Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography.
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.
Fitzgerald
R J
RJ
Department of Food Science, North Carolina State University, Raleigh 27695-7624.
Swaisgood
H E
HE
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
D002364:Caseins; D005456:Fluorescent Dyes; D007477:Ions; D007768:Lactalbumin; D002118:Calcium