Purification, primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin. Bfl. was purified from Bauhinia forficata seeds by ammonium sulfate fractionation. DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. the molecular homogeneity and purity of BfL were assessed by reversed-phase H PLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. Bit is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol-sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. Bfl hemagglutinating activity was stable at 100 degrees C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. the primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of alpha-helix and beta structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. Bfl also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties. (c) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniv Fed Pernambuco, Dept Bioquim, BR-50670901 Recife, PE, BrazilInst Clin Neuroimmunol LMU, Munich, GermanyMax Planck Inst Biochem, Dept Prot Analyt, D-82152 Martinsried, GermanyUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilFAPESP: 07/58929-4FAPESP: 09/53766-5Web of Scienc