Preparation of Concanavalin A Consisting Solely of Intact Subunits

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Abstract
Concanavalin A (Con A) consisting solely of intact subunits, i.e., fragment-free Con A, was prepared by the following procedure. Con A obtained by a modification of the method of Cunningham et al. (Abe, Y. & Ishii, S. (1974) Seikagaku (in Japanese) 46, 559) was dialyzed against 1 mM EDTA and chromatographed on a CM-cellulose column at pH 7.1 in the presence of 1 mM EDTA. The chromatography gave three major peaks, F1, F2, and F3. Fragment-free Con A was obtained in the peak eluted last (F3). This Con A gave a single band on poly-acrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS), but gave a minor band as well as the major band in PAGE at pH 4.5. The minor band was removed by gel filtration on a Bio-Gel P-150 column. By our method, fragment-free Con A can be prepared even from jack bean meal containing a large proportion of fragment-containing Con A. In the above CM-cellulose chromatography, a period of dialysis of Con A against a buffer containing EDTA was very important for the preparation of fragment-free Con A. When Con A was dialyzed overnight, the protein could be fractionated as described above. However, Con A which had been dialyzed for a week was not adsorbed at all on the column. In the presence of metals (Ca2+ and Mn2+), Con A was adsorbed on the column equilibrated with a metal-containing buffer and eluted as a single peak. Results in the three kinds of chromatography suggest that negatively charged Con A is more demetalized than positively charged Con A, that fragment-containing Con A loses bound metals more rapidly than fragment-free Con A, and that Con A is demetalized in discrete step(s).

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