The concept of two-dimensional electrophoresis-guided purification proven by isolation of rhodocetin from Calloselasma rhodostoma (Malayan pit viper)

Two-dimensional gel electrophoresis (2DE) is an important tool for investigating the complexity of snake venom proteomes. Apart from applications based on whole proteome analysis, we suggest that 2DE can be used as an assay to guide the progress of protein purification. The aim of this study was to prove the feasibility of this concept by using it to purify rhodocetin from Calloselasma rhodostoma venom. Rhodocetin (α subunit) spot on the 2DE profile of C. rhodostoma venom was first identified and confirmed by mass spectrometry, with a molecular mass of 16 kDa and calculated pI of 5.16. Rhodocetin was subsequently purified by successive anion-exchange and gel filtration chromatography. Every peak from both chromatography profiles was collected and tested on 2DE. The presence of rhodocetin (α subunit) spot in the 2DE profile of the peak DP2 indicated the presence of the protein. The purified compound was used to spike the crude venom. A spiked spot with a 1.6-fold increase in intensity was observed and its position matched to that of rhodocetin (α subunit) on the 2DE profile. Together, these spots confirmed the identity of the purified compound as rhodocetin. Hence, our results have demonstrated the effectiveness of the concept we now term 2DE-guided purification.(AU)

Functional and structural characterization of phospholipases A² isolated from Bothrops asper snake venom in Panamá

Envenoming by Bothrops snakes is the most serious type of envenoming from the medical and economic point of view in Central America. Bothrops asper is responsible for 90% of the snakebites registered in Panamá every year. Despite its medical and economic relevance, only the venom of Costa Rican and Guatemalan populations of this species has been studied to some detail, and there is very little information on intraspecies variability in venom composition and toxicity. In this study the crude venom of B. asper from Panamá was characterized and its pharmacological and biochemistry activities were investigated with standard laboratory assays. Furthermore, we described the isolation, functional and structural characterization of four basic phospholipases A2, namely MTX-I, MTX-II, MTX-III, MTX-IV, and a new acid phospholipase A2 called Basp-I-PLA2. The proteins were isolated from the crude venom by a combination of two chromatographic steps, using ion-exchange chromatography on CM-Sepharose (0.05 M NH4HCO3 pH 8.1 buffer), and hydrophobic chromatography on Phenyl-Sepharose (0.05 M Tris-HCl pH 7.4), followed by concentration gradient from 4 to 0 M NaCl at 25°C in the same buffer. Analyses of phospholipids hydrolyzed by these enzymes have shown that all phospholipases belong to type A2. The acidic isoform demonstrated more catalytic activity than basic PLA2s. This enzyme was more active on substrates such as phosphotidylcholine and phosphatidylglycerol.(AU)

Effect of gamma irradiation on the behavioral properties of crotoxin

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (l8O-220 g) were used. Crotoxin (100, 250, and 500 mug/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 mug/kg for the hole-board test, and at the doses of 1000 and 2500 mug/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05). Crotoxin decreased the social interaction time(s) at the doses of 1OO, 250 and 500 mug/kg (means + SEM) from 51.6 ñ 4.4 to 32.6 ñ 3.7,28.0 ñ 3.6 and 31.6 ñ 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoeletric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7 per cent of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysls. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an altemative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Effects of three fractions obtained from Naja haje venom on hemolysis and lipid metabolism in rabbits

Three fractions (F1, F2 and F3) were obtained from the venom of the Egyptian cobra Naja haje by gel filtration. F1 and F2 had indirect hemolytic effects on rabbit erytrocytes. The HU50 (the amount of the fraction causing 50 per cent hemolysis) values of F1 and F2 were 12.04 ñ 3.89 and 36.57 ñ 2.20 ug, respectively. The effects of the three cobra venom fractions on lipid metabolism were studied after 1, 2 3 and 4 hours. Serum total lipids and cholesterol were significantly decreased at almost all tested times, but variable changes were observed in the serum triglycerides. Serum inorganic phosphorus levels were significantly increased with F1 and F2 administration more than that with the F3 fraction. These results suggest that F1 and F2 possess phospholipase A2-like activities and their effects on the lipid metabolism is more intense than that of F3.

Aislamiemto y algunas propiedades de la proteinasa I del veneno de la serpiente peruana Lachesis muta / Isolation and various properties of proteinase I from the venom of the Peruvian snake Lachesis muta

Se ha purificado una enzima proteolítica con elevada actividad sobre caseína a partir del veneno de la serpiente Lachesis muta. Esta proteína denominada "Proteinasa I" fue obtenida mediante una cromatografía de filtración sobre sephadex G-100 a pH 6.5 con buffer acetato de amonio 0.1 seguida de una cromatografía de intercambio iónico con DEAE-celulosa a pH 7.5 y una recromatografía en DEAE-celulosa a pH 9.0 y 7.8. La proteína aislada mostró un banda homogenea en electroforeis en gel de poliacrilamida. El peso molecular calculado por filtración en gel fue 26,100 y su pH óptimo 8.4. La ctividad enzimática no fue alterada depués de un calentamiento a 45-C por 10 minutos, mientras que a 70- sólo se registró un 4%de actividad. La enzima no ataca ésteres sintético como TAME y BAEE, no es afectada por lo iones de calcio y carecen de actividad hemorrágica

Ofidiotoxicosis en el Hospital Universitario Ramón González Valencia de Bucaramanga / Snake's toxicosis in Hospital Universitario Ramon Gonzalez Valencia from Bucaramanga

Con el objetivo primordial de conocer las características clínico-epidemiológicas de la ofidiotoxicosis en Santander, se llevo a cabo un estudio retrospectivo en el cual se revisaron los 55 casos de mordedura de serpiente atendidos en el Hospital Universitario Ramon González Valencia de Bucaramanga en los últimos cinco años. El accidente ofídico ocurrió con mayor frecuencia en áreas rurales, afectando casi siempre campesinos (70 por ciento), especialmente de sexo masculino (69.1 por ciento) y en plena producción laboral (edad promedio 19.1 años). El 80 por ciento de las mordeduras ocurrieron en los miembros inferiores. Las serpientes del género Bothrops fueron responsables de 50 casos de ofidiotoxicosis (91 por ciento) y en todos ellos se observaron importantes signos locales como edema, dolor, hemorragia y necrosis que eventualmente llevo a perdida de tejido, a amputación o a ambos (siete pacientes); en 37 por ciento de los casos se presentaron manifestaciones sistémicas leves y en 27 por ciento manifestaciones severas. La más grave complicación fue la hemorragia del sistema nervioso central que se presentó en tres pacientes, uno de los cuales falleció (mortalidad general de 2 por ciento). Las pruebas de coagulación fueron muy sensibles para documentar la severidad del envenenamiento y para evaluar la eficacia del suero antiofídico, que fue muy efectivo incluso utilizandolo varios días después del accidente. La mordedura de serpiente constituye un importante riesgo ocupacional, espcialmente para los trabajadores del campo, hacia quienes se deben dirigir especialmente las campañas educativas enfocadas a la prevención. Se recomienda una adecuada provisión de suero antiofídico en los hospitales localizados en áreas de riesgo y profundizar en la investiogación toxicológica y clínica de este evento a nivel universitario