Preparation of Homogeneous Concanavalim A

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Abstract
Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6-8.degree. C for 16-18 h at pH 3.0-3.2 in 1 M NaCl, 0.08 M glycine and 3 mM each Ca+2 and Mn+2, heat treating at 45.degree. C for 2 h and centrifuging. 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 given. Con A solutions standing at room temperature at pH 7 for 10 days lose through precipitation only 5-8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCl. In the solution conditions of SDS [sodium dodecyl sulfate] and urea-SDS gels, Con A not only unfolds slowly and incompletely but it also forms high MW 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.