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Isolamento e caracterização parcial de lectinas em sementes de Capparis yco

by Pereira Rabêlo, Clébia Cleriane

Abstract (Summary)
Purification of multiple molecular forms from Capparis yco seed lectin (CySeL)was achieved by ammonium sulphate fractionation of extract, followed by ionexchange chromatography of fractions (F) on CM-Cellulose column. The protocolwas efficient to obtain from F20-40 and F40-80, CySeL1 (12 mg) and CySeL2 (9 mg),respectively; additionally revealed differences in isoforms charge properties.Chromatography on cation exchange Hitrap SP was not efficient to separeteisoforms. The lectins were non-specific to human erythrocytes and high specifichemagglutinating activities (SHA) were detected with rabbit cells. HA of CySeL1 wasinhibited with monosaccharides and glycoproteins, while CySeL2 recognized onlyglycoproteins. Affinity chromatography on fetuin-agarose column showed that F20-40 HA was more retained on column than F40-80 HA. The pH value and presence ofions interfered differently on isoforms HA; only CySeL2 remained active at pH 10 andwas stimulated by 40 mM Ca2+ and Mg2+. CySeL1 and CySeL2 had the sameelectrophoretic patterns. Upon native PAGE, three protein bands that wereextracted from gel showed HA; SDS-PAGE with amp;#946;-mercaptoethanol revealed twopolypeptides of Mr 15 and 14 kDa. Carbohydrate staining of gel showed theglycoprotein nature of 14 kDa subunit. The protocol used was able to isolate, inmilligram quantities, lectin isoforms from C. yco seeds. The CySeL1 and CySeL2structural differences detected in CM-Cellulose chromatography, inhibition of HAwith monosaccharide and effect of pH and ions on HA associated to high yield purification of proteins indicated their potencial use in structural and biotechnologicalapplications
This document abstract is also available in Portuguese.
Bibliographical Information:

Advisor:Patrícia Maria Guedes Paiva

School:Universidade Federal de Pernambuco

School Location:Brazil

Source Type:Master's Thesis

Keywords:Lectinas Capparis yco - sementes

ISBN:

Date of Publication:02/25/2005

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