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

Abstract

Concanavalin A yielded a monodiperse pattern in the analytical ultracentrifuge in the pH range 2-5. The sedimentation coefficient, so20,w in pH 5 acetate buffer containing 0.1 NaCl (total [Gamma]/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 s20,w of 3.9S; a D20,w of 5.43 x 10-7 cm2/second, and a iV of 0.73 ml/gm, a molecular weight of 68,000 was calculated (0.10 acetate, NaCl, [Gamma]r/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 +/- 0.1. Starch gel electrophoresis patterns of concanavalin A in borate buffer (pH 8.6) were characterized by an anodically migrating streak. Incorporation of -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, E1%1cm of 11.4 +/- 0.1, the molar ratio Mtyrosine/ Mtryptophan 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/105 gm protein). Amino terminal studies showed the presence of 1.5 moles DNP-alanine/ 68,000 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.