Vitamin D-dependent Calcium-binding Protein

A method of purification of a vitamin D-dependent calciumbinding protein from chick intestinal mucosa is described. The purification procedure involved the use of ammonium sulfate precipitation, gel filtration on Sephadex G-100, and preparative disc electrophoresis. The assay system for calcium-binding protein is based upon the competition between calcium-binding protein and an ion exchange resin for added 45Ca. An equation linearly relating binding protein concentration to binding activity is given. Sedimentation velocity and gel filtration behavior indicated that the product was homogeneous with respect to size. Two acrylamide gel systems gave single bands, whereas a third showed the presence of a possible degradation product in addition to calcium-binding protein. The molecular weight of calcium-binding protein was found to be about 2.8 x lo4 by calibrated gel filtration and about 2.5 X lo4 by equilibrium sedimentation (assumed partial specific volume = 0.72). The formation constants between calcium-binding protein and calcium, strontium, and barium were 2.6 X lo5 M-‘, 3.9 X lo4 M-l, and 5.8 X lo3 M-‘, respectively, and it appears that 1 mole of protein binds 1 atom of calcium. Preliminary analysis of the composition of the protein is reported.