Isolation, characterization, and subunit structures of multiple forms of Dolichos biflorus lectin.

The Dolichos biflorus lectin was isolated from seed homogenates by adsorption onto insoluble polyleucyl hog blood group A + H substance and subsequent elution with N-acetyl-D-galactosamine. Although the lectin was homogeneous as determined by discontinuous polyacrylamide gel electrophoresis, isoelectric focusing, sedimentation equilibrium, and immunodiffusion against rabbit antisera prepared against the crude seed extract, the lectin was fractionated into at least two electrophoretically distinquishable forms (A and B) by chromatography on concanavalin A-Sepharose. Approximately 12% of the original lectin sample did not bind to the concanavalin A and contains the B form. The bound lectin was eluted specifically and quantitatively as a biphasic peak from the concanavalin A-Sepharose with a gradient of methyl alpha-D-glucopyranoside. Carbohydrate analyses of lectin fractions obtained from different portions of the elution profile showed variation in the amount of mannose and N-acetylglucosamine, thus confirming the heterogeneity of the electrophoretic A form. Both the A and B forms of the lectin are active and are apparently present in the dry seeds. Once separated, the two electrophoretic forms of the D. biflorus lectin are distinguishable by electrophoresis. The separated A and B forms show a high degree of similarity in molecular weights (113,000 and 109,000, respectively), antigenic character, and amino acid compositions. Both forms have alanine as NH2-terminal residues and either leucine or valine as the only detectable COOH-terminal residues. The A and B forms specifically agglutinate and have similar titers for type A human red blood cells. They gave similar precipitin curves with hog blood group A + H substance and show similar inhibition curves with methyl alpha-N-acetyl-D-galactosamine and N-acetyl-D-galactosamine. Discontinuous polyacrylamide gel electrophoresis of the unfractionated D. biflorus lectin in 0.1% sodium dodecyl sulfate-8.0 M urea produced two major bands, corresponding to subunits IA and IIA of the A form of the lectin and two minor bands corresponding to subunits IB and IIB of the B form. Subunit molecular weight determinations by electrophoresis in 0.1% sodium dodecyl sulfate gels showed molecular weights of 26,500 for subunits IA and IIA and 26,000 for subunits IB and IIB, Thus indicating that each form of the lectin is composed of four subunits.