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Preparative biochemistry

Preparation of homogeneous concanavalin A.


PMID 7312834

Abstract

Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6-8 hr at pH 3.0-3.2 in 1 M NaCl, 0.08 M glycine and 3 mM each Ca2+ and Mn+2, heat treating at 45 degrees C for 2 hr and centrifuging. The supernatant was neutralized to pH 5 and stored in the cold. Te overall yield was 70-80%. Some of the properties of Con A at pH 5 are: The absorption coefficient of a l g/dl solution is 13.7 at 280 nm; the mean residue ellipticity at 224.5 nm is -9,300 degrees to -9,800 degrees; 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 NaCl. 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.