-2 was slower than that by the kininogenase-1, although both enzymes rapidly cleaved the peptide bonds in the kininogen molecule.     

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

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 μmole/min·mg, Kcat = 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 ± 12NIH thrombin units/mg protein enzyme.     

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.     

Some properties of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

A kininogenase (bradykinin-releasing enzyme) from the venom of A. caliginosus, is a single polypeptide-chain glycoprotein with a mol.wt of about 33,500, which contains 10.1% carbohydrate. The isoelectric point of the enzyme is 3.5 and the enzyme has 274 amino acid residues based on the mol.wt of 33,500. The enzyme hydrolyzed arginine esters more readily than lysine esters, but did not hydrolyze tyrosine ester. The activity of the enzyme on hydrolysis of arginine ester or on liberation of kinin from purified bovine high mol.wt kininogen was inhibited by diisopropylfluorophosphate, indicating that the serine hydroxyl group is involved in enzymatic activity. Moreover, the enzyme split N-alpha-carbobenzoxy-Gly-Pro-Arg-p-nitroanilide (PNA), H.D.Val-Leu-Arg-PNA, H.D.Pro-Phe-Arg-PNA, H.D.Phe-pipecolyl-Arg-PNA and Pro-Phe-Arg-4-methylcoumaryl-7-amide more readily than the other chromogenic or fluorogenic substrates. This result indicates that the substrate specificity of the enzyme is broader than that of mammalian serine proteinases.     

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.     

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.     

Enzymological characterization of FII(a), a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the enzymological characterization of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom. METHODS: The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII(a) was determined by atomic absorption spectroscopy. RESULTS: After incubation with FII(a) (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h , respectively. The molecular weights of major degradation products were 45,000 and 41,000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171+/-25 mg/kg), potassium (489+/-17 mg/kg) and calcium (319+/-13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA. CONCLUSION: FIIa can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FII(a) is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.     

Fibrinolytic enzyme from Agkistrodon halys brevicaudus (Korean mamushi) snake venom.

By means of DEAE-Sepharose CL-6B ion exchange chromatography and TSK-GEL G2000 SW high-performance gel filtration, a purified protein with fibrinolytic activity was obtained from the venom of Agkistrodon halys brevicaudus (Korean mamushi). The protein was homogeneous as judged by isoelectric focusing electrophoresis and high-performance gel filtration. Its mol. wt is 39,200 and its isoelectric point 4.12. The specific fibrinolytic activity of the protein was 3.2 times higher than that of the crude venom. The fibrinolytic activity of the purified principle was 33 units/mg protein (units of standard urokinase activity).     

Isolation and pharmacological characterization of a new alpha-neurotoxin (alpha-AgTx) from venom of the viper Agkistrodon halys (Pallas)

A hitherto unknown alpha-neurotoxin, alpha-agkistrodotoxin, was isolated from the venom of the pit viper Agkistrodon halys (Pallas). It's molecular weight was approx. 8000 +/- 80 (SDS-polyacrylamide electrophoresis). The toxin crossreacted with antiserum directed against alpha-bungarotoxin and inhibited binding of 125I-alpha-bungarotoxin to the nicotinic acetylcholine receptor of cultured myotubes (IC50 = 2 X 10(-9) M). The association and dissociation rates were 4.85 X 10(5) per mole per min and 3.55 X 10(-4) per min, respectively, giving a Kd of 7.3 X 10(-10) M. The toxin also inhibited carbachol-induced influx of cations through the nAChR (IC50 = 6 X 10(-8) M).     

白眉蝮蛇降纤酶的纯化及特性分析

目的从白眉蝮蛇蛇毒中提取降纤酶,并对其进行特性分析。方法采用一步亲和色谱法分离纯化降纤酶,用高效液相色谱分析其纯度,SDS-PAGE测定其相对分子质量。结果分离得到的降纤酶纯度高(99%以上),SDS-PAGE分析为一条带,相对分子质量为36 000,比活性为4 800 u/mg。结论用一步亲和色谱法从白眉蝮蛇蛇毒中纯化降纤酶的方法简单有效,所得产品纯度高、产量高。     

Purification and characterization of a phospholipase A2 from Cerastes cerastes (horn viper) snake venom

A single phospholipase A2 has been found in Cerastes cerastes venom, purified to homogeneity by a combination of chromatographic steps involving gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sephadex A-50. Its mol. wt, its amino acid composition and its partial amino acid sequence have been determined. High homologies between its sequence and those of other Viperid phospholipides A2 have been noticed. The phospholipase was non-lethal to mice up to a dose as high as 25 mg/kg by i.p. and i.v. injection. This non-toxic enzyme exhibited an acidic isoelectric point and hydrolyzed monolayers of different short chain phospholipids. Some kinetic parameters have been studied potentiometric titration (with or without Triton X-100) and the rate of catalysis seemed not to be affected by changes in the physical state of the substrate.     

Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom

A major phospholipase A2 (VRV PL-VIIIa) which constitutes 24% of the whole Vipera russelli venom was purified to homogeneity by CM-Sephadex C-25 column chromatography followed by gel filtration on Sephadex G-50. VRV PL-VIIIa is a basic protein with a molecular weight of 11,800 by SDS-PAGE. This enzyme contributes 45% of the total PLA2 activity of the venom, but it is least toxic compared to other purified basic PLA2 enzymes prepared from V. russelli venom. The LD50 value (i.p.) of VRV PL-VIIIa is 5.3 mg/kg body wt. It shows neurotoxic symptoms and damages vital organs such as lung, liver and kidney at LD50 doses. It induces myonecrosis when injected i.m. into the thigh muscle of mice and edema when injected into the foot pads.