Isolation, Characterization, and Subunit Structures of Multiple Forms of Dolichos biflorus Lectin*

The Dolichos bifloms lectin was isolated from seed homogenates by adsorption onto insoluble polyleucyl hog blood group A + H substance and subsequent elution with N-acetyl-n-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 distinguishable 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 o-n-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. bifloms lectin

The Dolichos bifloms lectin was isolated from seed homogenates by adsorption onto insoluble polyleucyl hog blood group A + H substance and subsequent elution with N-acetyl-n-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 distinguishable 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 o-n-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. bifloms lectin are distinguishable by electrophoresis.
The separated A and B forms show a high degree of similarity in molecular weights (113,000 and 108,000, respectively), antigenic character, and ammo acid compositions.
Both forms have alanine as NH,-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 a-N-acetyl-n-galactosamine and N-acetyl-n-galactosamine. Discontinuous polyacrylamide gel electrophoresis of the' unfractionated D. bifloms 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.
The seeds of the Dolichos bifloms plant contain a lectin that specifically agglutinates type A erythrocytes and precipitates blood group A substance (l-5). This lectin has been isolated by adsorption onto insoluble polyleucyl hog blood group A + H substance (6) and subsequent elution by N-acetyl-n-galactosamine (4,5), the immunodominant sugar of the blood group A substance (7). Inhibition studies of the isolated lectin have shown that its blood group A specificity is due to its ability to recognize terminal nonreducing a-N-acetyl-n-galactosamine residues (4,5).
The isolated lectin was found to be a glycoprotein, homogeneous by a number of criteria, including disc gel electrophoresis under acid and basic conditions, sedimentation velocity, isoelectric focusing, and immunodiffusion against rabbit antisera to the crude seed extract. The lectin was precipitated totally by human blood group A substance (4 in the above electrophoresis system as described by Wu and Bruening (25) except that 12.5% w/v sucrose was used in the sample buffer.
In the case of the urea-sodium dodecyl sulfate gels, the sample buffer also contained 2.0 mg/ml of sodium dodecyl sulfate and the sample was heated at 65' for 15 min prior to application to the gels. Subunit molecular weight determinations on sodium dodecyl sulfate gels were conducted as described by Weber and Osborn (27) with the exception that the protein reduction was carried out in 5 mM dithiothreitol as well as 1% mercaptoethanol. Molecular weights and charge to friction ratios of the lectins and subunits were determined by measurements of mobility retardation with increasing gel concentration as described by Hedrick and Smith (28).

Preparation of Concanavalin
A-Sepharose-The N-hydroxysuccinimide ester of succinyl-aminodipropylamino-Sepharose was prepared as described by Cuatrecasas and Parikh (29. 30). This ester was added to a 1. ture was 250". Flow rates of H, and air through the detector were 30 and 300 ml/min, respectively.

Physical Characteristics
and Criteria of Homogeneity-The isolated Dolichos biflorus lectin formed a single peak with a p1 of 5.05 upon isoelectrofocusing in pH 3 to 10 and pH 4 to 6 gradients in glycerol. Immunodiffusion of the lectin with rabbit antisera prepared against the crude seed extract produced a single precipitin band which formed a line of identity with the lectin preparation described in previous work (4). The lectin formed a single, diffuse band in discontinuous polyacrylamide gel electrophoresis on pH 9.7 glycine gels. The diffuseness of the band was independent of the gel concentration and quantity of lectin applied to the gel. An apparent molecular weight range of 107,000 to 112,000 was estimated from measurements of retardation of lectin mobility in increasing concentrations of pH 9.7 glycine gels.  (34). Of 158 mg of lectin chromatographed on concanavalin A-Sepharose, 12.1% of the lectin did not bind to the column and 87.6% of the lectin was bound and specifically eluted with a concave gradient of 0 to 0.3 M methyl Lu-n-glucopyranoside (35,36) in PBS (Fig. 1). The biphasic character of the elution profile of the bound lectin indicates a variation in number of concanavalin A receptor sites on the D. biflorus lectin. A similar elution profile was obtained with gradients of methyl a-n-mannopyranoside. The elution profiles of the unbound and bound lectin fractions did not change upon rechromatography of the various fractions. All of the lectin was recovered in the column flow through upon chromatography on wheat germ agglutinin-Sepharose or on unmodified Sepharose.

Characterization of Multiple
Forms of Lectin-The eluates of D. biflorus lectin from the concanavalin A-Sepharose column were divided into seven fractions, I -VII (Fig. l), and analyzed for carbohydrate content by gas-liquid chromatogra-phy of alditol acetate derivatives of the sugars obtained after acid hydrolysis.
As shown in Table I, each of the fractions contain mannose and N-acetylglucosamine but differ from one another in relative quantities of these sugars. Fractions ZZ to VZZ had the same electrophoretic mobility as the unfractionated lectin on pH 9.7 glycine gels whereas Fraction I had a different electrophoretic mobility (Fig. 2). These two electrophoretic forms of the lectin, designated as Forms A and B, have apparent molecular weights of 116,000 and 103,000, respectively, as estimated from measurements of mobility retardation with increasing gel concentration. A similarity in retardation plot intercepts indicated very similar charge to friction ratios for the two forms.
Although Form B of the lectin could not be detected in electrophoresis of the unfractionated lectin on pH 9.7 glycine gels regardless of the amount of protein applied (Fig. 2), its presence in the unfractionated lectin could be verified by discontinuous electrophoresis on sodium dodecyl sulfate-urea gels in which subunits of Form B have different mobilities than subunits of Form A (Fig. 3). To test the possibility that Form B of the lectin originated as a degradation product of Form A during the 8-to lo-day purification procedure, small quantities of the lectin were isolated batchwise from a common seed   The two fractions were identical when tested in immunodiffusion against antisera prepared against the seed extract and had very similar amino acid compositions (Table II). Fractions I and VII and unfractionated lectin were labeled with dansyl-chloride and hydrolyzed in acid. Dansyl-alanine co-chromatographed with the dansylated NH,-terminal residue from each of the samples in Solvents 1 to 3.
COOH-terminal amino acids were determined by hydrolysis with carboxypeptidase A. The kinetics of released COOH-terminal amino acids indicated an essentially simultaneous release of both leucine and valine residues for both the A and R forms.
Fractions I and VII had similar titers for type A human erythrocytes and showed similar precipitin curies with hog blood group A + H substance (Fig. 4). Similar inhibition curves were obtained for both fractions with methyl tr-N-acetyl-D-galactosamine3 and N-acetyl-o-galactosamine ( Fig. *5).

Subunit
Characterization-Sodium dodecyl sulfate-urea gel electrophoresis of the isolated A and B forms of the lectin showed an increased proportion of subunits designated IA and IIA in Form A and of subunits designated IB and IIB in Form H (Fig. 3). Analysis of the two predominant subunit:, of unfractionated lectin on pH 9.7 glycine gels in urea by the technique $The methyl n-N-acetyl-n-galactobamine of Hedrick and Smith (28) indicated that IA and IIA subunits do not represent monomer-dimer states of the protein; rather they are similar in molecular weight but different in charge to friction ratios. Subunit molecular weight determinations on Forms A and B were carried out in 0.1% sodium dodecyl sulfate as described by Weber and Osborn (27). Under these conditions, only one electrophoretic band was observed for each lectin form (Fig. 6 form (Form A) has been further subfractionated on the basis of its affinity for concanavalin A. All the forms are active.
Concanavalin A has a specificity for the cu-methylglycosides of n-mannose, n-glucose, and N-acetyl-n-glucosamine (35,36 Etzler and Kabat (4). Since the seeds were obtained from a different supplier, the discrepancies detected in molecular weight, isoelectric pH, and methionine content may represent variation in lectin character resulting from differences in the seed source. Despite these minor differences the lectins from both seed suppliers had similar amino acid compositions and specificities and were found to be antigenitally identical when tested with antisera prepared against extracts from each of the two seed sources. Kabat, E. A. (1956)