Imobilização de lectinas em rede interpenetrada à base de álcool polivinílico e polianilina para a construção de colunas de cromatografia de afinidade

Abstract

The fabrication technique of affinity chromatography columns using lectins has required the development of more specific matrix, which results in a good immobilization capacity systems with high activity.This study aims to propose a lectin-immobilized carrier system allowing the separation of glycoproteins, including affinity chromatography applications.An interpenetrated network of PVAand PANI was synthesizedand activated with GA, and different proportionswere tested in accordance with a statistical design central compound 22.Then, tests were performed to define the optimal conditions for the immobilizationof two different lectins individually: the partially purifiedfraction of Brosimum gaudichaudii(Moraceae)lectin, a Cerrado plant known as mama-cadela, which showed a high concentration of lectin in the seeds;and concanavalin A (ConA), a commercial lectin. The mama-cadela crude extractwas obtained from the millingof seeds and was partially purified by a series of molecular exclusionand bioaffinity chromatography. The chromatographic fractions of proteins were quantified and hemagglutinating activity was determinated. Characterization tests by inhibition of sugars, showsthat the lectin has selectivity for lactose andβ-galactose showing anomeric selectivity.Other tests demonstrated that the lectin in question does not depend on Ca2+and Mn2+ions and the presence of EDTA does not affect itsactivity. Both lectins were successfully immobilized in PVA-PANIGsupport, however, the optimal conditions of immobilization were different. The best immobilization resultsfor Brosimina were obtainedafter2 h with a concentration of 240 μgmL-1offered lectin at pH 6.0; The ConAalso showed the best results in2 h with a concentration of 240 offered μg mL-1lectin supplied, but the optimum pH was 8.0. This result shows that the PVA-PANIGsupport is promising for the manufacture of bioaffinity lectin columns and an option to the study of glycoconjugates in general.