Red Rattlesnake Research Articles

Isolation and biochemical characterization of rubelase, a non-hemorrhagic elastase from Crotalus ruber ruber (Red Rattlesnake) venom.

A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen. However, hemorrhagic activity was not observed. By ESI/Q-TOF and MALDI/TOF mass spectrometry combined with Edman sequencing procedure, the molecular mass of rubelase was determined to be 23,266 Da. Although its primary structure was similar to rubelysin (HT-2), a hemorrhagic metalloprotease isolated from the same snake venom, the circumstances surrounding putative zinc binding domain HEXXHXXGXXH were found to be different when the three-dimensional computer models of both metalloproteases were compared. The cytotoxic effects of rubelase and rubelysin on cultured endothelial and smooth muscle cells were also different, indicating that the substitution of several amino acid residues causes the changes of active-site conformation and cell preference.

Primary Structure of a Hemorrhagic Metalloproteinase, HT-2, Isolated from the Venom of Crotalus ruber ruber1

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.

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

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

Comparative study of two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber (red rattlesnake)

1. 1. Two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber were isolated and characterized. 2. 2. E-I and E-II have molecular weights of 32,000 and 33,000, and isoelectric points of 5.2 and 4.6, respectively. 3. 3. E-I and E-II are active upon the glandular kallikrein substrate, but neither enzyme was shown to have plasma kallikrein substrate hydrolytic activity. 4. 4. E-I has minimal fibrinogen-clotting activity. It was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the Aα-chain of fibrinogen.

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. 1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. 4. Specific esterolytic activities of the kallikrein-like enzyme on N- tosyl- l- arginine methylester (TAME) and N- benzoyl- l- arginine ethylester (BAEE) are 109.5 and 23.6 μmol/min/mg, respectively. 5. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.

Biochemical characterization of hemorrhagic toxins with fibrinogenase activity isolated from Crotalus ruber ruber venom

Three hemorrhagic toxins with proteolytic activity were isolated from the venom of Crotalus ruber ruber (red rattlesnake). Molecular weights of HT-1, HT-2, and HT-3 were 60,000, 25,000, and 25,500, respectively. Although HT-3 was a basic protein, HT-1 and HT-2 were slightly acidic proteins. Total amino acid residues were 482, 207, and 221 for HT-1, HT-2, and HT-3, respectively. Protease activity of all the toxins was inhibited in the presence of EDTA or o-phenanthroline, suggesting that the toxins are metalloproteins. Analyses for various metals by inductively coupled plasma-atomic emission spectrometry indicated that sodium, potassium, zinc, and calcium atoms were present in significant quantities. With all three toxins, there was roughly 1 mol of zinc to 1 mol of protein; the results for calcium were not consistent. All three hemorrhagic toxins degraded the Aα chain of fibrinogen, while HT-1 also degraded the Bβ chain. Although fibrinogen was degraded by the three toxins, no clots were observed, indicating that the proteolytic specificities of the three toxins were different from those of thrombin. The hemorrhagic toxins increased creatine kinase activity in mice serum, indicating muscle damage, which was substantiated by histological examination.

Field Notes on a Red Rattlesnake in Lower California

Field Notes on a Red Rattlesnake in Lower California