BINDING OF VITAMIN-D AND CHOLESTEROL TO BETA-LACTOGLOBULIN

beta-Lactoglobulin was isolated directly from acidic whey by bioselect ive adsorption on N-retinyl-Celite(TM), yielding preparations of great er than or equal to 96% purity. Interactions of these preparations wit h vitamin D-2, vitamin D-3, ergosterol, cholesterol, and 7-dehydrochol esterol were examined by following changes in the fluorescence spectra . Both the excitation and emission spectra indicated that energy was t ransferred between the tryptophanyl residues of the protein and the ch romophore of the ligand. Analyses of the fluorescence changes that occ urred upon titration of beta-LG with the various ligands allowed deter mination of the dissociation constant for the complex and the number o f moles bound per mole of protein. The affinity for vitamin D-2 (disso ciation constant of 4.91 nM) was 10-fold higher than that of the other compounds. except for ergosterol, which was 5-fold larger than the ot hers. Also, the affinity was 10-fold higher than that typically report ed for the retinoids. Furthermore, the value obtained for the number o f moles bound per mole of protein was 2 mol.mol(-1) for each of the li gands examined in this study; it has been well established that all of the retinoids are bound with a stoichiometry of 1.0. These results su ggest that beta-LG may be a better carrier of vitamin D than of vitami n A.