Binding of retinoids to beta-lactoglobulin isolated by bioselective adsorption.
Binding of the retinoids, all-trans-retinol, all-trans-retinal, all-trans-retinyl acetate, and all-trans-retinoic acid, to beta-lactoglobulin (LG) (96% purity) that had been prepared by bioselective adsorption on N-retinyl-Celite was determined from changes in the fluorescence quenching (332 nm) of the protein tryptophanyl residues. High affinity binding of all of these compounds occurred at pH 7.0, and the apparent dissociation constant ranged from 1.7 to 3.6 x 10(-8) M. Furthermore, 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 were 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 obtained, indicating that a large number of the sites already had bound lipid or that the protein had been denatured. As the pH was lowered toward 5.15, the affinity decreased about fourfold, but the stoichiometry of binding was unchanged. Far UV circular dichroism spectra indicated that the secondary structure of the protein was not significantly affected by ligand binding; however, the near UV spectra were changed, indicating 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.
Wang
Q
Q
Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
Allen
J C
JC
Swaisgood
H E
HE
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods / Q000662:veterinary*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008892:Milk / Q000737:chemistry; D008894:Milk Proteins / Q000032:analysis; D011485:Protein Binding; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000662:veterinary*; D000067816:Whey Proteins
D007782:Lactoglobulins; D008894:Milk Proteins; D012176:Retinoids; D000067816:Whey Proteins