Preparation of Homogeneous Concanavalim A

Abstract

Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6–8°C for 16–18 hr at pH 3.0–3.2 in 1 M MaCl, 0.08 M glycine and 3 mM each Ca⁺² and Mn⁺², heat treating at 45°C for 2 hr and centrifuqing. The supernatant was neutralized to pH 5 and stored in the cold. The overall yield was 70–80% Some of the properties of Con A at pH 5 are: The absorption coefficient of a 1 g/dl solution is 13.7 at 280 nm; the mean residue elliptic-ity at 224.5 nm is −9,300° to −9,800°; by sedimentation equilibrium, its molecular weight is 53,000 between pH 3.0 and pH 5.2. Con A solutions standing at room temperature at pH 7 for ten days lose through precipitation only 5–8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCI. In the solution conditions of SDS and urea-SDS gels, Con A not only unfolds slowly and incompletely, but it also forms high molecular weight aggregates. Thus, electrophoresis of Con A in such gels is unsuitable for tests of homogeneity. However, as judged by sedimentation equilibrium in 6.5 M quanidine at pH 8.1, purified Con A was monodisperse.