BINDING OF RETINOIDS TO BETA-LACTOGLOBULIN ISOLATED BY BIOSELECTIVE ADSORPTION

Binding of the retinoids, all-trans-retinol, all-transretinal, all-tra ns-retinyl acetate, and all-trans-retinoic acid, to beta-lactoglobulin (LG) (96% purity) that had been prepared by bioselective adsorption o n N-retinyl-Celite(TM) was determined from changes in the fluorescence quenching (332 nm) of the protein tryptophanyl residues. High affinit y binding of all of these compounds occurred at pH 7.0, and the appare nt dissociation constant ranged from 1.7 to 3.6 x 10(-8) M. Furthermor e, a stoichiometry of 1.0 mol.mol(-1) of protein was obtained for each case, indicating that all of the sites in the protein preparation wer e available. When beta-LG in whey protein isolate (57.4% beta-LG) was studied, a stoichiometry of 0.65 to 0.82 mol.mol(-1) of protein was ob tained, indicating that a large number of the sites already had bound lipid or that the protein had been denatured. As the pH was lowered to ward 5.15, the affinity decreased about fourfold, but the stoichiometr y of binding was unchanged. Far UV circular dichroism spectra indicate d that the secondary structure of the protein was not significantly af fected by ligand binding; however, the near UV spectra mere changed, i ndicating that the flexibility of tryptophanyl residues decreased. The latter effect is consistent with the change in fluorescence quenching and suggests that a tryptophan is in the binding site.