PurificaÃÃo, caracterizaÃÃo e aplicaÃÃo biotecnolÃgica da(s) lectina(s) presente(s) em Bauhinia membranacea Benth

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Lectins are proteins or glycoproteins of non-immune origin which bind biospecifically carbohydrates with agglutinant cell capacity of a reversible way. Plant leaves of Bauhinia genus have been used in popular medicine with properties said hypoglicemiant and diuretic. Several leaf extracts of Bauhinia membranacea Benth showed lectin presence, indicated by hemagglutinating activity (HA). The aim of this work was the purification of B. membranacea leaf lectin (BmeLL) and its immobilization on Sepharose CL-4B to isolate glycoproteins. A chosen extract (10 %, w/v) was prepared in 10 mM citrate-phosphate buffer, pH 6.5, containing 0.15 M sodium chloride (selected buffer) by 16 h at 4 oC. The extract was treated with ammonium sulphate and a 0-80 % fraction (F0-80%) was used to isolate the lectin since it showed the highest specific hemagglutinating activity (SHA). Chromatography on guar gel column was used to purify the lectin; the matrix was equilibrated with 0.15 M sodium chloride. Protein elution was performed with 0.02 M sodium hidroxide, pH 11. SDS-PAGE (8.5 %, w/v) revealed pure BmeLL. The lectin agglutinated at higher titers rabbit and human erythrocytes; it was not stimulated in the presence of ions (Ca++, Mg++). BmeLL showed better SHA with 0.01 M citrate-phosphate buffer, pH 6.0, containing 0.15 M sodium chloride. A temperature assay revealed that BmeLL was thermostable. The gel filtration chromatographic profile on a Sephacryl S-300 column showed a main protein peak. Casein, fetuin, thyroglobulin, azocasein and asialofetuin abolished lectin activity. BmeLL immobilized on Sepharose CL-4B was efficient to bind avidin and thyroglobulin as well as glycoproteins from egg white and hog thyroid. Preparations containing BmeLL will be utilized in biological assays which permit to evaluate its use as hypoglicemic agent; BmeLL purified will be utilized to characterize cell surfaces

ASSUNTO(S)

bioquimica purificaÃÃo biotecnolÃgica bauhinia membranacea

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