Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins)

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Abstract
The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) was isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (.apprx. 58%) of purified binding protein [BP] was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialyzed and analyzed. These eluates contained the BP as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step was also effective in the isolation of the BP from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analyzed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified BP which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein.

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