J.R. Giglio

Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.

A myotoxin, bothropstoxin (BthTX), showing no detectable phospholipase A2 activity, was purified to homogeneity from the venom of the Brazilian snake Bothrops jararacussu by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-25. Four phospholipases (Sm-SP1 to Sm-SPIV) were also isolated, the latter showing, similarly to BthTX (Sm-SPv) myonecrotic activity. Approximate mol. wts, as determined by SDS-PAGE, and pI of Sm-SPI to Sm-SPIV are: 22,400-4.2; 15,500-4.8; 13,800-6.9; and 13,200-7.7, respectively. BthTX is a single chain protein, approximate mol. wt 13,000, with 16 half-cystine residues, pI = 8.2 and LD50 = 7.5 mg/kg (i.p.) and 4.8 mg/kg (i.v.) for 20 g mice. The ten first N-terminal amino acid residues show a significant homology to other toxins with phospholipase structure. BthTX is specifically myotoxic, contrary to crude B. jararacussu venom which, although also myotoxic, affects intramuscular arteries and veins leading to thrombosis. BthTX and Sm-SPIV also differ from toxins isolated from the venom of other Brazilian snakes which are strongly hemorrhagic.

Bothropstoxin-I : amino acid sequence and function

The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated fromBothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (Mr=13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations—such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys49 for Asp49; and Asp71 for Asn71—which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II fromBothrops asper venom, but five extra changes—namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132—have been found relative to myotoxin II.

Isolation and characterization of toxic proteins from the venom of the Brazilian scorpion Tityus serrulatus.

Through gel filtration on Sephadex G-25 and chromatography on CM-cellulose-52 five toxic proteins, electrophoretically pure, were isolated from the venom of the Brazilian scorpion Tityus serrulatus and partially characterized, as follows: 1. Toxin T1 VIII, with 61 amino acid residues, mol. wt 6675 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-Met-Asx-His-Glx-Gly-Cys-Lys-; 2. Toxin T1VI with amino acid residues, mol. wt. 7549 and amino terminal sequence Gly-His-Phe-Gly-Lys; 3. Toxin T2III(I), with 63 amino acid residues, mol. wt. 7216 and amino terminal sequence Lys-Lys-Asx-Gly-Tyr-Pro-Val-Cys-Cys-Ser-; 4. Toxin T2IV, which is apparently identical to toxin T1VIII above, since it showed the same elution volume in chromatography on CM-cellulose-52, the same N-terminal Lys and the same electrophoretic mobility as T1VIII; 5. Toxin T1IV, a not previously described toxin from the venom of T. serrulatus, with 45 amino acid residues, mol. wt. 5188 and amino terminal sequence Lys-Glx-Gly-Tyr-Leu-, identical to the first five residues of T1VIII, although with a lower molecular weight. The pharmacological study of T1VIII in guinea pig vas deferens showed a pre-junctional sensitizing action, evidenced by a decrease of the dose-response curves to adrenaline and acetylcholine, with no increase of the maximum. This effect may be due to the liberation of noradrenaline.

Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae).

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The alpha-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity

Bothtrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate Mr of 14000 and 27000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 14 half-cystine residues. Its isoelectric point and extinction coefficient (E(1.0 mg/ml) (1.0 cm) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A2 (PLA2) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK... revealed high homology with other Lys 49 PLA2-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI, which diffracted to a maximal resolution of 1.6 A, were obtained and indicated the presence of a dimer in the asymmetrical unit.

Biochemical and histopathological alterations induced in rats by Tityus serrulatus scorpion venom and its major neurotoxin tityustoxin-I

Intravenous injection into the rat of sublethal doses of Tityus serrulatus scorpion venom (100 micrograms protein/kg) or its major neurotoxin tityustoxin-I (TsTX-I, 20 micrograms/kg) caused, 30-180 min after injection, statistically significant increases in the serum levels of aspartate aminotransferase, amylase, creatine kinase and lactate dehydrogenase, as well as hyperglycemia, a high level of plasma free fatty acids and a low level of liver glycogen. The in vitro serum levels of the above enzymes did not change. For alanine aminotransferase, gamma-glutamyl transferase and alkaline phosphatase, neither in vitro nor in vivo alterations were observed. The whole venom and TsTX-I caused hepatic congestion with hemolysis and hydropic degeneration. Other histological lesions included edema and congestion with subpleural hemorrhage in the lungs, hypertrophy of fibers with degeneration areas in the heart, and congestion and hemorrhage in the kidneys. In the salivary glands, alterations to the acini and ductules were visible. In the adrenal glands no morphological alterations could be detected at the studied doses. The results suggest that the in vivo enzymatic and histopathological alterations are due to tissue lesions evoked by the whole venom and TsTX-I. An indirect effect, however, induced by stimulation of acetylcholine and catecholamine release in the postganglionic nerve terminals, cannot be excluded.

Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom

Bothrops insularis snake venom was fractionated by gel filtration on Sephadex G-150 followed by ion-exchange chromatography on SP-Sephadex C-25. Two active fractions were purified to homogeneity: (1) SIII-SpI, approximate mol. wt 32,000 and N-terminal amino acid residue Val. This fraction showed esterase activity on TAME, edema-inducing activity on the rat hind paw and contractile activity on the isolated guinea pig ileum. The latter two activities were antagonized by benadryl plus methysergide; (2) SIII-SpVI, a myonecrotic and edema-inducing phospholipase, approximate mol. wt 29,000, N-terminal amino acid residue pyro-Glu, consisting of two chains of approximately 15,000 mol. wt each linked by disulphide bridge(s). The induction of edema by this fraction was not antagonized by benadryl plus methysergide, indomethacin, BW755C or BN52021, but it was antagonized by dexamethasone. Three highly purified hemorrhagic heterodimeric fractions, SIII-SpIII-3, SIII-SpIII-4 and SIII-SpIII-5, of approximate mol. wts 26,000, 29,000 and 26,000, and having N-terminal residues of Asx, Asx and Gly, respectively, were further isolated by preparative polyacrylamide slab gel electrophoresis. SIII-SpIII-4 and SIII-SpIII-5 increased the recalcification time of citrated rat plasma. None of the five isolated fractions showed any proteolytic (on casein) or kininogenase activity.

A simplified procedure for the fractionation of Tityus serrulatus venom: Isolation and partial characterization of TsTX-IV, a new neurotoxin

Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.

The Amino Acid Sequence of Bothropstoxin-II, an Asp-49 Myotoxin from Bothrops jararacussu (Jararacucu) Venom with Low Phospholipase A2 Activity

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2s and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2 activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA2s have been considered to be devoid of this enzymatic activity.

Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations.

Bothropstoxin, a 13,700 mol. wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca(2+)-free Krebs solution and was completely abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA.