Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil)

1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. Purified preparation hydrolyzed the Asn(3)--Gln(4), His(10)--Leu(11), Ala(14)--Leu(15), Tyr(16)--Leu(17), Arg(22)--Gly(23) and Phe(24)--Phe(25) bonds of oxidized insulin B. chain. 6. The A alpha chain of fibrinogen was first split and B beta chain was cleaved later by this toxin. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.     

Presence of zinc in proteolytic hemorrhagic toxin isolated from Agkistrodon acutus venom

1. Hemorrhagic toxin (Ac1-proteinase) was isolated from the venom of Agkistrodon acutus (Formosa) and the zinc content was determined (1.15 mol/mol protein). The results we extensively compared with hemorrhagic toxin e of Crotalus atrox venom (U.S.A.). Comparable results were obtained for zinc content, hemorrhagic and proteolytic activities for native hemorrhagic toxins from two different venoms. It is of interest that the zinc content of hemorrhagic toxins is identical even though the venoms are obtained from snakes inhabiting totally different continents. 2. Zinc content, hemorrhagic and proteolytic activities were compared before and after the removal of zinc. It was found that both hemorrhagic and proteolytic activities disappeared upon removal of the zinc. 3. Zinc content of all hemorrhagic toxins with proteolytic activity reported so far were also compared and it is concluded that regardless of their geographic origin, zinc is present in venom hemorrhagic toxins on a unimolar basis. 4. When zinc is removed there is a drastic change in the low-frequency region of the Raman spectrum suggesting the presence of a zinc ligand co-ordination. The decrease of alpha-helical content and increase of random coil are reflected in the amide I and III bands of Raman spectroscopy after the removal of zinc. Increase of random coil and the loss of zinc are probably responsible for the loss of hemorrhagic and proteolytic activities.     

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies.The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.     

Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (western diamondback rattlesnake)

Hemorrhagic toxin f (HT-f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT-f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT-f possesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT-f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the gamma chain of fibrinogen without affecting either the A alpha or B beta chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT-f was shown to differ immunologically from other hemorrhagic toxins such as HT-a and HT-c. HT-f also possesses lethal toxicity. When zinc was removed the apo-HT-f lost its lethal toxicity. HT-f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.     

Isolation and characterization of horridum toxin with arginine ester hydrolase activity from Heloderma horridum (beaded lizard) venom

A hemorrhagic toxin with lethal and arginine ester hydrolytic activities was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel, and Q-Sepharose column chromatography. The hemorrhagic toxin was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis and immunodiffusion. Its molecular weight is approximately 31,000 with an isoelectric point of 3.9. Hemorrhagic, lethal, and benzoyl-L-arginine ethyl ester hydrolytic activities of this preparation were inhibited by diisopropyl fluorophosphate (DFP), N-bromosuccinimide, and beta-mercaptoethanol, suggesting that serine, tryptophan, and disulfide bonds are involved in these activities. Also there was an increase in creatine kinase activity in mice serum which is an indicator that the toxin is involved in muscle damage. This protein was stable to heat and pH ranges between 2 and 11. The Michaelis constant (Km), for benzoyl-L-arginine ethyl ester, and inhibition constant (Ki), for DFP, were found to be 6.9 X 10(-3) and 1.93 X 10(-4) M, respectively.     

Hemorrhagic toxins from Western diamondback rattlesnake (Crotalus atrox) venom: isolation and characterization of five toxins and the role of zinc in hemorrhagic toxin e.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.     

Proteolytic specificity of hemorrhagic toxin b from Crotalus atrox (western diamondback rattlesnake) venom

In our effort to identify the proteolytic specificity of various hemorrhagic toxins isolated from western diamondback rattlesnake venom, hemorrhagic toxin b was isolated in homogeneous form by previously published methods. Hemorrhagic toxin b hydrolyzed glucagon, producing six fragments. The proteolytic sites were identified as Thr(5)-Phe(6), Thr(10)-Ser(11), Asp(15)-Ser(16), Asp(21)-Phe(22) and Try(25)-Leu(26). When oxidized insulin B chain was used, proteolysis occurred at four sites: Asn(3)-Gln(4), His(10)-Leu(11), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The proteolytic specificity of hemorrhagic toxin b is quite different from those of the nonvenom proteases such as thermomycolin, aspergillopeptidase c, alkaline protease from Aspergillus flavus, elastase, subtilisin and papain.     

Regional variation in the presence of canebrake toxin in Crotalus horridus venom

Reverse-phase HPLC was used to isolate the PLA complex neurotoxin “canebrake toxin” from the venom of Crotalus horridus from northern Florida. Individual venoms from 107 specimens of C. horridus throughout its range were investigated for the presence of the toxin. The distribution of canebrake toxin was limited to two separate regions, including a region of Louisiana, Arkansas and Oklahama, and a separate region from southeastern South Carolina through eastern Georgia to northern Florida. Four distinct venom types were found and designated Venoms A (neurotoxic), B (hemorrhagic), A + B (neurotoxic and hemorrhagic) and C (lacking in both neurotoxic and hemorrhagic activities).     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Disintegrin, hemorrhagic, and proteolytic activities of Mohave rattlesnake, Crotalus scutulatus scutulatus venoms lacking Mojave toxin

Venom from the Mohave rattlesnake, Crotalus scutulatus scutulatus, has been reported to be either: (1) neurotoxic; (2) hemorrhagic, or both (3) neurotoxic and hemorrhagic. In this study, 14 Mohave rattlesnakes from Arizona and Texas (USA) were analyzed for the presence of disintegrins and Mojave toxin. All venom samples were analyzed for the presence of hemorrhagic, proteolytic and disintegrin activities. The venoms were each chromatographed by reverse phase and their fractions tested for disintegrin activity. All specimens containing Mojave toxin were the most toxic and lacked proteolytic, hemorrhagic and disintegrin activities. In contrast, the venoms containing these activities lacked Mojave toxin. Two disintegrin genes, scutustatin and mojavestatin, were identified by PCR of genomic sequences. Scutustatin is a highly conserved disintegrin, while mojavestatin shows low conservation to other known disintegrins. Venoms with the highest LD50 measurements lacked both disintegrin genes, while the specimens with intermediate and low LD50 contained both genes. The intermediate LD50 group contained Mojave toxin and both disintegrin genes, but lacked hemorrhagic and disintegrin activity. Our results raise the possibility that scutustatin and mojavestatin are not expressed in the intermediate LD50 group, or that they may not be the same disintegrins responsible for the disintegrin activity found in the venom. Therefore, it is possible that Mohave rattlesnakes may produce more than two disintegrins.     

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 μg, respectively. These purified hemorrhagic factors were not lethal at 15 μg/g in mice. Factor a hydrolyzed the Bβ chain of fibrinogen, while factor b hydrolyzed the Aα chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.     

Protease activity increases in plasma, peritoneal fluid, and vital organs after hemorrhagic shock in rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable - possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock.     

Characterization of two hemorrhagic zinc proteinases, toxin c and toxin d, from western diamondback rattlesnake (Crotalus atrox) venom

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.     

Two toxins from the venom of Trimeresurus tokarensis

1. Two toxins, Tokaratoxin-1 (TT-1) and Tokaratoxin-2 (TT-2), were isolated from the venom of Trimeresurus tokarensis using gel filtration on a Sephadex G-100 column, followed by chromatography on DEAE-Sephacel and carboxymethyl-cellulose. TT-1 possessed both hemorrhagic and proteolytic activities. However, TT-2 did not show hemorrhagic activity. 2. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing and SDS-PAGE. 3. Molecular weights of TT-1 and TT-2 were 71,000 and 25,400, respectively. Although TT-2 is a basic protein, TT-1 is an acidic protein. 4. Biological activities of TT-1 and TT-2 were inhibited by EDTA, EGTA and o-phenanthroline, suggesting that the toxins are metalloproteins. Atomic absorption analyses indicated that TT-1 contains 2.79 mol Ca/mol protein and TT-2 contains 1.04 mol Ca/mol protein and 1.07 mol Zn/mol protein, respectively. 5. The two toxins degraded the A alpha and B beta chains of fibrinogen. 6. TT-1 induced necrosis in addition to its hemorrhagic activity while TT-2 induced necrosis only.     

Uroprotective Effect of Pentoxifylline in Cyclophosphamide‐Induced Hemorrhagic Cystitis in Rats

The role of phosphodiesterase inhibitor, pentoxifylline, in the prevention of cyclophosphamide‐induced hemorrhagic cystitis was evaluated in a rat model. Hemorrhagic cystitis was induced in rats by an intraperitoneal (i.p.) injection of a single dose of cyclophosphamide (150 mg/kg). Pentoxifylline (150 mg/kg/day/ip) was administered for 10 days followed by cyclophosphamide. Hemorrhagic cystitis was well characterized macroscopically, microscopically, and biochemically. Cyclophosphamide induced bladder injury including acute severe inflammation, vascular congestion, severe edema, hemorrhage, inflammatory cell infiltration in the lamina propria, and epithelial denudation; as well as it notably elevated serum inflammatory cytokines (tumor necrosis factor‐α, interleukin‐6, and interleukin‐1β), bladder content of malondialdehyde and total nitrate, accompanied with depletion of bladder antioxidant enzymes activities (glutathione peroxidase, superoxide dismutase, glutathione‐S‐transferase, and catalase). Prior administration of pentoxifylline improved all biochemical and histologic alterations induced by the cytotoxic drug cyclophosphamide. In conclusion, pentoxifylline has proven uroprotective efficacy in the cyclophosphamide‐induced hemorrhagic cystitis model, possibly through modulating the release of inflammatory cytokines and nitric oxide and restoring the oxidant/antioxidant balance. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:343‐350, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21494     

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta)

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－ＳｅｐｈａｄｅｘＣ－２５离子交换层析的步骤,从湖南产尖吻蝮（Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）蛇毒中纯化出两个出血毒素（ＤａＨＴ－１和ＤａＨＴ－２）．ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸（Ａｓｘ,Ｇｌｘ）分别占２３％和２４％,ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具蛋白水解酶活性,无对ＴＡＭＥ,ＢＡＥＥ的水解活性和ＰＬＡ２酶活性．两者的蛋白水解酶活力与出血活性并非正相关．ＤａＨＴ－１和ＤａＨＴ－２的最适温度分别为３５℃和４０℃,最适ｐＨ为６－９,对热均不稳定,温度高于６０℃活性完全丧失。金属离子的分析显示每摩尔毒素蛋白约含０．５ｍｏｌ的Ｚｎ,１ｍｏｌ的Ｃａ,较多的Ｎａ、Ｋ、Ｍｇ,不含Ｃｏ。     

Isolation and characterization of hemorrhagic metalloproteinase from Vipera berus berus (common viper) venom

1. Hemorrhagic metalloproteinase (HMP) was isolated by gel filtration on a Sephadex G-100 (superfine) and affinity chromatography on agarose HPS-7. 2. Hemorrhagic metalloproteinase is a glycoprotein with mol. wt 56.3 kDa. It contains 1 zinc atom per molecule of protein. 3. Hemorrhagic metalloproteinase hydrolyzes casein, fibrinogen and splits the insulin B chain at positions Ala14-Leu15, Tyr16-Leu17, His10-Leu11. It digests A alpha chain of fibrinogen.     

Purification and characterization of a lethal toxin from the venom of Heloderma horridum horridum

A lethal toxin was isolated from the venom of Heloderma h. horridum by gel filtration and ion-exchange chromatography. Molecular weight of the purified toxin was determined to be 28 kDa under reducing and nonreducing conditions. Biological activity, assayed by i.v. routes of injection, shows an LD50 for this preparation of 0.135 micrograms/g. Additionally, the toxin possesses an inhibitory effect on direct electrical stimulation of the isolated mouse hemi-diaphragm. However, neither hemorrhagic nor hemolytic activities were detected. Phospholipase A2 activity, proteolytic activity and arginine esterolytic activity were absent. The amino acid composition of the lethal toxin and the NH2-terminal sequence up to residue number 33 were determined. Neither show similarities to other components from H. h. horridum venom.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.