Venom Proteases Research Articles

Isolation and characterization of a protein C activator from tropical moccasin venom

A protease from the venom of the tropical moccasin (Agkistrodon bilineatus) that activates protein C was purified to homogeneity by ion-exchange and gel permeation chromatography. The purified protease is a glycoprotein, and exhibited a molecular weight of 35,000 and 38,000 in SDS-PAGE under non-reducing and reducing conditions, respectively. The purified protease readily activated human protein C and steady-state kinetic parameters indicated an apparent Km for human protein C of 1.7 μM and an apparent kcat of 0.02 sec −1. Calcium inhibited the activation of human protein C by the venom protease (K 1=93 μM). Amino-terminal sequence analysis revealed that the tropical moccasin protein C activator was highly homologous to the protein C activator isolated from Southern copperhead venom.

Effect of king cobra venom on α2-macroglobulin and proteases in human blood plasma

Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro-Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0.1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α2-macroglobulin. On chromatography on Sephadex G-200, α2-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with aVe/Vo value of 2.5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α2-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the α 2 − acroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasmain situ causes limited cleavage of α2-macroglobulin as well as α2-macroglobulin bound proteases, inactivating them.

Effect of a Crotalus basiliscus ( Mexican West Coast rattlesnake) venom proteinase (B2) on complement : Armando Varela-Ramirez1 and Eppie D. Rael2 ( 1Departamento de Investigacion, Facultad de Medicina, Universidad Autonoma de Chihuahua, Mexico, and 2Départment of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U.S.A.)

Effect of a Crotalus basiliscus ( Mexican West Coast rattlesnake) venom proteinase (B2) on complement : Armando Varela-Ramirez1 and Eppie D. Rael2 ( 1Departamento de Investigacion, Facultad de Medicina, Universidad Autonoma de Chihuahua, Mexico, and 2Départment of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968, U.S.A.)

Proteolytic and Immunologic Comparison of Human and Bovine von Willebrand Factor

The structures of bovine and human vWF were compared by proteolysis with Staphylococcus aureus V8 protease and rattlesnake venom Protease I. Fragments were analyzed for chain composition, heparin binding, collagen binding, platelet agglutinating activity and recognition by a panel of monoclonal antibodies which reacted with both bovine and human vWF. Similar large fragments from the C-terminal domain of vWF were seen in each case. The N-terminal domain resulting from cleavage of bovine vWF was much smaller than that seen upon digestion of human vWF with V8 protease. Protease I destroyed the heparin binding domain in human vWF. Bovine vWF was much less sensitive to proteolysis than was human vWF.

Isolation of human blood coagulation α-factor X a by soybean trypsin inhibitor-Sepharose chromatography and its active-site titration with fluorescein mono- p-guanidinobenzoate

A method based on active-site affinity chromatography on soybean trypsin inhibitor (SBTI)-Sepharose was developed for isolation of human factor X a in primarily the undegraded α-form. The chromatography procedure separated factor X a from factor X, the Russell's viper venom proteinase used to activate factor X, and traces of contaminating thrombin. α-Factor X a was unstable at pH 7.6 and 25 °C, undergoing slow proteolytic degradation to functionally heterogeneous products as evidenced by the greater loss of coagulation assay activity compared to activity measured with a chromogenic substrate. The results of monitoring factor X a degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with proteolysis of the light chain as a major component reaction occurring in parallel with slower proteolysis of the heavy chain. The decreased rates of these reactions at pH 6.0 enabled isolation and storage of factor X a in ⩾88% α-form and minimized the heterogeneity due to proteolytic degradation. Characterization of the reaction of fluorescein mono- p-guanidinohenzoate (FMGB) with human and bovine factor X a isolated by SBTI-Sepharose chromatography demonstrated its utility as a sensitive reagent for continuous fluorometric active-site titration. Analysis of the reaction kinetics as a function of FMGB and human factor X a concentrations in Γ/2 0.3, pH 7.4, buffer at 25 °C indicated that the ratio of acylation to deacylation rate constants was >200 and that the K m for FMGB was 0.06–0.11 μ m, predicting pre-steadystate burst amplitudes of ⩾96–98% of the active-site concentration at FMGB concentrations ⩾5 μ m. Human factor X a active-site concentrations were consistent with 82–99% active preparations when compared with the protein concentrations determined from the 280-nm absorbance. Concentrations of human α-factor X a as low as 20 n m could be measured with FMGB, indicating a sensitivity approximately 50 times greater than that measured by spectrophotometric active-site titration with p-nitophenyl p′-guanidinobenzoate.

Effects of venom proteases on peptide chromogenic substrates and bovine prothrombin

C.-M. Teng, J.-P. Wang, T.-F. Huang and M.-Y. Liau. Effects of venom proteases on peptide chromogenic substrates and bovine prothrombin. Toxicon 27, 161–167, 1989.—Eighteen proteases were isolated from six hemorrhagic venoms of snakes belonging to the families of Crotalidae and Viperidae. According to their actions, they are classified as thrombin-like enzymes, α-fibrinogenases, β-fibrinogenases, Factor X activator, prothrombin activator, hemorrhagins and esterases. Thrombin-like enzymes, β-fibrinogenases, hemorrhagins and esterase hydrolyzed Phe-Pip-Arg- pNA (S-2238, substrate for thrombin) more strongly than CBZ-Ile-Glu-Gly-Arg- pNA (S-2222, substrate for Factor Xa), CBZ-Phe-Val-Arg- pNA (B-7632) or CBZ-Pro-Phe-Arg- pNA (B-2133). Thrombin-like enzymes, β-fibrinogenase and esterase hydrolyzed tosyl- l-arginine methyl ester and benzoyl- l-arginine ethyl ester. S-2238 is the most susceptible chromogenic substrate for most venom proteases. Thrombin-like enzymes degraded prothrombin molecule progressively down to prethrombin 2 while α-and β-fibrinogenases degraded it only to prethrombin 1. Factor X activator of Vipera russelli venom and esterase of T. mucrosquamatus venom did not have any effect on prothrombin. Thus, the effects of venom proteases on prothrombin are not parallel to their amidolytic or esterolytic effects.

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. 1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe- Ser-Arg-MCA. 2. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. 3. The Bβ chain of fibrinogen was first split and Aα chain was cleaved later. Pancreatic kallikrein hydrolyzed only the Aα chain without affecting the Bβ chain. 4. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH 2) was also cleaved at the site of the Arg-Ser bond. 6. 6. Its NH 2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe- Leu-Val-Ala-Leu-Tyr-Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.

Thrombolysis with a snake venom protease in a rat model of venous thrombosis

A fibrin(ogen)olytic protease isolated from the venom of Crotalus atrox (the western diamondback rattlesnake) was tested for thrombolytic activity. The protease, called atroxase, solubilized fibrin when tested on fibrin plates and hydrolyzed fibrinogen rendering it incoagulable with a specific fibrinogenolytic activity of 42 mg fibrinogen/min/mg protein. Atroxase was unable to activate plasminogen. In vivo , fibrinolytic activity was tested on artificial thrombi induced in the posterior vena cava of Sprague-Dawley rats. Thrombolysis was then characterized by angiographic techniques over a period of three hours. Intravenous administation of the protease, at a dosage of 6.0 mg/kg, resulted in thrombolysis within one hour followed by recanalization of the originally occluded vein within two hours. Fibrinogenolytic activity resulted in a 60% decrease in the rat's plasma fibrinogen level. Histological examination of kidney, liver, heart and lung tissue showed no necrosis nor hemorrhage. These results are the first step in evaluating the thrombolytic potential of anticoagulant proteases within C. atrox venom using laboratory animals.

A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor.

The generation of two cleavage products of human C3, termed C3o and C3p, by incubation with a C3-cleaving protease isolated from cobra venom (Naja naja siamensis) is described. The venom protease removes the C3p fragment (Mr approximately 33,000) from the C3dg region of the C3 alpha-chain. The major cleavage fragment C3o (Mr approximately 140,000) contains the unaltered beta-chain of C3 and two alpha-chain-derived polypeptides of Mr approximately 29,000 and Mr approximately 38,000, respectively. Amino-terminal amino acids sequence analysis of C3p and the three chains of C3o allowed the identification of the exact location of the two alpha-chain-derived fragments of C3o and the three cleavage sites of the venom protease. The chain structure of C3o resembles those of C3c and cobra venom factor. In contrast to C3c but like cobra venom factor (and C3b), C3o was found to support the activation of the serine protease Factor B by cleavage in the presence of Factor D and Mg2+ into Bb and Ba, generating an enzymatically active complex that is able to cleave a fluorogenic peptide substrate for C3 convertases. Since the only stretch of amino acid residues of C3o not present in C3c is the carboxyl terminus of the Mr approximately 29,000 chain of C3o, it is suggested that this region is important for the interaction with Factor B and convertase formation.

Effect of cobra and viper venoms on α2-macroglobulin activity in human, bovine, and goat sera

Incubation of human serum with cobra or viper venoms ( 10 μ g 0.1 ml serum) caused negligible decrease in total protease inhibitory activity whereas α 2-macroglobulin activity was reduced by 67.0–82.0% in 16 hr. The action of venoms on MG activity was time dependent. Human α 2-macroglobulin activity was reduced to a much greater extent than goat or bovine factors by the venoms. While 25 μ g venoms 0.1 ml serum caused 60–100% inhibition of human α 2-macroglobulin activity, the bovine factor was not affected under similar conditions. Goat α 2-macroglobulin was affected to the extent of 0–20%. Evidence is provided to show that venom proteases generate endogenous proteases in situ in human plasma or serum which in turn bind to α 2-macroglobulin. The venom-mediated action was abolished by prior dialysis of the serum or its dilution. Ethylenediaminetetraacetate at 10 −3 m concentration also blocked the reaction. While phenylmethylsulfonyl fluoride had no effect, pepstatin in the concentration range 10 −2 to 10 −3 m caused partial inhibition of the venom-mediated inhibition of α 2-macroglobulin activity in human serum.

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom.

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.

Purification, characterization and substrate specificity of a basic proteinase in the venom of Habu ( Trimeresurus flavoriridis)

A basic proteinase was purified and characterized from the venom of Habu ( Trimeresurus flavoviridis). Its molecular weight, isoelectric point and optimum pH were approx. 24 000, 9.2 and 9, respectively. Susceptibility to several reagents was examined. The proteinase had endopeptidase activity cleaving the Gly-Leu bond in synthetic peptides but no exopeptidase activity. It did not hydrolyze a peptide, Z-Gly-Pro-Leu-Gly-Pro, which had been a good substrate for the major proteinase in the venom. The proteinase cleaved oxidized insulin B chain at five positions: His 10-Leu 11, Ala 14-Leu 15, Tyr 16-Leu 17, Gly 23-Phe 24 and Phe 24-Phe 25. From the disappearance of intermediate peptides and the peptides accumulated, the order and the intensity of cleavage of these positions were determined, and the substrate specificity was compared with those hitherto described for hemorrhagic and nonhemorrhagic venom proteinases.

Purification and characterization of α2-, α2- β- and β-macroglobulin inhibitors in the hedgehog, Erinaceus europaeus: β-macroglobulin identified as the plasma antihemorrhagic factor

C. A. de Wit and B. R. Weström. Purification and characterization of α 2-, α 2- β- and β-macroglobulin inhibitors in the hedgehog, Erinaceus europaeus: β-macroglobulin identified as the plasma antihemorrhagic factor. Toxicon 25, 1209 – 1219, 1987. — Three macroglobulin inhibitors were purified from hedgehog ( Erinaceus europaeus) plasma by sequential chromatography on Cibacron Blue Sepharose, Sephacryl S-200 and preparative agarose gel electrophoresis. Each macroglobulin was characterized for proteinase inhibiting activity, molecular weight by polyacrylamide gel electrophoresis (PAGE), subunit size by sodium dodecyl sulfate (SDS)-PAGE, immunological cross-reactivity to other macroglobulins and antihemorrhagic activity against European viper ( Vipera berus) venom. Hedgehog α 2-macroglobulin is a tetramer ( M r 800,000) composed of identical monomers ( M r 200,000) that inhibits all proteinases tested and is the homologue of human α 2-macroglobulin, rat α 2-acute phase globulin, dog α 1-macroglobulin and swine α 2-macroglobulin fast. Hedgehog α 2- β-macroglobulin is a dimer ( M r 450 – 550,000) composed of identical monomers ( M r 200,000) that inhibits all proteinases tested and appears to be structurally similar to other animal ‘;half-molecule’ macroglobulins. Hedgehog β-macroglobulin ( M r 700,000) gave subunits of 34,000 and 39,000 after SDS-PAGE and showed cross-reactivity with swine α 2-macroglobulin slow. It inhibits all proteinases tested and is the only macroglobulin with antihemorrhagic activity against V. berus venom. This antihemorrhagic activity may be due to β-macroglobulin's different structure as compared to other macroglobulins, which may make it less susceptible to inactivation by venom proteinases.

Inhibition of trypsin-like snake venom proteinases by derivatives of benzamidine: J. Stürzebecher (Institute of Pharmacology and Toxicology, Medical Academy Erfurt, DDR-5010 Erfurt, G.D.R.)

Inhibition of trypsin-like snake venom proteinases by derivatives of benzamidine: J. Stürzebecher (Institute of Pharmacology and Toxicology, Medical Academy Erfurt, DDR-5010 Erfurt, G.D.R.)

Inhibition of batroxobin, a serine proteinase from Bothrops snake venom, by derivatives of benzamidine

Benzamidine derivatives which are competitive inhibitors of trypsin-like serine proteinases also inhibited the enzymatic activity of batroxobin, a thrombin-like snake venom proteinase. Structure — activity relationships showed that primary amides of 4-amidinophenyl-α-aminobutyric acid have pronounced, relatively selective antibatroboxin activity. Identical effects were found on batroxobin isolated from the venoms of Bothrops atrox or Bothrops moojeni. Esters containing a benzamidine moiety acylated the active centre serine hydroxyl of either batroxobin, however, the inhibition was temporary. Such compounds, especially 4-amidinophenyl esters of substituted benzoic acids, are a particularly useful tool for designing acyl-batroxobin intermediates with different deacylation rates. With 4-nitrophenyl 4′-guanidinobenzoate, the acyl enzyme was formed so rapidly that titration of the active site of batroxobin was possible. Irreversible inhibition of batroxobin was caused only by the selective thrombin inhibitor d-Phe-Pro-ArgCH 2Cl.

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin.

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.

Enzymatic digestion of human plasma inter-α-trypsin inhibitor by snake venom metalloproteinases

1. 1. Incubation of human plasma inter-α-trypsin inhibitor with crotalid, viperid, colubrid or elapid venoms resulted in random cleavage of the intact inhibitor (200,000 mol. wt) and formation of inhibitor of 130,000, 77,000, 58,000, and 38,000 mol. wt, along with several minor products. 2. 2. The overall patterns of digestion varied among the venoms studied. However, a 77,000 mol. wt inhibitor cleavage product was formed by all venoms tested, and this fragment was resistant to proteolysis even after a 24 hr incubation with the venoms. 3. 3. Venom pre-treated with phenylmethylsulfonyl fluoride digested inter-α-trypsin inhibitor; however, pre-treatment with EDTA completely stopped the reaction, indicating that venom metalloproteinases were responsible for the inhibitor digestion. 4. 4. The inhibitor cleavage products retained the ability to inhibit trypsin, but had no inhibitory activity against venom proteinases.

Chromogenic proteinase substrates as possible tools in the characterization of Crotalidae and Viperidae snake venoms

J.Meier, K. Stocker, L. G. Svendsen and M. Brogli. Chromogenic proteinase substrates as possible tools in the characterization of Crotalidae and Viperidae snake venoms. Toxicon 23, 393 – 397, 1985. — Proteinase activities have been determined photometrically in 25 different Crotalidae and Viperidae snake venoms by using five different chromogenic proteinase substrates. The activity profiles obtained by listing the identity numbers of substrates hydrolyzed by the venoms in a decreasing potency order remained quite stable within a given snake population. Submission of a venom to various physical and chemical treatments did not alter its activity profile. It is therefore concluded that this simple method of determining proteinase activities could be of help in snake venom characterization, as well as in studying the influence of metallic ions and inhibitors of snake venom proteinases.

Enzymatic inactivation of human [formula omitted]-antichymotrypsin by metalloproteinases in snake venoms of the family elapidae

1. 1. Incubation of dialyzed Elapid venoms with the human plasma proteinase inhibitor, α 1 -antichymotrypsin, resulted in enzymatic inactivation of the inhibitor by metalloproteinases in the crude venoms. 2. 2. Dendroaspis angusticeps venom exhibited the highest activity on α 1 -antichymotrypsin. However, venoms from seven genera inactivated the inhibitor, indicating that the metalloproteinases responsible for the inactivation are widespread among snakes of the family Elapidae. 3. 3. Electrophoretic analysis revealed that intact α 1 -antichymotrypsin (64,000 daltons) was converted to a 60,000 dalton inactive inhibitor. No stable complexes between α 1 -antichymotrypsin and venom proteinases were observed, and no random proteolysis of the inhibitor occurred. 4. 4. The Elapid venoms showed little or no proteolytic activity on casein or hide powder azure, confirming observations from other laboratories. However, all venoms tested completely inactivated native α 1 -antichymotrypsin by limited proteolysis.

Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, α1-antichymotrypsin and α2-antiplasmin

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human α 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human α 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on α 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.