Biochemical characterization of a lectin from Delonix regia seeds |
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Authors: | Pando S C Macedo M L R Freire M G M Toyama M H Novello J C Marangoni S |
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Institution: | (1) Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brasil;(2) Departamento de Ciências Naturais, Laboratório de Purificaçäo de Proteínas e suas Funções Biológicas, CEUL, Universidade Federal de Mato Grosso do Sul, Três Lagoas, MS, Brasil |
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Abstract: | A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn2+ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL. |
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Keywords: | Delonix regia glucose/mannose lectin Leguminosae seeds N-terminal sequence |
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