PurificaÃÃo e caracterizaÃÃo da lectina da vagem de Caesalpinia ferrea (cfepl): aplicaÃÃo biolÃgica

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Lectins are proteins that bind specifically and reversibly to carbohydrates. Caesalpinia ferrea is a leguminous tree widely distributed in Brazil used in popular medicine. The aim of this work was the purification and characterization of C. ferrea pod lectin (CfePL). Pod extract (E) in 0.15 M NaCl was partially purified on activated charcoal followed by ammonium sulphate fractionation (0 â 80%, F80). Hemagglutinating activity (HA) of E and F80 were evaluated using different erythrocytes. F80 was chromatographed on chitin column and washed with 0.15 M NaCl followed by 1 M NaCl; CfePL was eluted with 1 M acetic acid (pH 4.0). CfePL HA was evaluated with ions (Ca++ and Mg++), pH values (2 - 12), carbohydrates, glycoproteins and temperatures (30Â - 100Â C, 30 min). Molecular mass of native protein was determined in a ÃKTAFPLC system using a Sephacryl column; CfePL preparations were evaluated by PAGE for acidic and basic native protein, as well as under denatured and reduced conditions. CfePL antimicrobial activity was performed with strains of Gram-positive (5) and Gram-negative (3) bacteria or fungi (4). CfePL did not show specificity to human erythrocytes; rabbit erythrocytes (512-1) were used to HA evaluation. CfePL HA was stimulated by ions (65536-1); at different pH values, the best HA (2048-1) were obtained with citrate-phosphate (pH 4.5, 5.0 and 5.5) and phosphate (pH 7.5) buffer, been almost abolished at pH 9.0 (2.0-1). CfePL, active even after heating at 100ÂC, was partially inhibited by carbohydrates (threalose, fucose, N-acetyl-Dglucosamine, mannose, fructose, galactose, ramnose, saccharose) and ovalbumin, fetuin, casein and glycoproteins from rabbit, human and fetal bovine serum. CfePL, a basic protein, showed a main band by SDS-PAGE. ÃKTAFPLC system resolved two protein peaks with 43 and 31 kDa. CfePL (1,5μg) inhibited growth of tested microorganisms; best results (17 mm halo) were obtained with Escherichia coli and Colletotrichum gloesporioides and presented minimum inhibitory concentration (MIC) of 10 μg/ml for both microorganism. In conclusion, CfePL, purified in milligram quantities, was a powerful antimicrobial agent of low cost, with wide spectrum of action

ASSUNTO(S)

caesalpinia ferrea eritrÃcitos lectina bioquimica

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