PurificaÃÃo, caracterizaÃÃo de uma lectina e compostos fenÃlicos da entrecasca de Sebastiania jacobinensis (Muill. Arg.): efeito de radiaÃÃo gama sobre estrutura-atividade protÃica e proteÃÃo de flavonÃides isolados

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

A lectin from Sebastiania jacobinensis bark (SejaBL) was isolated using a combination of acetone precipitation, fractionation by ammonium sulphate, ion exchange and gel filtration chromatographies. The molecular mass of SejaBL was determined as approximately 52.0kDa by SDS-PAGE and 50.0kDa by gel filtration. The lectin is a glycoprotein with a neutral carbohydrate content of 6.94%, composed of two subunits with the same molecular mass of 24kDa. The purified lectin hemagglutinated rabbit and human erythrocytes. Glycoproteins inhibited agglutination of rabbit erythrocytes. SejaBL showed maximum activity over the pH range 3.0-7.5, heat stability up to 70ÂC and a potential trypsin inhibitory activity. Fluorescence spectroscopy indicated the existence of hydrophobic surface. Lectin inhibited the mycelial growth of Fusarium moniliforme and Fusarium oxysporum with an IC50 value of 123Â0.5μg and 303Â0.9μg, respectively. Non-target organisms, Artemia salina Leach and embryo of Biomphalaria glabrata, were not affected, indicating low environmental toxicity. As an experimental model, the lectin purified from S. jacobinensis bark (SejaBL) was used as probe that changes on the hydrophobic center, by gamma radiation, structurally alter proteins. To elucidate the effect of free radicals in the molecular properties of SejaBL activity, tertiary structure (intrinsic and bis-ANS fluorescence), molecular weights and reversed phase chromatography were examined after -irradiation at various doses (0.020 to 35kGy). Specific Hemagglutinating Activity (SHA) in irradiated lectin showed significant loss (p<0.05), above 12.5kGy, and a significant increase (p<0.05) in 0.1, 0.8 and 1kGy. Fluorescence spectroscopy indicated suppression of emission intensity when excited at 280 and 295 nm, with high binding of bis-ANS, after high doses. SDS-PAGE and RP-HPLC showed polypeptide fragmentation. Significant change in the hydrophobic surface indicated hydrogen abstraction in amino acids, causing structural disorganization from the lectin. Flavonoids are ubiquitous plant secondary products with radical scavenger ability. The S. jacobinensis Bark Lectin (SejaBL), protein capable of binding to carbohydrates, has their hydrophobic surface modified by free radicals produced by water radiolysis. The present study shows the protective effect on protein and the antioxidant stability of S. jacobinensis bark flavonoids (SejaBF), after gamma irradiation. Two flavonoids isolated were present with fast scavenger ability measured by 2.2-diphenyl-1-picrylhydrazyl radical (DPPH) to 0.5mg/mL, after a high dose gamma radiation. The damage caused by irradiation on protein activity was reduced by 55% in the presence of the flavonoids. The results showed the radiostability of the antioxidant capacity of SejaBF, to protect the lectin from free radicals after irradiation

ASSUNTO(S)

bis-ans toxicidade ambiental capacidade de capturar radical dpph fluorescÃncia bis-ans fluorescence radiaÃÃo gama atividade antifÃngica flavonÃides antifungal activity bioquimica flavonoids dpph radical scavenger capacity environmental toxicity, gamma radiation lectina lectin

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