Bothrops Neuwiedi Research Articles

Antigenic relationship of hemorrhagic factors and proteases isolated from the venoms of three species of Bothrops snakes

F. R. Mandelbaum and M. T. Assakura. Antigenic relationship of hemorrhagic factors and proteases isolated from the venoms of three species of Bothrops snakes. Toxicon 26, 379 – 385, 1988. By comparative studies of the immunological properties of the metalloproteins (hemorrhagic factors and proteases) isolated from the venoms of Bothrops jararaca, Bothrops neuwiedi and Bothrops moojeni, it was found that the hemorrhagic factors contain common antigenic determinants and the proteases were immunologically distinct entities. The rabbit antisera raised for the hemorrhagic factors not only neutralized the hemorrhagic activities of the respective factors but also activities of the other hemorrhagic factors. Although the homology among these proteins are not yet known, these studies have shown that the hemorrhagic factors must have a similar partial structure which includes the catalytic hemorrhagic active site.

Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (jararaca pintada)

Two hemorrhagic factors were isolated from the venom of Bothrops neuwiedi (jararaca pintada) by ammonium sulfate precipitation followed by chromatography on DEAE-Sephadex A-50 and DEAE-cellulose DE-32, gel filtration on Sephadex G-100 and polyacrylamide-gel electrophoresis. These factors were named neuwiedi hemorrhagic factors NHF a and NHF b. They are acidic proteins of pI 4.2–4.3 and consist of single polypeptide chains of molecular weights 46,000 and 58,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The hemorrhagic activity of NHF b is 23 times stronger than that of NHF a. Both hydrolyse casein, although NHF a is about 20 times more active than NHF b. They are metalloproteins inhibited by EDTA and 1,10-phenanthroline. NHF a and NHF b are serologically closely related antigens. These two factors are recognized as identical antigens by horse serum against crude Bothrops neuwiedi venom. However, the rabbit specific antiserum was able to differentiate NHF a from NHF b showing, nevertheless, that they have common determinants apart from specific determinants for each one.

Fauna reptiliana do norte da grande Porto Alegre, Rio Grande do Sul, Brasil

This paper reports the preliminary results of two years of herpetogeographic studies in the region called «Grande Porto Alegre» at its northern portion. The physiognomy shows, at North, slopes of the Brazilian Southern Plateau, more or less forested in a general view; these elevations graaually decrease to the South until the levei of the sea to Atlantic littoral by the eastern border, and to Rio Grande do Sul savanah, which extends to Uruguay and Argentina. The biota is transitional from the highland prairies and forests of the plateau to lowland prairies of the pampas. Three faunistic Provinces are included: Guarani, Pampean, and Tupi. The most conspicuous dispersion is shown by the taxa proceeding from the plateau to savanah (Dispersion Centre Guarani); the pampean species occur in low percentage (Dispersion Centre Uruguay), and an inappreciable account of Tupi fauna (Dispersion Centre of «Serra do Mar»). The new records are: presence of Chironius pyrrhopogon, Hydrodynastes gigas, Bothrops neuwiedi para-naensis, Micrurus corallinus; intergradation zone of Guarani and Pampean geographic roces; and new taxa of snakes, which will be described brieflly. The systematic composition presents: Chelonia, 4 (3 Chelidae, 1 Testudinidae); Sauria, 10 (1 Gekkonidae, 2 Iguanidae, 1 Anguidae, 3 Teiidae, 3 Amphisbaenidae); Serpentes, 45 (35 Colubridae, 4 Elapidae, 6 Viperidae); Crocodylia (1 Crocodylidae).

Effect of temperature upon inhibition by substrate of lactate dehydrogenase isoenzymes from a poikilotherm

The effect of temperature upon the inhibition by substrate (pyruvate and lactate) and the values of K 1 for NAD + and pyruvate have been studied comparatively on lactate dehydrogenase ( l-lactate : NAD + oxidoreductase, EC 1.1.1.27) isoenzymes purified from tissues of a poikilotherm (the snake Bothrops neuwiedii) and of a homeotherm (Ox). Inhibition of the ophidian isoenzyme B 4 by high concentrations of pyruvate is suppressed at the upper limits of the temperature of the habitat. The data presented suggest that the temperature increment affects only the forces binding the pyruvate in the abortive ternary complex Enzyme-NAD +-pyruvate.

Effect of temperature upon catalytic properties of lactate dehydrogenase isoenzymes from a poikilotherm

Lactate dehydrogenase isoenzymes A 4 and B 4 ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27) have been purified from muscle and heartof the snake Bothrops neuwiedii. Catalytic properties at different temperatures and pH values have been studied compartively with those of the same isoenzymes from beef. Values of K m for ophidian isoenzymes were markedly reduced when the temperature decreased. At low substrate concentrations, the increased enzyme-pyruvate affinity compensates for the lower thermal energy and the reaction rate appears to be independent of temperature. Under the same conditions, the activity of beef isoenzymes was closely related to thermal energy. These findings suggest that lactate dehydrogenases from the poikilotherm are able to function with the same efficiency at all temperatures within the range of habitat and to adjust immediately to thermal changes.

Isolation and properties of an inhibitor of phospholipase A from Bothrops neuwiedii venom

An inhibitor of phoapholipase A has been isolated from Bothrops neuwiedii venom after gel filtrations through Sephadex G-50 (pH 4.5), Sephadex G-25 (pH 7.6), Sephadex G-15 (pH 4.0), and chromatography on SE-Sephadex C-25 (pH 4.2–4.5). When subjected to paper electrophoresis, the inhibitor migrates as a simple compound with isoelectric point near pH 6.8. Aminoacid composition, sensitivity toward proteases, and the absorption spectrum fit in well with a polypeptide structure lacking tyrosine and tryptophan. In the absence of EDTA, an inactive, anionic derivative appears in inhibitor preparations; the reaction can be reversed by 2-mercaptoethanol. Direct interaction of enzyme and inhibitor is proved by the inhibition of enzyme activity and the chromatography of enzyme-inhibitor mixtures. Titration of inhibitor with venom phospholipases A (isoenzymes P-1 and P-2) yields sigmoid-shaped concentration-inhibition curves, with P-1 far more sensitive than P-2. The enzyme-inhibitor interaction depends on pH since it is tight at pH 4.5 but does not occur at pH 7.5. Presence of thiol groups in inhibitor is consistent with ( a) characteristic spectral changes after reaction of inhibitor with PMB 4 4 4 Abbreviations: PMB, p-chloromercuribenzoate; NEM, N-ethylmaleimide; IM-venom, B. neuwiedii venom, from the Instituto Nacional de Microbiologia, Buenos Aires; IB-venom, same, from the Institute Butantan, Brasil. and NEM; ( b) the inhibitor inhibition by PMB, NEM, iodoacetate, and Hg 2+, and ( c) the reversal of PMB inhibition with reduced glutathione. Since phospholipase A is insensitive towards Hg 2+, addition of Hg 2+ to enzyme-inhibitor mixtures (or crude venom samples) causes an apparent enzyme activation (deinhibition). When substrate (egg-yolk lipoprotein) is added to enzyme-inhibitor mixtures, the reaction kinetics show an initial “lag-period” which is proportional to the inhibitor concentration. The “lag-period” does not occur in the absence of inhibitor or in the presence of Hg 2+, that inactivates the inhibitor.

Isolation and purification of two phospholipases A from Bothrops venoms

Two proteins with phospholipase A activity (P-1 and P-2) were isolated from Bothrops neuwiedii venom by a procedure involving filtration through Sephadex G-50 at pH 4.5 (Step 1); filtration through Sephadex G-25 at pH 7.6 (Step 2); and chromatography on DEAE-Sephadex A-50 (or DEAE-cellulose) at pH 7.6 (Step 3). The developed method was adequate for 50–100 mg dried venom samples. When DEAE-Sephadex A-50 was used for Step 3, two symmetrical peaks of activity were eluted with constant specific activity throughout each peak (phospholipases A P-1 and P-2). P-1 and P-2 migrated as homogeneous monodisperse materials when subjected to polyacrylamide electrophoresis. When DEAE-cellulose was used for Step 3, three peaks of activity were eluted. The second and third peaks corresponded to P-1 and P-2 and represented 95% of the recovered enzyme activity; the first eluted fraction proved to be an artifact. Assay of P-1 or P-2 on pure ovolecithin as substrate yielded lysolecithin and fatty acid as sole reaction products, in accordance to the predicted stoichiometry. After Step 2, the phospholipase A activity recovered was higher than the total activity measured in the crude venom which is explained by the separation of an inhibitor during the enzyme purification. The inhibitor was responsible for the initial lag period that characterized the kinetics of the enzyme reaction with the crude venom. After Step 3, the minimal recovery of phospholipase A (P-1 plus P-2 activities) was about 80% and the purification achieved was 23-(P-1) and 19-fold (P-2) as compared with the crude venom activity. A purification method involving (1) an acidheat treatment, (2) a pH 9 fractionation, followed by (3) the Sephadex G-25 step, and (4) the DEAE-cellulose step gave also two different active proteins but both the total recovery of activity and the specific activity of fractions were less than those obtained with the standard method. Venoms of B. jararaca, B. jararacussu, and B. atrox were also fractionated and two phospholipases A fractions resembling P-1 and P-2 were obtained.

Immunological (Ouchterlony method) identification of intrasubspecies qualitative differences in snake venom composition

The Ouchterlony method of gel double-diffusion was employed to study the individual venoms of 298 snakes of six Bothrops neuwiedi subspecies. This species is widely distributed and its subdivision permits a more detailed comparative study between the morphology of these animals and the composition of their venoms. The venoms were compared to five standards: three venom pools and two sera. The results indicate that the venom of B. neuwiedi has five basic immunological variants with twenty different well-defined venoms. A single variant can be present in several subspecies; or several variants may be found in a single subspecies. These findings show that a very close relationship between morphology and venom composition does not exist in this species. No attempt was made to see if these immunological variations were correlated with specific biological activities. The geographical distribution of some of the snakes with these variations supports the existence of the variations, and seems to indicate that these variants are probably determined genetically. Staining and photographic improvements for immunoprecipitation are described.

Campanha Antiofídica em Minas Gerais

O A. descreve as fases da campnha antiofídica em Minas Gerais (1918 a 1940). Registra as seguintes espécies de ofídios venenosos encontrados: Bothrops cotiara, Bothrops alternata, Bothrops atrox, Bothrops neuwiedii, Bothrops jararaca, Bothrops inaequalis, Bothrops jararacussú, Crotalus terrificus terrificus, Lachesis mutus, Micrurus lemniscatus, Micrurus frontalis, Micrurus corallinus corallinus e numerosas espécies não venenosas, fazendo um mapa para a distribuição geográfica dos ofídios venenosos em Minas Gerais. Apurou que dominam no Estado as espécies do gênero Bothrops e não do gênero Crotalus. Registrou diferenças morfológicas, principalmente de tamanho dos Crotalus terrificus terrificus do Norte do Estado e do Centro e Sul. Relata 2 casos de alergia, de certa gravidade, pelo veneno ofídico, líquido ou sêco, curando-se os pacientes pelo afastamento dos trabalhos da seção antiofídica do Instituto Ezequiel Dias. Registra, ainda, 22 anos de trabalhos, numerosas observações pelas picadas de ofídios venenosos e não venenosos, sendo que estas últimas, em gravidade. Das 980 primeiras observações, 921 foram de acidentes humanos e, destas, 531 tomaram sôro, com a mortalidade de 1,30%; 390 não tomaram sôro e tiveram mortalidade variável, conforme a dose, a época do emprêgo do sôro e a espécie do ofídio, entre 7,6 e 100% dos casos, sendo a média geral de 33%. nenhum acidentado pela Lachesis mutus se salvou.