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Isolation and biochemical characterization of a toxin from \"Rhinella schneideri\" poison with action on the complement system.

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Author(s):
Fernando Antonio Pino Anjolette
Total Authors: 1
Document type: Master's Dissertation
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto
Defense date:
Examining board members:
Eliane Candiani Arantes Braga; Cleni Mara Marzocchi Machado; Wagner Ferreira dos Santos
Advisor: Eliane Candiani Arantes Braga
Abstract

Important studies focused on amphibians secretions analysis are based on large amount of biologically active components present in them, such as biogenic amines, steroids, polysaccharide amine, glycosides, protease inhibitors and several other compounds, responsible for complex symptomatology observed in the envenomation by Bufo paracnemis. The genus Bufo presents several molecules in their excretions that can be divided into categories such as biogenic amines, bufadienolides (Bufogenin), steroids (Bufotoxins), alkaloids, peptides and proteins. Marongio (2006) found in one of his studies the toad poison of Bufo paracnemis, now classified as Rhinella schneideri, has an active component of the classical pathway of the complement system (SC), which needs further studies and characterization. The purification process of this study was accomplished through cation chromatography (CM-Cellulose-52), and seven fractions were obtained, called C1, C2, C3, C4, C5, C6 e C7. The fraction C1 was chromatographed on anion-exchange resin (DEAE- SepharoseTM) resulting in 4 subfractions referred to as D1, D2, D3, and D4. The subfraction D3 showed activity on complement system and was subjected to gel filtration (SephacrylTMS-200) giving 5 subfractions termed S1, S2, S3, S4, and S5. The subfractions S2 and S5 induced reduction of the hemolytic activity of the classical/lectin pathway. Both showed positive results in the assays of migration of neutrophils and bidimensional immunoelectrophoresis. In the assay of generating capacity of SC5b-9, the subfraction S2 presented greater significance when compared to the other used subfractions. Aiming to clarify the action mechanism of the active subfractions on the complement system, tests of determination of the proteolytic or inhibitory activity of proteases (trypsin, chymotrypsin and elastase) had been done. However, in the used concentrations, the samples had not shown proteolytic or inhibitory activity of protease. The isolated compounds also had been submitted to the initial amino-terminal sequence. The identification of the first 15 aminoacids of the major proteinic band of the polyacrylamide gel and 10 and 5 aminoacids of the subfractions S2 and S5 was possible, respectively. However, the sequence of N-terminal results had presented low trustworthiness, due to the low amount of used material. In this work, were isolated and characterized two capable compounds to induce the activation of the complement system. This action was evidenced, after exposition of the normal human serum to the subfractions, for inducing to the formation of the SC5b-9 complex and will increase the migration of neutrophils, probably because they can induce the formation of chemotactic factors. This study allowed a better evaluation of some components present in the complex mixture which is the poison of Rhinella schneideri, that, in the future, important pharmacological tools for the study of diverse pathologies related the complement system. (AU)