Characterization of two arginine ester hydrolases from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0-8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-N-benzoyl-L-arginine methyl ester (BAME), p-tosyl-L-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The Km values for each enzyme with alpha-N-benzoyl-L-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.     

Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.     

Studies on the chemical modification of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom

The chemical modification of hemorrhagic toxin I (AaHI) from Agkistrodon acutus has been studied. Inactivation was observed upon modification of 3 out of 7 histidine residues with diethyl pyrocarbonate. The His residues are deblocked, accompanied by a return of activity, upon treatment with neutral hydroxylamine. The circular dichroism and fluorescence spectra of diethyl pyrocarbonate inactivated toxin and the native toxin are the same, indicating that modification with diethyl pyrocarbonate does not cause any gross change in the structure of the protein. At least one His residue may thus play an essential role in the enzyme activity. Reaction of the toxin with N-bromosuccimide abolished the enzyme activity, with modification of Trp, Tyr and His residues. The loss of Trp did not parallel the inactivation. Hydrogen peroxide, dioxane and 2-hydroxy-5-nitrobenzyl-bromide treatment damaged the Trp residues, but did not affect the activity. Therefore, the modified tryptophan side chains are not essential for activity. Modification of 2.5-3.0 Tyr residues out of 9 with acetylimidazole did not affect the enzyme activity, nor did nitration of the toxin with tetranitromethane. The reactive tyrosines are apparently not essential.     

尖吻蝮蛇蛇毒中毒镇痛成分的研究

目的 利用国产尖吻蝮蛇蛇毒筛选阿片受体激动剂,从中寻找无成瘾性新型镇痛药物。方法 通过层析分离方法,从国产尖吻蝮蛇蛇毒中获得电泳纯样品,采用微生理测定仪测定样品对CHO－μ受体细胞的活性,同时进行动物热板法镇痛试验,小鼠竖尾、跳跃式成瘾性试验。结果 从尖吻蝮蛇蛇毒中获得一分子量为1．2万的电泳纯蛋白质,且与CHO-μ受体细胞亲和力活性很高,该成分对小鼠具有较高的镇痛作用,无成瘾性,无毒性。结论 该成分具有较好的临床应用价值。     

Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutus)

Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight.     

烙铁头蛇毒中一个具有抗补体活性金属蛋白酶的纯化和性质研究

目的从烙铁头蛇毒中筛选分离抗补体活性蛋白,并对其部分生物学活性开展研究,以了解其在蛇伤中的病理生理作用和潜在的应用价值。方法采用蛋白层析技术分离纯化抗补体活性蛋白,测定其分子量、等电点、抗补体作用、多种蛋白水解酶活性、水肿活性及出血活性。结果从烙铁头蛇毒中分离纯化出一个抗补体活性蛋白TMAC-1,表观分子量约为25ku,等电点为9.0。TMAC-1能够抑制补体经典途径和替代途径的溶血,预孵条件下,其IC50分别为62、29mg·L-1;不预孵条件下,其IC50为263、246mg·L-1。TMAC-1能够裂解C3、C5,但裂解产物不能诱导内皮细胞P-selectin的表达。TMAC-1能依次降解纤维蛋白原的Aα、Bβ、γ链,该活性能被EDTA、1,10-phenanthroline、EGTA完全抑制,不受PMSF、SBTI的抑制。TMAC-1具有水肿活性和微弱的偶氮酪蛋白水解活性,没有精氨酸酯酶水解活性和皮下出血活性。结论 TMAC-1是一个非出血性的金属蛋白酶,它可通过酶切补体特定成分抑制补体激活。     

Isolation of coagulant and anticoagulant principles from the venom of Agkistrodon acutus

By means of zone electrophoresis on starch, the venom of Agkistrodon acutus could be separated into six fractions. The thrombin-like and esterase activities migrated toward anode and concentrated in Fraction 6. Both activities were about eleven times stronger than those of the crude venom and were inhibited by DFP. There were three anticoagulant principles which could prolong plasma prothrombin time. One migrated toward anode and two migrated toward cathode.

By means of DEAE Cellulose column chromatography, seven fractions were obtained. The thrombin-like and esterase activities were concentrated in Fraction 7, while anticoagulant activity was located in Fractions 1, 5 and 6.

By means of DEAE Sephadex column chromatography, the venom could be separated into twelve fractions. The thrombin-like and esterase activities were concentrated in Fraction 10. Both activities were about sixteen times stronger than those of the crude venom. There were also three anticoagulant principles which were found in Fractions 1, 6 and 7.

The thrombin-like activity was more heat stable than the anticoagulant activity. The caseinolytic activity was found in Fractions 1, 9 and 11. Other fractions had only weak caseinolytic activity.     

Purification and physiological study of a hypotensive factor from the venom of Vipera aspis aspis (aspic viper)

A hypotensive factor was isolated from the venom of Vipera aspis aspis by Sephadex G-75, S-Sepharose column chromatography, and reverse phase HPLC using a Develosil 300 ODS-7 column. The purified factor was a basic protein with a mol. wt of 25,000 and an isoelectric point of 7.95. Intravenous injection of hypotensive factor induced an immediate fall in blood pressure of rats, whose duration depended on the dose employed. The hypotensive response was not affected by dithiothreitol, beta-mercaptoethanol, EDTA, p-tosyl-L-phenylalanine chloromethylketone, p-chloromercuribenzoic acid, or diisopropyl fluorophosphate, and was resistant to heat-treatment at 100 degrees C for 30 min, however, it disappeared after incubation with antivenom prepared against the hypotensive factor. The factor is devoid of proteinase, esterase, phospholipase A2 and kallikrein-like activities, and lethal, hemorrhagic and capillary permeability increasing activities are also absent. Compared to the hypotension induced by the hypotensive factor in normotensive rats, a more potent response was observed when it was administered to 11-week-old spontaneously hypertensive rats.     

Species difference in the fibrinogenolytic effects of alpha- and beta-fibrinogenases from Trimeresurus mucrosquamatus snake venom

Alpha- and beta-fibrinogenases prepared from Trimeresurus mucrosquamatus venom digested specifically the alpha(A) and beta(B) chains of the fibrinogen molecule, respectively. alpha-Fibrinogenase digested bovine fibrinogen more markedly than human fibrinogen, while beta-fibrinogenase digested human fibrinogen more markedly than bovine fibrinogen. Human fibrin was also digested by both enzymes. Plasma fibrinogens of 4 animal species were digested by alpha-fibrinogenase to the same degree, while those by beta-fibrinogenase in the following order: human greater than dog greater than guinea-pig greater than rabbit. The fibrinogenolytic effects of alpha-fibrinogenase on human fibrinogen were strongly inhibited by sera of the 4 animal species, while those of beta-fibrinogenase were inhibited in the following order: rabbit greater than guinea-pig greater than dog greater than human. It was concluded that the different activities of the protease inhibitors in the plasma of animal species are mainly responsible for the sensitivity differences.     

Purification and characterization of anticoagulation factors from the venom of Agkistrodon acutus.

Two anticoagulants from five-pace snake (Agkistrodon acutus) venom, anticoagulation factor I (ACF I) and anticoagulation factor II (ACF II), have been purified by a multiple-step chromatography procedure of anion-exchange chromatography, gel permeation chromatography and cation-exchange chromatography. Each of them is shown to be homogeneous as judged by PAGE, SDS-PAGE and mass spectrometry. In vitro, both proteins show equivalent anticoagulant activity, and are devoid of proteolytic, esterolytic, L-amino acid oxidase, phospholipase A, thrombin-like, fibrinolytic, hemorrhagic and lethal activities. They have similar amino acid compositions with similar absorption coeffecients (A(1%)(280)) (30.5 for ACF I and 30.0 for ACF II). Both are disulfide-linked consisting of two 14.7 kD chains for ACF I and two 14.6 kD chains for ACFII. ACF I has a molecular mass of 29,604+/-8 atomic mass units (amu) compared to 29,468+/-6 amu for ACF II, determined by mass spectrometry. The isoelectric points of ACF I and ACF II are 5.7 and 7.0, respectively. We conclude that the two isoforms possess equivalent biological activities with similar amino acid compositions and molecular masses, but different isoelectric points.     

Hemorrhagic activity and mechanism of FⅡa′a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the local hemorrhagic activity of a fibrinolytic enzyme (FⅡa) from Agkistrodon acutus venom and its mechanism. METHODS: The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FⅡa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M 199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively. RESULTS: The minimum hemorrhagic dose (MHD) of FⅡa was 89 μg.In vitro, FⅡa (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50,0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FⅡa, HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.CONCLUSION: FⅡa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X,prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.     

Characterization of mucrotoxin A from the venom of Trimeresurus mucrosquamatus (the Chinese habu snake)

Mucrotoxin A from the venom of Trimeresurus mucrosquamatus was isolated in homogeneous form by a previously published method. Mucrotoxin A did not hydrolyze casein, however, when dimethylcasein was used as a substrate, the toxin cleaved the substrate. This toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The sites of cleavage in the oxidized B chain of insulin were identified as Ser(9)-His(10), His(10)-Leu(11), Ala(14)-Leu(15), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The toxin digested the A alpha chain of fibrinogen first, followed by hydrolysis of the B beta chain. The fact that no fibrin clot formed indicates that the sites of cleavage in the A alpha and B beta chains of fibrinogen by the toxin must be different from those cleaved by thrombin. Mucrotoxin A produced systemic hemorrhage in internal organs such as the heart and stomach.     

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon15, 161–167, 1977.—In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon acutus venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did ε-aminocaproic acid. Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due-to a direct action on fibrin. Both EDTA (5 × 10^-4 M) and cysteine (5 × 10^-3 M) completely inhibited the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the fibrinolytic principle.     

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism

Aim:

To evaluate the effects of the fibrinolytic enzyme FII_a from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models.

Methods:

Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography.

Results:

Intravenous administration of FIIa (0.1–5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FII_a infusion. FII_a (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FII_a (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways.

Conclusion:

FII_a could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.     

Comparison of kinin-forming and amidolytic activities of four trimucases, oedema-producing and kinin-releasing enzymes, from Trimeresurus mucrosquamatus venom.

Four kinin-releasing enzymes, trimucase I, II, III and IV, isolated from Trimeresurus mucrosquamatus venom (TMV) caused rat hind-paw swelling. Trimucase I and III were less potent than trimucase II and IV in this effect. Pretreatment with diphenhydramine or methysergide significantly reduced trimucase-induced paw swelling, while aspirin had no effect. Cellulose sulphate pretreatment suppressed the oedematous responses elicited by trimucases. The residual response was further depressed by diphenhydramine and methysergide. Trimucases also caused kinin generation in-vitro from rat plasma. This kinin-forming activity was in the order of trimucase II greater than IV greater than or equal to III greater than I greater than TMV. All trimucases hydrolysed chromogenic peptides N-benzoyl-Pro-Phe-Arg p-nitroanilide, N-benzoyl-Phe-Val-Arg p-nitroanilide and DL-Val-Leu-Arg p-nitroanilide; the order of this amidolytic activity was trimucase I greater than II greater than III greater than or equal to IV. These data indicate that the effects of venom kinin-releasing enzymes on plasma kininogen are not parallel to their amidolytic effects.     

Purification and characterization of L-amino acid oxidase from the venom of Trimeresurus mucrosquamatus (Taiwan habu snake)

L-Amino acid oxidase (EC.1.4.3.2) was purified to homogeneity via four steps consisting of Sephadex G-100, CM-Toyopearl 650M, and first and second granulated hydroxyapatite column chromatographies. The mol. wt of the enzyme was 140,000 when estimated by analytical gel filtration and was 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. The enzyme has an absorption spectrum characteristic of flavoprotein, contains 2 moles of FMN per mole of enzyme and has an isoelectric point of 5.4. The enzyme oxidatively deaminated hydrophobic amino acids such as Leu, Met, Phe, and Tyr while basic amino acids except for Lys were also oxidized though at slower rates. This specificity was generally similar, with some exceptions, to that of the enzyme from Trimeresurus flavoviridis venom. For oxidative deamination of Leu, Km and maximum velocity of the enzyme were 1.17 mM and 9.9 units/mg, respectively, at pH 7.6. The activity was inhibited almost completely by heavy metal ions, some aromatic benzoates and sulfhydryl reagents but not by metal-chelating agents.     

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用

尖吻蝮蛇毒和蝮蛇毒凝血酶样酶促凝的协同作用魏文利１，孙家钧，陈家树（中山医科大学药理教研室，广州５１００８９，中国）关键词尖吻蝮蛇；蝮蛇；蛇毒；凝血酶类；血液凝固目的：尖吻蝮蛇（ＤＡ）和蝮蛇（ＡＨ）蛇毒凝血酶样酶（ＴＬＥ）对促凝作用的协同效果．方法：...     

五步蛇蛇毒纤溶组分Ⅱ的分离和若干药效学的特征

五步蛇蛇毒经DEAE—sephadex A—50柱层析.获得15个蛋白组分。sephadex G—50柱再层析组分Ⅱ,呈单一个蛋白峰,聚丙酰胺凝胶电泳结果呈单一区带。狗血浆纤维蛋白平板法证实五步蛇蛇毒组分Ⅱ有溶解纤维蛋白的作用,同时狗血浆纤维蛋白热平板法还证实其溶解纤维蛋白的作用系直接性的。体外试管实验显示组分Ⅱ虽对纤维蛋白和纤维蛋白原均有溶解作用.但在浓度17.8mg·L~(-1)以下时.其溶解纤维蛋白活性相对较高。兔scO.1ml(500mg·L~(-1)组分Ⅱ未发现类似五步蛇全毒的出血反应。小白鼠腹腔注射纯化后的组分Ⅱ,LD_(50)为11.105 mg,kg~(-1).