Isolation, purification, and physicochemical characterization of a d-galactose-binding lectin from seeds of Erythrina speciosa

Abstract

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose–Sepharose. The lectin content was about 265 mg/100 g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5 mM, and then p-nitrophenyl α-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two p I values of 5.8 and 6.1 and E 1% 1 cm of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS–PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca 2+ and Mn 2+ ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 °C for more than 90 min the lectin was fairly stable; however, when heated at 70 °C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 °C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered λ max position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar d-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical β-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.