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 characterization of arginine ester hydrolases from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

An arginine ester hydrolase (ME-5) was isolated from the venom of Trimeresurus mucrosquamatus by column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel and by isoelectric focusing, obtaining 1.4 mg of purified enzyme from 1 g of crude venom. The enzyme was homogeneous by SDS and non SDS disc electrophoresis on polyacrylamide gel at pH 8.3. ME-5 is a glycoprotein which possesses both TAME hydrolase and capillary permeability-increasing activity, but it did not show clotting or bradykinin-releasing activities. Its molecular weight is approximately 33,000 and its isoelectric point is 6.48. The enzyme is stable to heat treatment and to pH changes between 5 and 9. Trimeresurus mucrosquamatus venom contains five arginine ester hydrolases, designated as ME-1, 2, 3, 4 and 5. Three of the five (ME-3, 4 and 5) are inactivated by DFP, suggesting that the serine hydroxyl group is involved in enzymatic activity. All five arginine ester hydrolases showed capillary permeability-increasing activity, but none of the enzymes showed clotting activity. Their amino acid compositions were determined and all appear to be unique and distinct from those of other snake venoms.     

Physiological and biochemical properties of A kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper)

and . Physiological and biochemical properties of a kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper), Toxicon 26, 1193–1204, 1988.—A kallikrein-like enzyme was isolated from the venom of Vipera aspis aspis by Sephadex G-75, Q-Sepharose and Heparin-Sepharose CL-6B column chromatography. The purified enzyme is a glycoprotein with a mol.wt of 43,000 and an isoelectric point of 4.1. The enzyme possesses arginine ester hydrolase activity, but no proteolytic activity against either dimethylcasein or fibrinogen. The reaction mixture of the enzyme and bovine plasma induced contraction of the isolated rat uterus, suggesting that the enzyme releases kinin from the plasma constituent. The amount of enzyme, which releases an equal amount of kinin corresponding to 1 nmole of bradykinin per min, is 2.36 mg. Additionally, the kallikrein-like enzyme demonstrated capillary permeability-increasing activity and hypotensive activity. A synthetic kininogen analog, Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser, was cleaved by the enzyme to release bradykinin and kallidin, also indicating that the enzyme has a kallikrein-like activity. Uterine contraction, capillary permeability-increasing activity and arginine ester hydrolase activity were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is essential for enzymatic and biological activities. Antithrombin III and heparin, serine-protease inhibitors found in plasma had no inhibitory effect on these activities of the purified enzyme. The amino acid sequence of the NH₂ terminal region of the enzyme has similarities with kallikrein-like enzynes from other snake venoms and with porcine pancreatic kallikrein.     

Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish (Acanthaster planci)

A lethal factor in venom of the crown-of-thorns starfish (Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an LD50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present.     

Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi)

Two thrombin-like enzymes were purified from the venom of Agkistrodon caliginosus. Both were homogeneous on polyacrylamide gel electrophoresis, hydrolyzed $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester and did not show any caseinolytic activity. The molecular weights of the enzymes were estimated to be about 34,000–37,000 and 42,000–43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel-filtration on Sephadex G-100 column. One enzyme was purified from the venom by gel-filtration on Sephadex G-100 column, CM-Sephadex C-50 and DEAE-Sephadex A-50 chromatographies, affinity chromatography on p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B and gel-filtration on Sephadex G-100 column. The second enzyme was separated from the first enzyme by the DEAE-Sephadex A-50 chromatography mentioned in the above procedure and was isolated by affinity chromatography on arginine-Sepharose 4B and gel-filtration on Sephadex G-100 column. From 4 g of the venom, 0.88 mg of the first enzyme and 1.18 mg of the second enzyme were obtained, with specific activities of 118 and 139 NIH units per mg of protein, respectively.     

蜂毒素的纯化方法及体外抗肿瘤作用研究

目的从蜜蜂毒中分离纯化蜂毒素 ,观察其体外对多种肿瘤细胞的生长抑制作用。方法用SephadexG 2 5、SephadexG 5 0、SephadexG 75进行 3步色谱法从蜜蜂毒中分离纯化蜂毒素 ,以高效液相色谱法进行含量测定 ;溴化四唑蓝比色法观察蜂毒素对SMMC 772 1、BEL 74 0 2和Hep 3B等 3种肿瘤细胞的生长抑制作用。结果高效液相色谱测定结果显示供试品为单峰 ,保留时间与对照品一致 ;蜂毒素剂量依赖性地抑制 3种受试肿瘤细胞的生长。结论以凝胶过滤色谱可制得高纯度的蜂毒素 ,蜂毒素在体外呈现出明显的抗肿瘤作用     

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 and properties of a lethal,hemorrhagic protein, “Mucrotoxin A”, from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Mucrotoxin A was purified from the lyophilized venom of Trimeresurus mucrosquamatus using gel filtration on a Sephadex G-100 column, followed by chromatography on CM-Sephadex C-50 and DEAE-Sephadex A-50. By these procedures, 14 mg of purified preparation could be obtained from 1 g of crude venom. The purified preparation was homogeneous by disc electrophoresis on polyacrylamide gel at pH 8.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. Mucrotoxin A possessed both lethal and hemorrhagic activities, but it did not show caseinolytic activity. Its molecular weight was approximately 94,000 and the isoelectric point was 4.3. Mucrotoxin A contains approximately 3 moles of Ca and 2 moles of Zn per mole of toxin. The amino acid composition of Mucrotoxin A was determined. No carbohydrate was present.     

Properties of NAD glycohydrolase purified from five-pace snake (Agkistrodon acutus) venom

NAD glycohydrolase (NADase) (E.C. 3.2.2.5) from five-pace snake (Agkistrodon acutus) venom was purified to electrophoretic homogeneity through a 4-step isolation procedure, including column chromatography using DEAE-Sephadex A-50, Sephadex G-75, CM Sephadex C-50 and Sephadex G-100. The final product was 11.8-fold purified with a 3.9% yield. The pure enzyme showed maximal activity at about 40 degrees C with optimal pH at 7.5. It was a glycoprotein with a pI of 7.6. Its mol. wt was respectively 98,000 as measured by gel filtration and 50,000, by SDS-PAGE. There was only one N-terminal residue, proline. NADase is thus composed of two identical subunits in each molecule. The enzyme contained copper ions. NADase activity was lost when the copper enzyme complex was treated with EDTA. The Km of the enzyme for beta-NAD, NADP and beta-NGP were 0.50 mM, 0.13 mM and 0.16 mM respectively.     

Thrombin-like enzyme, flavovilase, with kinin-releasing activity from Trimeresurus flavoviridis (habu) venom

A thrombin-like enzyme, flavovilase, with kinin-releasing activity was isolated, purified, and characterized from the venom of Trimeresurus flavoviridis (habu) using Sephadex G-100, DEAE-Cellulose, and CM-Cellulose column chromatographies. The final preparation was homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing electrophoresis. The enzyme possesses a molecular weight of 26,500, an isoelectric point of 5.0, and consists of 247 total amino acid residues. Specific electrolytic activities of this enzyme on N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE) were determined to be 50.9 and 17.4 micromol/min/mg, respectively. The enzyme was inhibited by p-APMSF (p-amidinophenylmethanesulfonyl fluoride hydrochloride), beta-mercaptoethanol, and N-bromosuccinimide. Additionally, the enzyme was found stable to heat treatment. It was also observed that the enzyme cleaved a kininogen analog with the release of bradykinin.     

Characterization of the anticoagulants from Taiwan cobra (Naja naja atra) snake venom

Taiwan cobra (Naja naja atra) snake venom was separated into 19 fractions by means of CM-Sephadex C-50 column chromatography. Anticoagulant Fractions V-VII were refractionated by gel filtration on Sephadex G-50 and the purified component possessed phospholipase A2 activity and an inhibitory effect on collagen-induced platelet aggregation. The anticoagulant action could be antagonized by phospholipid or platelet factor 3. Anticoagulant Fraction XVII was also further refractionated by gel filtration on Sephadex G-50 and the purified component was shown to be cardiotoxin. It was a weak anticoagulant, caused direct hemolysis and potentiated collagen-induced platelet aggregation. Thromboelastographic studies showed that the anticoagulant action of cobra venom is due to the synergistic effects of phospholipase A2 and cardiotoxin.     

Isolation and some biochemical properties of a paralysing toxin from the venom of the wasp Microbracon hebetor (Say)

A toxin with paralysing activity was isolated from crude preparations of Microbracon hebetor (Say) venom by ion exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, electrophoresis on polyacrylamide gel and gel chromatography on Sephadex G-75. The active component is very labile. A 28-fold purification was obtained with a recovery of about 2.5% of the original biological activity. The toxin shows one single protein band after disc electrophoresis. The molecular weight was assessed at 61,000 by gel chromatography, and at 62,700 by the sedimentation equilibrium method. The isoelectric point was at pH 6.8. The purified toxin was inactivated by a number of proteolytic enzymes.     

A potent platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

By means of gel filtration on Sephadex G-75, DEAE-Sephadex A-50 column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon halys snake venom and shown to be a single peptide chain, as judged by SDS-polyacrylamide gel electrophoresis. The purified platelet aggregation inhibitor was an acidic protein with a molecular weight of 14,000 and possessed phospholipase A₂ activity. Its inhibitory activity on platelet aggregation was heat stable (at 96°C, 30 min) in an acidic medium (pH 5.5), while its phospholipase A enzymatic activity was heat labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium arachidonate, collagen or ionophore A-23187 was non-competitive and dose-dependent with a similar id₅₀ (～ 11 μg/ml). It exerted its inhibitory action without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min).     

Fractionation of bulldog ant venom

The venom of an Australian Bulldog Ant, Myrmecia pyriformis, has been fractionated by means of low voltage starch gel electrophoresis and gel filtration on Sephadex G50 and G75 columns. The aims of the study were (a) to establish whether the biological activities which had previously been described resided in separate venom components, and (b) to isolate, in a purified form, the component possessing non-histamine smooth muscle stimulant activity. Starch gel electrophoresis demonstrated the presence of seven protein components. Sephadex gel filtration resulted in the separation of a fraction which appeared homogeneous on starch gel electrophoresis. This fraction, which was enzyme-free, possessed smooth muscle stimulant, red cell lysing and histamine-releasing activities and resembled the major peptide from bee venom, melittin.     

Extraction and Purification of Curcain,a Protease from the Latex of Jatropha curcas Linn

Abstract— A proteolytic enzyme, curcain, has been extracted from the latex of Jatropha curcas Linn. The enzyme was purified by chromatography on carboxymethyl cellulose and gel filtration on Sephadex G-200. The homogeneity of protein associated with curcain was established by non-denatured polyacrylamide gel electrophoresis using a discontinuous buffer system. The molecular weight of curcain was estimated by Sephadex G-100 gel filtration using a calibration curve of standard proteins to be around 22000 daltons.     

The antihemorrhagic factor of the Mexican ground squirrel, (Spermophilus mexicanus).

The Mexican ground squirrel (Spermophilus mexicanus) has a natural resistance to western diamondback rattlesnake venom (Crotalus atrox). The LD50 for the Mexican ground squirrel is 53 mg/kg body weight, which is 13 times higher than that of BALB/c mice. An antihemorrhagic factor from serum of the Mexican ground squirrel was isolated using Sephadex G-200 gel filtration, ion exchange A-50, G-75 gel filtration and HPLC DEAE 5PW ion exchange chromatography. The purified factor neutralized proteolytic and hemorrhagic activity of crude C. atrox venom. The results of this research suggest that the antihemorrhagic factor in the serum of the Mexican ground squirrel is not an antibody and neutralizes hemorrhagic activity of C. atrox venom.     

Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu)

, , , , , and . Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu). Toxicon 26, 1205–1208. — The hemorrhagic factor, HR2b, was purified from the venom of Trimeresurus flavoviridis (habu) by a combination of gel filtration, cation exchange column chromatography and high performance liquid chromatography. The purified HR2b was homogeneous by the criteria of ultracentrifugation and SDS-disc electrophoresis. The mol. wt of HR2b was 18,000 and 18,500 by gel filtration on Sephadex G-50 and by SDS-disc electrophoresis, respectively, indicating a monomer structure for the hemorrhagic factor. Crystals of HR2b, taking the form of thin plates, were obtained in the presence of ammonium sulfate.     

Purification and properties of hyaluronidase from Palamneus gravimanus (Indian black scorpion) venom.

Scorpion venoms are a rich source of enzymes. Some of the enzymes such as phospholipase A2, proteolytic enzymes and phosphodiesterase are well characterized. However, hyaluronidase has not been studied extensively. In this paper we describe the purification and characterization of hyaluronidase (Hyaluronate lyase, E.C.3.2.1.35) from the Palamneus gravimanus scorpion venom by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The optimal pH and temperature for its maximum activity of the isolated enzyme were 4.5 and 37 degrees C, respectively, and its K(m) was 47.61 microg/ml at 37 degrees C and its specific activity was 6411.7 +/- 117TRU/min per mg against 250 +/- 4.0 TRU/min per mg for the whole desiccated venom suggesting 25-fold purification. The molecular weight of the isolated enzyme was 52 +/- 1 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography on Sephadex G-75. The enzyme was stable for 30 days in the presence of NaCl; no loss of activity was observed up to 37 degrees degrees C and showed a sharp decrease in its activity at 40 degrees C. Heparin inhibited the enzyme activity.     

Coagulant proteinase from Bothropscolombiensis venom

The venom of $\text{Bothrops}$$\text{colombiensis,}$ like other Crotalidae venoms, contains thrombin-like activity. We purified a mixture of isoenzymes by chromatography of the crude venom on DEAE-Sephacel where coagulant proteinases were separated from other proteolytic enzymes. By subsequent chromatography on Sephadex G-100 we obtained coagulant proteinase as a single band on acrylamide gel electrophoresis at pH 7.5 which showed 4 major protein bands when subjected to flat gel isoelectric focusing. This heterogeneity is presumably due to carbohydrates present in this glycoprotein. The native molecular weight of the coagulant proteinase was found to be over 90 000 by gel filtration. SDS electrophoresis showed, however, that the monomer molecular weight is around 67 000. The specific coagulant activity of the purified enzyme was increased 13 fold by purification and was 231 NIH units/mg. The optimal pH for coagulation of bovine fibrinogen was at pH 7.0. The enzyme shows maximal stability in the pH range 5–6 when incubated for 1 hr at 37 °C. The intraperitoneal LD₅₀ for white mice was 4.0 mg/kg. The enzyme is similar to other known coagulant proteinases from snake venoms and thus potentially useful as a therapeutic agent.     

Purification and partial characterization of an arginine ester hydrolase from the venom of the bushmaster snake, Lachesis muta noctivaga

An arginine esterase was purified from the venom of Lachesis muta noctivaga by gel filtration on Sephadex G-100 and by affinity and DEAE cellulose chromatography. The purified enzyme preparation had an arginyl esterase specific activity of 181 mumoles/min/mg, which was 10.9-fold higher than the esterase activity found in the crude venom. The enzyme is free of kinin-releasing activity (kininogenase) and thrombin-like activity (fibrinogenase). The purified fraction showed a single band on polyacrylamide gel electrophoresis. The Km for Bz-L-Arg-O-Et is 1.14 X 10(-3)M, Vm 181.7 mumoles/min/mg and Kcat 90.9 sec-1. The pH profile indicates that the enzyme has an active region centered at pH 8.1. L. muta noctivaga arginyl esterase is reversibly inhibited by benzamidine (Ki 8.9 X 10(-4)M) and irreversibly inhibited by diisopropyl fluorophosphate.