Estabelecimento de um protocolo eficiente para a purificação de uma lectina da alga marinha vermelha, Acanthophora spicifera (M. Vahl) Børgesen

Abstract

Lectins are proteins or glycoproteins that bind specifically and reversibly to carbohydrates without altering their covalent structure. Algal lectins, particularly those from red algae, share some common characteristics such as: monomeric forms, low molecular weight, thermostability, hemagglutination independent of divalent cations, and affinity for glycoprotein oligosaccharides but not for monosaccharides. In addition, they are rich sources of lectins that are potentially anticancer, antiviral, and drug carriers. Understanding their mechanism of action depends on structural studies, which are restricted to a few species of algae. Few macroalgal lectins have been isolated and characterized compared to other sources, such as terrestrial sources. Therefore, the present study aimed to evaluate the most effective protocol to purify a lectin present in the marine algae Acanthophora spicifera. The lyophilized and macerated algae specimens were subjected to four different extractions: two extracts in 20 mM phosphate buffer with 150 mM NaCl, pH 7.0 (PBS) in a 1:40 w/v ratio for 16 h at 25 oC. The extracts with PBS were called extract 1 (E1), without acidification with HCl, and extract 3 (E3) with acidification until reaching pH 1.0. The other two extracts were obtained by treating the algae with 70% ethanol (1:20 w/v) for 30 min, three times. The resulting material was subjected to extraction with 20% ethanol (1:20 w/v) for 4 h at 4 oC. Extracts 2 (E2) and 4 (E4) correspond to the ethanolic extracts without and with acidification, respectively. The soluble protein content of the extracts was determined by the Bradford method and the hemagglutinating activity was performed in order to determine the specific activity. The specific activity (U.H/mgP) of the crude extracts were: E1 with 1.8x107 , E2: 1.5x10, E3: 3.5x109 and E4: 1.9x106 . Only E1 and E3 were purified, both were precipitated at 60% ammonium sulfate and the fractions subjected to ion exchange chromatography separately. The proteins that did not bind to the matrix (P1) were eluted with 20 mM phosphate buffer (PB), a second peak was eluted with PB containing 0.5 M NaCl (P2) and the last peak was eluted with PB containing 1 M NaCl (P3). The purity and molecular mass estimates of the peaks from the chromatography of E1 and E3 were assessed by 15% SDS-PAGE, which revealed only a single band, P2 of E1 and P1 of E3, both peaks with a relative mass of 15 KDa. It was concluded that even after chromatography, there were still contaminants in E1 and that, with the acid treatment, E3 presented the best profile for purification of a lectin present in the algae Acanthophora spicifera.