Purification, some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Biochimie. 2002 Oct;84(10):1035-43. doi: 10.1016/s0300-9084(02)00003-2.

Abstract

Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.

MeSH terms

  • Amino Acid Sequence
  • Cross Reactions / immunology
  • Erythrina / chemistry*
  • Fluorescence
  • Galactose / metabolism*
  • Hemagglutination
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Molecular Weight
  • Plant Leaves / chemistry
  • Plant Lectins / antagonists & inhibitors
  • Plant Lectins / chemistry*
  • Plant Lectins / immunology
  • Plant Lectins / isolation & purification*
  • Protein Subunits / chemistry
  • Seeds / chemistry*
  • Sequence Analysis, Protein
  • Spectrometry, Fluorescence
  • Thermodynamics
  • Titrimetry

Substances

  • Plant Lectins
  • Protein Subunits
  • erythrina lectin
  • Galactose