Crotalidae Venoms Research Articles

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. L ebetina venom having Chymo Trypsin Like Proteolytic activity ( VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin–agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl- l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.

ChemInform Abstract: Studies on Hemorrhagic Toxins from the Venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus and Arginine Ester Hydrolases from Trimeresurus mucrosquamatus Venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

Inhibition of Hemorragic Snake Venom Components: Old and New Approaches

Snake venoms are complex toxin mixtures. Viperidae and Crotalidae venoms, which are hemotoxic, are responsible for most of the envenomations around the world. Administration of antivenins aimed at the neutralization of toxins in humans is prone to potential risks. Neutralization of snake venom toxins has been achieved through different approaches: plant extracts have been utilized in etnomedicine. Direct electric current from low voltage showed neutralizing properties against venom phospholipase A2 and metalloproteases. This mini-review summarizes new achievements in venom key component inhibition. A deeper knowledge of alternative ways to inhibit venom toxins may provide supplemental treatments to serum therapy.

FACTOR X ACTIVATING PROTEASES FROM SNAKE VENOMS

A variety of factor X activators have been detected in snake venoms. About 10 activators have been isolated from Viperidae, Crotalidae, and Elapidae venoms. Viperidae and Crotalidae venom activators are mainly metalloproteases. Only two factor X activators are characterized from Elapidae venoms, both belonging to serine proteases. Most thoroughly investigated snake venom factor X activators are from Vipera russelli (now renamed Daboia russelli; RVV-X) and Vipera lebetina (now renamed Macrovipera lebetina; VLFXA). VLFXA is cloned and sequenced, and its primary structure is deduced from the cDNA sequence. Both activators consist of a heavy chain and two C-type lectin–like light chains that are held together by disulfide bonds. Primary stuctures of the heavy chain and one light chain are similar for both activators. The primary structure of the second light chain is not detected in RVV-X. Heavy chains of RVV-X and VLFXA contain metalloprotease and disintegrin-like and cysteine-rich domains. All chains of VLFXA are synthesized from different genes. The primary structures of factor X activating snake venom serine proteases remain unknown.

Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

Structure-Function Relationship of Phospholipase A2From Snake Venoms

AbstractThe enzyme phospholipase A2 (EC 3.1.1.4; PLA2) is widely distributed among various species in the animal kingdom, notably in the pancreatic tissues of mammals and animal venoms of reptiles and insects. It catalyzes efficiently and specifically the hydrolysis of the 2-acyl ester bond in 3-sn-phosphoglycerides. The primary sequences of more than 100 PLA2 from various tissues of different animal families have been solved. Based on their primary structure, PLA2s are classified into two groups. Group I comprises those from mammalian pancreas and the venoms of snake families Elapidae and Hydrophidae. The Group II PLA2s those from the venoms of Crotalidae and Viperidae, have a short extension at the carboxyl terminus that terminates in a half-cystine linked to a half-cystine near the active site at position 50. Despite these structural differences, the PLA2S are homologous proteins which show considerable sequence similarity, particularly in the Ca2+-binding and active site regions.Besides a catalyst for...

Antagonism of the myotoxin and hemorrhagic effects of crotalidae venoms by Eclypta prostrata extracts and constituents

Antagonism of the myotoxin and hemorrhagic effects of crotalidae venoms by Eclypta prostrata extracts and constituents

Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae snake venoms

F. Kornalík and E. Táborská. Cross-reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae snake venoms. Toxicon 27, 1135–1142, 1989.—The lethal, local and defibrinating effects of two African Viperidae and four Crotalidae were assayed. Two commercial antivenoms (Behringwerke Nord Africa and Wyeth anticrotalic) and γ-globulins from hyperimmune rabbit serum were tested for their ability to neutralize the toxic effects of the venoms. Cross neutralization of the lethal effect (antiviperidae antivenom against Crotalidae venom and vice versa) was evident with Behringwerke against Crotalidae as well as with Wyeth against Viperidae. No cross-reactivity was observed with monovalent antivenoms. Practically complete cross-reactivity was observed with polyvalent, but less with monovalent antivenoms in the neutralization of skin hemorrhage. The local effect was neutralized to 0–20% if the antivenom was mixed with the venom. An i.v. injection of antivenom had no effect after intradermally injected venom. Only Echis and Bothrops venoms cause defibrination in vivo. The ability of the two polyvalent antivenoms to neutralize defibrination was very weak. One half milliliter of antivenom neutralized 1 μg ( Echis carinatus) or 10 μg ( Bothrops asper) of defibrinating venom if injected simultaneously. If antivenom was administered 3 hr after the venom, the fibrinogen level increased to about 50% of the normal values after 24 hr. Cross-reactivity in the neutralization of the defibrinating activity was observed with both polyvalent antivenoms.

Antigenic relationships of fractionated western diamondback rattlesnake ( Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies

R. A. Martinez, S. Y. Huang and J. C. Perez. Antigenic relationships of fractionated western diamondback rattlesnake ( Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies. Toxicon 27, 239–245, 1989.—Seven hemorrhagic factors have been isolated from Crotalus atrox venom, but their antigenic relationships have not been well studied. In this study, two different monoclonal antibodies, C. atrox peak 8 (CA-P-8) and C. atrox subclone 5 (CA-5+), were produced against two C. atrox venom hemorrhagic fractions and used in an ELISA (enzyme-linked immunosorbent assay) to determine if the hemorrhagic factors in C. atrox venom are antigenically related. The same ELISA test was used to determine cross-reactivity of seven other crude Crotalidae venoms. The two monoclonal antibodies were tested for their ability to neutralize each hemorrhagic HPLC fraction separated from C. atrox venom. C. atrox venom was fractionated into 22 fractions using HPLC analytical DEAE ion exchange. Fractions 4–17 were hemorrhagic. The CA-P-8 monoclonal antibody reacted strongly with hemorrhagic fraction 8; CA-5+ had a broader reactivity and reacted with several HPLC hemorrhagic and non-hemorrhagic fractions. Crude venoms of C. adamanteus, C. scutulatus scutulatus and C. viridis lutosus reacted with CA-P-8, while C. viridis lutosus, C. viridis oreganus, C. scutulatus scutulatus and C. horridus horridus reacted with CA-5+. C. molossus molossus and C. lepidus lepidus did not react with CA-P-8 and CA-5+. Hemorrhagic HPLC fractions 6, 7, 8, were completely neutralized by monoclonal antibody CA-P-8; fraction 9 was partially neutralized. The present study indicated that some C. atrox venom HPLC hemorrhagic fractions have both common and unique epitopes. Antigenic determinants were also found to be shared among different Crotalus species. The availability of a panel of monoclonal antibodies would be useful in elucidating the antigenic components and biological functions related to venom toxins.

Neutralization of crotalidae venom induced platelet aggregation by affinity chromatography isolated IgG to Crotalus viridis helleri venom : Bar-Or D, Sullivan JB, Black E, et al Clin Toxicol 22:1–9 Nov 1984

Neutralization of crotalidae venom induced platelet aggregation by affinity chromatography isolated IgG to Crotalus viridis helleri venom : Bar-Or D, Sullivan JB, Black E, et al Clin Toxicol 22:1–9 Nov 1984

Phospholipase A 2 electrophoretic variants in reptile venoms

The venoms of poisonous reptiles provide rich sources of phospholipase A 2. We have investigated the potential usefulness of this enzyme in the characterization of venoms and in biochemical taxonomy of venomous reptiles using a recently developed electrophoretic technique. Venoms from Elapidae (18 species examined), especially Australian elapids (6), exhibited high phospholipase A 2 activity and complex electrophoretic patterns. Dendroaspidae venoms (3) exhibited low or non-detectable phospholipase A 2 activity in simple patterns. Viperidae venoms (5) yielded phospholipase A 2 patterns with intermediate complexity and activity. Sea snake venoms (3) exhibited low phospholipase A 2 activity and patterns of variable complexity. Crotalidae venoms (16) exhibited low or no detectable phospholipase A 2 activity in simple patterns. The single colubrid venom examined yielded low phospholipase A 2 activity and a simple electrophoretic pattern. Helodermatidae venoms (2) showed high activity with characteristic, complex patterns. In general, the venoms of snakes widely regarded as being more advanced exhibited phospholipase A 2 patterns of lower complexity and activity. The complexity of the patterns obtained precludes extensive usefulness of the technique for taxonomy at the family and subfamily level, but it may make the technique useful for taxonomy at the subspecies level.

37] Proteinases from the venom of Agkistrodon halys blomhoffii

Snake venoms contain several types of proteinases including en'dopeptidases, nonproteolytic arginine ester hydrolases, thrombinlike enzymes and kininogenase. The venom endopeptidase is mainly distributed in families of Crotalidae and Viperidae. A common feature of this proteinase from the Crotalidae venoms is that they all hydrolyze peptide bonds with amino groups contributed by leucine and phenylalanine residues, and require more than a hexapeptide sequence for hydrolysis of this bond. Another common characteristic point is that they are easily inactivated by EDTA and cystcine. In these properties, they differ from well known mammalian endopeptidase. This chapter aims at examining the assay principle: The enzyme is assayed with 2.0% casein solution in Tris-HC1 buffer, pH 8.5, at 37 °. Undigested casein is precipitated with trichloroacetic acid and filtered or centrifuged. Digested casein in the supernatant is determined with the Folin–Ciocalteau reagent. It also details the purification procedure, and properties such as homogeneity, physical and chemical properties, pH profile and biological activities.

Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae: Walter F. Kocholaty, Edith B. Ledford, Joyce G. Daly and Thomas A. Billings, Toxicon, 1971, 9, 131. (Biochemistry Division, U.S. Army Medical Research Laboratory, Fort Knox, Kentucky 40121, U.S.A.)

Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae: Walter F. Kocholaty, Edith B. Ledford, Joyce G. Daly and Thomas A. Billings, Toxicon, 1971, 9, 131. (Biochemistry Division, U.S. Army Medical Research Laboratory, Fort Knox, Kentucky 40121, U.S.A.)

Toxicity and some enzymatic properties and activities in the venoms of Crotalidae, Elapidae and Viperidae

Venoms from the Elapidae were found to be the most toxic, followed by the Viperidae and Crotalidae. Venoms of the latter showed the highest variation in toxicity among the various species. The Elapidae venoms were practically void of proteolytic activity, while those of the Crotalidae were by far the most potent in this respect with the Viperidae venoms in the intermediate position. Of the three families investigated, the Crotalidae venoms were the richest sources of over-all enzymatic activity.

Proteinase, arginineester hydrolase and a kinin releasing enzyme in snake venoms.

Abstract The distributions of proteinase, arginineester hydrolase and kinin releasing enzyme in 32 commercially available snake venoms were studied. The endopeptidase activity in Crotalidae venoms was considerably higher than that in Elapidae venoms, but significant differences in potency were observed even between different samples of the same species. The crude venoms of the families Crotalidae and Viperidae had arginineester hydrolytic activity independent of proteinase activity. There are significant differences of the arginineester hydrolytic activities even in the venoms of different species of the Crotalidae and Viperidae, and the venoms of Elapidae had no esterolytic activity. The kinin releasing activity like arginine ester hydrolytic activity was greatest in venoms of Crotalidae. However, some venoms of Crotalidae, such as the venom of Agkistrodon actus and the venom of Trimeresurus flavoviridis showed slight kinin releasing activity.

Effects of snake venoms on mammalian cells in tissue culture.

Abstract The effects of snake venoms on two types of cells in tissue culture were investigated. Venoms of Crotalidae, Viperidae, Elapidae and Hydrophiidae, from widely separated geographical areas, were used. Venoms of the three Crotalidae and one Viperidae showed very strong cytolytic activity, while those of the Elapidae and Hydrophiidae had little effect. These results are in agreement with the clinical observation that Crotalidae and Viperidae snakes cause necrosis and hemorrhage on envenomation while Elapidae and Hydrophiidae show little local tissue damage. Addition of antivenin to Crotalidae venoms slowed down the cytolytic effect of venoms.

Selected Crotalidae venom properties as a source of taxonomic criteria

Intraspecific variations in protein content, protein complexity, and cell lysing ability were demonstrated in the venoms of the copperhead Agkistrodon contortrix mokeson and the rattlesnakes Crotalus atrox and Crotalus scutulatus. The quantitative differences in these venom properties seem to have limited value as taxonomic criteria.

Some biochemical evidence in support of the classification of venomous snakes

Proteolytic enzyme activities of 22 poisonous snake venoms were investigated by using the synthetic substrates, p-toluene-sulfonyl-L-arginine methyl ester and N-benzoyl-L-arginine ethyl ester. Eleven crotalid, three viperid and eight elapid venoms were used. Venoms of Crotalidae and Viperidae showed proteolytic enzyme activity but none of the Elapidae venoms investigated showed this enzyme activity.