ISOLAMENTO, PROPRIEDADES BIOQUÍMICAS E ESTUDOS BIOLÓGICOS DA LECTINA DE SEMENTES DA Macrotyloma axillare (E. Meyer)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

Two fractions of a 2-N-acetilgalactosamine (2-NacGal) specific lectin was isolated of Macrotyloma axillare seeds (LMA) using an alternative methodology to affinity chromatography. The active fractions was purified by thermal precipitation of the gross extract at 90C for 30 minutes; precipitation with 20-60% cold ethanol, followed by ion exchange chromatography pH 8,3 and elution in step-wise NaCl gradients. The 0,2M and 0,3M NaCl elution intervals presented hemagglutination activity on red blood cells A1. A @29KDa single band was revealed in the 12% SDS-page electrophoresis analysis. The stability assays in different pH was demonstrated that 0,2M LMA is completely stable in 6,0-8,0 and the 0,3M LMA, of 5,0-9,0. The tendency to form tetramers was demonstrated in both isoforms 0,2M LMA and 0,3M LMA in alkaline pH (8,3) and dimmer in acid and neutral pH (5,0 and 7,0), it was verified in exclusion chromatography of molecular mass. The especificificity of both fractions was demonstrated for red blood cells A1, using activity assays in native red blood cells (A1, A2, B and O blood group). However, the 0,3M LMA had hemagglutination activity at all groups of the trypsinizated red blood cells, losing its specificity in contrast with the 0,2M LMA that stayed A group specific, but without qualitative distinction of A1 and A2. The 0,2M LMA presented positive agglutination in @8% of one sample with 103 Escherichia coli strains in agglutination assays. Differentiated agglutinative titles were observed, indicating that 2-NacGal is not very distributed in the tested strains. In biodistribution assays, the radioactivity was detected on kidneys and in the urine after 180 minutes after the 0,2M 99mTc-LMA injections in Swiss mice, indicating a probable specific binding of the in the kidneys and intact 0,2M 99mTc-LMA transport to the mices urine. The proposed alternative method purified the two active fractions (0,2 and 0,3M) from the seeds of the Macrotyloma axillare.

ASSUNTO(S)

bioquimica purificação de proteínas macrotyloma axillare (e. meyer) lectinas

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