Protein-carbohydrate interaction. VII. Physical and chemical studies on concanavalin A, the hemagglutinin of the jack bean.

Concanavalin A yielded a monodiperse pattern in the analytical ultracentrifuge in the pH range 2-5. The sedimentation coefficient, sOZ~,~ in pH 5 acetate buffer containing 0.1 M NaCl (total I’/2 = 0.20) was 4s. At pH 7 and above, a two-peak schlieren pattern was observed, the faster component (about 7s) probably representing a dimer. On the basis of an s~o.~ of 3.9s; a D 20.~ of 5.43 X 10-T cm2/second, and a P of 0.73 ml/gm, a molecular weight of 68,000 was calculated (0.10 M acetate, NaCl, I?/2 = 0.45) for the homogeneous component at pH 5. From free-boundary electrophoresis data the isoelectric point of concanavalin A was determined to be 7.1 f 0.1. Starch gel electrophoresis patterns of concanavalin A in borate buffer (pH 8.6) were characterized by an anodically migrating streak. Incorporation of n-glucose into the gel gave a single sharp band which migrated slowly toward the cathode, the glucose probably inhibiting protein-starch interaction. Gel filtration experiments with Biogel P-100 suggested that at pH 5 the molecular weight of concanavalin A is in the vicinity of 50,000; at pH 7.5 concanavalin A is completely excluded, an indication that a high molecular weight species probably is formed by the association of subunits. Concanavalin A has an extinction coefficient, E&, of 11.4 f 0.1, the molar ratio Mtyrasine/ Mtryptophsn being 1.78. Unlike other phytohemagglutinins, concanavalin A does not contain cysteine (or cystine) or carbohydrate. Aspartic acid and serine constitute the most abundant residues, and the protein has a high amide content (68.6 groups/106 gm protein). Amino terminal studies showed the presence of 1.5 moles DNP-alanine/ 68,006 gm protein together with very small amounts of DNP-serine and DNP-glycine. The manganese content of concanavalin A was found to be 0.029 and 0.036%, by activation analysis and emission spectroscopy, respectively.