title: Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography.
abstract: The technique of analytical affinity chromatography was extended to characterize binding of ions and hydrophobic probes to proteins. Using the immobilized protein mode of chromatography, alpha-lactalbumin and kappa-casein were covalently attached to 200-nm-pore-diameter controlled-pore glass beads and accommodated for high-performance liquid chromatography. The existence of a high affinity binding site (Kdiss = 0.16 microM) (site I) for calcium ion in alpha-lactalbumin was confirmed by chromatography of [45Ca2+]. In addition, chromatography of the hydrophobic probes, 1-(phenylamino)-8-naphthalene-sulfonate (ANS)2 and 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) indicated that Ca2+ bound to a second site (presumably the zinc site or site II) with weaker affinity. Dissociation constants obtained for apo-alpha-lactalbumin were about 80 microM for ANS and 4.7 microM for bis-ANS in the absence of sodium ion. Addition of Ca2+ initially caused a reduction in surface hydrophobicity (lowered affinity for the probe dyes) followed by an increase at higher Ca2+ concentrations (greater than 0.5 mM), suggesting that occupancy of site II restores an apo-like conformation to the protein. Moreover, the effect of Zn2+ was similar to that observed in the higher Ca2+ concentration range, whereas Na+ apparently bound to site I. A calcium binding site of moderate affinity also exists in kappa-casein (Kdiss = 15.6 microM). A cluster of negative charges, probably including the orthophosphate group, most likely comprise this binding site. By preventing self-association, analytical affinity chromatography permits microscale characterization of ligand equilibria in proteins that are unaffected by protein-protein interactions.
authors.affiliation: Department of Food Science, North Carolina State University, Raleigh 27695-7624.
authors.forename: R J
authors.initials: RJ
authors.lastname: Fitzgerald
authors.forename: H E
authors.initials: HE
authors.lastname: Swaisgood
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D007477:Ions; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D002364:Caseins; D005456:Fluorescent Dyes; D007477:Ions; D007768:Lactalbumin; D002118:Calcium