ExtraÃÃo lÃquido-lÃquido da lectina da entrecasca de Crataeva tapia l. utilizando micelas invertidas

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

The lectins are ubiquitous protein in the nature that reversibly bind to mono, oligo, polysaccharides and glycoconjugates. They do not present catalytic activity and unlike antibodies, are not products of immune reply. The aim of the present work was to evaluate the extraction and back-extraction of a lectin purified by ionic exchange chromatography (CrataBL) and of crude extract (CE) from Crataeva tapia bark using the reversed micelles system of sodium di(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. The Crataeva tapia bark was collected in the region of Recife city (Pernambuco, Brazil) and the extract [10 % (w/v) in 150 mM NaCl] was obtained by trituration and agitation for 16 h at 4 oC, filtered on gaze and centrifuged (4.000 x g for 15 min). The supernantant obtained was called crude extract (CE). The factors that affect the protein extraction, such as: agitation contact time (5 - 20 min), ionic strength, salt type (NaCl, KCl e CaCl2) and concentration (30 â 300 mM) included, pH of aqueous phase (pH 3.0 â12.0) and surfactant concentration (5 - 100 mM AOT), were investigated. For the back-extraction the following parameters were evaluated: pH of aqueous phase (pH 5.0 â 7.0) and ionic strength (50 - 1000 mM of KCl) being added to the system 5 % of Butanol. The parameters, agitation speed (900 rpm), temperature (25oC) and protein concentration (0.374 mg/ml), were maintained constant in all experiments. The best results for the extraction were obtained with 5 min of contact time between the two phases, 30 mM of NaCl, citrate/phosphate buffer, pH 5.5 and 5 mM of AOT, where it was possible to obtain 100% and 70% of protein extraction for CrataBL and CE, respectively. For the back-extraction, the best condintions were; 10 mM citrate/phosphate buffer, pH 5.5 added by 1000 mM of KCl, where it was possible to obtain a protein recovery of 45.25% (CrataBL) and 80.65% (CE) with 50% of hemagglutinating activity for both samples. Samples from the best conditions of extraction and backextraction applied to the CE revealed only one single PAGE band for basic protein and two SDS-PAGE bands. The gel filtration chromatography (AKTA) showed two protein peaks: one of 40 KDa and other of 29 KDa. The oligomeric nature of lectin was detected by SDSPAGE and gel filtration chromatography. The comparison of the chromatographyc profile in gel filtration showed the efficiency of the reversed micelles system in the lectin purification

ASSUNTO(S)

bioquimica lectina crataeva tapia micelas

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