Binding of ions and hydrophobie probes to α-lactalbumin and k-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, α-lactalbumin and K-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 (K_diss = 0.16 μM) (site I) for calcium ion in α-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-α-lactalbumin were about 80 βm for ANS and 4.7 μm 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 (>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 K-casein (K_diss = 15.6 μM). 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.