Biochemical characterization of a lectin from Delonix regia seeds.
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 degrees 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.
Pando
S C
SC
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brasil. scpando@unicamp.br
Macedo
M L R
ML
Freire
M G M
MG
Toyama
M H
MH
Novello
J C
JC
Marangoni
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017124:Conserved Sequence; D007887:Fabaceae / Q000737:chemistry*; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D008970:Molecular Weight; D051381:Rats; D012639:Seeds / Q000737:chemistry; D020539:Sequence Analysis, Protein; D013696:Temperature
D000596:Amino Acids; D037102:Lectins