Purification and characterization of patagonfibrase, a metalloproteinase showing alpha-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom.

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aalpha-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca(2+) and inhibited by Zn(2+), cysteine, dithiothreitol and Na(2)EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins.

R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx-yy, 2000. - Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A(2) (PLA(2)) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS-PAGE revealed 7-20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA(2) activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A(2) activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.

Effects of temperature and storage conditions on the electrophoretic, toxic and enzymatic stability of venom components.

Rattlesnake venoms are complex biological products containing potentially autolytic components, and they provide a useful tool for the study of long-term maintenance of enzymes in a competent state, both in vivo and in vitro. To evaluate the stability of venom components, 15 aliquots of freshly extracted venom (from Crotalus molossus molossus) were subjected to 15 different temperature and storage conditions for 1 week and then lyophilized; conditions varied from storage at -80 degrees C (optimal preservation of activities) to dilution (1:24) and storage at 37 degrees C (maximal degradation potential). Effects of different storage conditions were evaluated using SDS-PAGE, metalloprotease zymogram gels, a cricket LD50 assay and enzyme assays (metalloprotease, serine proteases, phosphodiesterase, L-amino acid oxidase and phospholipase A2). Venom samples were remarkably refractive to widely varying conditions; enzyme activities of some samples were variable, particularly L-amino acid oxidase, and one sample treatment showed higher toxicity, but electrophoretic results indicated very little effect on venom proteins. This study suggests that most venom activities should remain stable even if stored or collected under potentially adverse conditions, and freezing samples is not necessarily advantageous. Proteins in the crude venom are not as labile as has been previously thought, and endogenous mechanisms present in the venoms likely inhibit autolysis during long-term storage that occurs in vivo in the gland.

Venom yields from several species of colubrid snakes and differential effects of ketamine.

The composition of rear-fanged colubrid snake venoms is largely unknown due primarily to the difficulty involved in venom collection. Several different methods have been used to maximize the yield of Duvernoy's secretions. The method proposed by Rosenberg in 1992, which includes the use of ketamine hydrochloride anesthetic and pilocarpine to induce Duvernoy's glands secretion, was used in the present study to collect venom from eight species of colubrids. Protein concentrations, using a dye-binding microassay technique, were determined for the venoms collected. Average protein concentrations ranged from 49.8 to 96.4%. Most yields (dry weight/snake) obtained from specimens in this study were significantly greater than yields previously reported. There was a wide range of effects that occurred due to the ketamine injections; however, all snakes recovered from the effects of the ketamine hydrochloride/pilocarpine with no apparent ill effects. Recommended doses of ketamine hydrochloride have thus been adjusted, depending on previous reactions to the drug. The use of ketamine/pilocarpine in the collection of Duvernoy's secretion has proven to be highly effective in increasing yields. Some caution should be observed when administering ketamine to various species of colubrids, as effects do not necessarily scale to body mass.

Characterization of the major metalloprotease isolated from the venom of the northern pacific rattlesnake, Crotalus viridis oreganus.

Rattlesnake venoms typically contain several different metalloproteases, some of which are hemorrhagic toxins. Metalloproteases contribute significantly to the often severe necrotic changes in tissues following envenomation, and these prominent components are important to the predigestive role of venoms. Venom of the northern Pacific rattlesnake (Crotalus viridis oreganus) contains at least five distinct metalloproteases, and the dominant protease (trivial name, CVO protease V) has been isolated and characterized as being a single polypeptide chain acidic protein with a molecular mass of 61 kDa and a pH optimum of approximately 9.0. It catalyzes the hydrolysis of several protein substrates, including casein, and is inhibited by metal chelators such as EDTA, EGTA and 1,10-phenanthroline but not by serine protease inhibitors such as PMSF. Calcium is present at a molar ratio of approximately 1:1, but, unlike other described venom metalloproteases, this protease does not appear to contain zinc. Caseinolytic activity is not significantly inhibited by citrate (at pH 9.0) at levels up to 2.0 mM; at 100 mM citrate (at pH 9.0) more than 65% of activity is retained. It is partially inhibited by nanomolar concentrations of ATP, but higher amounts (micromolar) do not result in further inhibition of activity. The protease shows fibrinolytic and fibrinogenolytic activity, but is only weakly hemorrhagic in rats. When stored in solution for long periods it undergoes autolytic degradation. This protease or a homolog appears to be present in venoms from several rattlesnake species but is not present in venoms from juvenile C.v. oreganus. The presence of this component in venoms from adult Pacific rattlesnakes is responsible for the age-related increase in metalloprotease activity of the crude venom.

An aqueous endpoint assay of snake venom phospholipase A2.

Phospholipase A2 (PLA2), an enzyme found in most snake venoms, catalyzes the hydrolysis of phospholipids in biological membranes, and some have presynaptic neurotoxic activity. A synthetic substrate, 4-nitro-3-(octanoyloxy)benzoic acid, was synthesized and purified on a silica gel column using a published method. This substrate was used to develop an endpoint assay which is rapid and requires a minimum of equipment. This aqueous assay system allowed enzyme activity to be examined without the use of radioactive substrates or organic solvents, minimizing waste disposal concerns. Whole venoms, partially purified enzyme isolated from Crotalus mitchelli pyrrhus venom, tissue extracts and commercial preparations were employed as sources of PLA2. Results show that this method is a convenient and specific assay for PLA2 from several sources and is particularly suited for assaying large numbers of fractions generated during purification procedures.

Complete primary structure and biochemical properties of gilatoxin, a serine protease with kallikrein-like and angiotensin-degrading activities.

The activity and the complete primary structure of gilatoxin, a glycoprotein component from the venom of the Mexican beaded lizard (Heloderma horridum horridum) has been elucidated. Gilatoxin, a serine protease, showed kallikrein-like activity, releasing bradykinin from kininogen; toxin-treated kininogen also produced lowered blood pressure in rats and contraction of isolated rat uterus smooth muscle. Gilatoxin catalyzed the hydrolysis of various arginine ester substrates for trypsin and thrombin and degraded both angiotensin I and II by cleavage of the dipeptide Asp-Arg from the NH2-terminal end. Fibrinogen was degraded but a fibrin clot was not produced, indicating that gilatoxin has specificities different from thrombin and snake venom thrombin-like proteases. The complete amino acid sequence of gilatoxin (245 residues) was deduced from NH2-terminal sequencing of overlapping peptide fragments cleaved from the reduced and alkylated toxin by enzymatic and chemical methods. The toxin is extensively glycosylated, containing approximately 8 mol of monosaccharide/mol of toxin, but appears to lack O-glycosylation sites. Amino acid sequence alignment of gilatoxin with batroxobin, crotalase, kallikrein, thrombin, trypsin, and several partial sequences of other Heloderma toxins reveals that there is considerable homology between these enzymes, particularly in the regions of the presumed catalytic site. Gilatoxin contains an additional 7 residues in the highly conserved catalytic region of serine proteases (including Asp-96, in the basic specificity pocket of thrombin) which may contribute to the unusual substrate specificity of the toxin.

Fibrinogenolytic proteases from the venoms of juvenile and adult northern Pacific rattlesnakes (Crotalus viridis oreganus).

1. Venoms of Crotalus viridis oreganus show marked ontogenetic variation in protease activity. Adult venoms are approximately five-fold higher in protease (caseinolytic) activity. 2. Of seven potential protease inhibitors, only EDTA and 1,10-phenanthroline caused a significant decrease in protease activity. Responses of juvenile and adult venoms were essentially equivalent, and attempts at recovery of protease activity of EDTA-treated venoms by the addition of Ca2+ or Zn2+ were unsuccessful. 3. Gel filtration resolved two proteases from juvenile and subadult venoms with approximate M(r) of 100,000 and 78,000. Four proteases were resolved from adult venom, and M(r) estimates were 78,000, 61,000, 35,000 and 19,000. 4. Proteases from juvenile and adult venoms showed fibrinogenolytic activity, each producing some unique degradation products. 5. The occurrence of three "new" proteases in adult venom produced the ontogenetic increase in activity seen in the crude venoms.

The unique Duvernoy's secretion of the brown tree snake (Boiga irregularis).

Recently, bites by the colubrid Boiga irregularis (brown tree snake) in infants and young children on Guam have produced severe systemic reactions which bear some resemblance to classical manifestations of neurotoxic venom poisoning. This study demonstrates that the Duvernoy's secretion which elicits these reactions is a remarkably simple venom secretion with comparatively low toxicity and generally weak enzymatic activity. The intravenous LD50 for Swiss-Webster mice was approximately 80 mg/kg; significant neurotoxic manifestations were not observed in mouse trials. Deaths of lethally challenged mice occurred within minutes of injection, and appeared to result from cardiopulmonary crises. Duvernoy's secretion yields, protein content, enzyme activities, electrophoretic data and toxicity characteristics of the secretion are presented.

Electric shocks are ineffective in treatment of lethal effects of rattlesnake envenomation in mice.

Electrical shocks, even crudely delivered from 'stun guns' and gasoline engine spark plugs, have been reported to be effective in the treatment of snake bite. We thus applied similar electric shocks to mice artificially injected with reconstituted rattlesnake venom at various LD50 multiples. Those envenomated mice treated with electric shock survived no better than the controls. We thus found no evidence that electric shocks crudely administered had any life saving effect in mice.

Fractionation of red diamond rattlesnake (Crotalus ruber ruber) venom: protease, phosphodiesterase, L-amino acid oxidase activities and effects of metal ions and inhibitors on protease activity.

Crotalus ruber ruber venom contains several different proteases, and the proteolytic activity of the crude venom is 6-15 times greater in adult than in juvenile venom. Venom samples were assayed for proteolytic, phosphodiesterase, L-amino acid oxidase and elastinase-like activities and were subjected to gel filtration on BioGel P-100. Two major size classes of proteases were resolved (mol. wt 67,000 and 20,500). EDTA, N-ethylmaleimide (N-EM) and 1,10-phenanthroline inhibited proteolytic activity of crude venom, and EDTA, Zn2+ and Cu2+ inhibited proteolytic activity of the fractionated venom.