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.     

Isolation and characterization of arginine ester hydrolase from Heloderma horridum (beaded lizard) venom.

1. An arginine ester hydrolase was isolated from Heloderma horridum (beaded lizard) venom by Sephadex G-75, DEAE-Sephacel and Q-Sepharose column chromatography, resulting in 5.4 mg of purified enzyme from 320.0 mg of crude venom. 2. The enzyme was shown to be homogeneous by both SDS and non-SDS disc electrophoresis on polyacrylamide gel at pH 8.3. 3. The enzyme possesses arginine ester hydrolase and transglutaminase-like activities, but did not exhibit clotting activity. 4. Molecular weight was determined to be ca 29 kDa, with an isoelectric point of 4.4. 5. The enzyme was stable to heat treatment (95 degrees C, 10 min) and to pH changes over the range 2-11. 6. The arginine ester hydrolase was inactivated by diisopropylfluorophosphate (DFP), beta-mercaptoethanol and N-bromosuccinimide, suggesting that serine, disulfide bonds and tryptophan are involved in enzymatic activity. 7. Amino terminal sequences were determined and appear to be similar to porcine pancreatic kallikrein.     

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.     

Exendin-4, a new peptide from Heloderma suspectum venom, potentiates cholecystokinin-induced amylase release from rat pancreatic acini.

We examined the actions of exendin-4, a new peptide isolated from Heloderma suspectum venom, on dispersed acini from rat pancreas. Exendin-4 caused a 3-fold increase in cAMP but did not alter cellular calcium concentration. Exendin-4-induced increases in cAMP were inhibited by an exendin-receptor antagonist, exendin (9-39)NH2, but not by VIP-receptor antagonists. Whereas up to 1 microM exendin-4 alone did not alter amylase release, potentiation of enzyme release was observed when the peptide (greater than 30 pM) was combined with cholecystokinin. Potentiation of amylase release was also observed when exendin-4 was combined with carbamylcholine, bombesin or a calcium ionophore, A23187. These results indicate that stimulation of exendin receptors on rat pancreatic acini causes an increase in cellular cAMP. Although this increase in cAMP alone does not result in amylase release, combination of exendin-4 with agents that increase cell calcium results in potentiation of amylase release.     

Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas.

The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.     

Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist.

Exendin-3 increased cellular cAMP levels and amylase release from dispersed acini from guinea pig pancreas. Low concentrations (0.1-3 nM) caused a 12-fold increase in cAMP, whereas higher concentrations (0.3-3 microM) caused an additional 24-fold increase in cAMP. Maximal cAMP with the highest concentration tested was the same as the maximal response with secretin, vasoactive intestinal peptide (VIP), peptide histidine isoleucine, helodermin, or helospectin-I. In terms of amylase release, exendin-3 had the same efficacy but was the least potent of these peptides. Exendin-3-induced increases in amylase release were inhibited by VIP receptor antagonists and the new peptide (greater than 0.1 microM) competed with radiolabeled VIP for binding sites on dispersed acini. Increasing concentrations of an exendin-3 fragment, exendin-3(9-39) amide, did not increase cAMP or amylase release but inhibited the increase in cAMP observed with 0.1-3 nM exendin-3. The fragment did not alter the effects of other peptides that are known to increase acinar cAMP. We conclude that exendin-3 interacts with at least two receptors on guinea pig pancreatic acini; at high concentrations (greater than 100 nM) the peptide interacts with VIP receptors, thereby causing a large increase in cAMP and stimulating amylase release; at lower concentrations (0.1-3 nM) the peptide interacts with a putative exendin receptor, thereby causing a smaller increase in cAMP of undetermined function. Exendin-3(9-39) amide is a specific exendin receptor antagonist.     

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann)

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.     

5-O-methylbiochanin a,a new isoflavone from Echinospartum horridum

Five known isoflavones (daidzein, formononetin, genistein, 5-O-methylgenistein and biochanin A) have been isolated from the leaves and stems of Echinospartum horridum. A sixth compound has been characterised by chemical and spectroscopic methods as the new isoflavone, 5-O-methylbiochanin A.     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.     

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.     

Primary structure of helodermin, a VIP-secretin-like peptide isolated from Gila monster venom

The inhibitory regulatory component of adenylate cyclase (Ni) was highly purified from rat brain synaptic membranes. A low Km GTPase activity was always associated with Ni through the purification, and the recovery of GTPase activity correlated well with that of Ni. Purified Ni was hardly ADP-ribosylated by islet-activating protein (IAP). A heat-labile factor in the fraction of the stimulative regulatory component (Ns) restored ADP-ribosylation and also activated the GTPase about 2-fold. NaF which was reported to interact with Ni markedly reduced GTPase activity. The purified Ni fraction inhibited adenylate cyclase only in the presence of a heat-stable factor found in the partially purified regulatory component. GTPase and inhibitory activities were weak in myelin which contained only a small amount of Ni. These findings support the view that GTPase activity is an intrinsic activity of Ni and some factors are necessary for the function of Ni.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)⁵-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.     

Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.

A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.     

Purification and characterization of a fibrinogenolytic and hemorrhagic metalloproteinase isolated from Vipera lebetina venom

Serious clinical problems such as hemorrhage, edema and tissue necrosis are observed following viperid envenoming. A proteinase (VLH2) was isolated from Vipera lebetina by combination of two chromatographic steps of gel filtration on Sephadex G-75 followed by DEAE Sephadex A-50. This acidic proteinase, with a molecular mass of about 55 kDa and isoelectric point of 5.4, displayed a fibrinogenolytic and hemorrhagic activities. VLH2 hydrolyses rapidly the Aα-chain of fibrinogen, followed, more slowly, by the Bβ-chain, leaving the γ-chain unaffected. The proteolytic and hemorrhagic activities of VLH2 were inhibited by EDTA, EGTA and 1–10 phenanthroline. However, these activities were not affected by AEBSF, Aprotinine, and E64, suggesting that VLH2 is a metalloproteinase with an α-fibrinogenase activity, requiring calcium and zinc for its activity. The enzyme VLH2 did not have proteolytic activity towards extracellular components gelatin, laminin and fibronectin. The hemorrhagic metalloproteinase VLH2 has a myotoxic activity, as determined by serum CK level and histological observation of muscle tissue. Furthermore, VLH2 is able to induce apoptosis of C2C12 myotubes. These results indicate that VLH2 is implicated in the local and systemic bleeding, contributing thus in the toxicity of V. lebetina venom.     

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.     

Purification and characterization of phosphodiesterase from Crotalus venom.

A procedure for the purification of phosphodiesterase from Crotalus venom on DEAE-cellulose at alkaline pH is described. The enzyme gives a single band in polyacrylamide gels and is free of contaminating nucleolytic enzymes. The molecular weight is about 115000. Concentration in an Amicon ultrafiltrator gave a highly concentrated active enzyme. Phosphodiesterase is relatively stable and can be stored at 4 degrees C in the presence of Mg2 and serum albumin for years. For the detection of contaminating endonuclease, an assay was used in which tRNA was the substrate and possible internal breaks were detected in polyacrylamide gel after denaturation. With bis(p-nitrophenyl) phosphate as substrate, 15mM Mg2 was necessary for optimal activity. The reaction remained linear for at least 15 min at 22 degrees C. At 45 degrees C, the liberation of p-nitrophenol was highest within 25 min of incubation. At 75 degrees C, inactivation of the enzyme occurred after 4 min.     

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.     

Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.