Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Contrary to earlier findings, monomeric native α-lactalbumin does bind naphthalene dyes such as ANS and TNS with marked enhancement of their fluorescence. Nanosecond decay measurements indicate there to be two dye binding sites per protein molecule with lifetimes of ca. 2 and 15 ns for ANS and 5 and 11 ns for TNS. The fluorescence titrations curves of α-lactalbumin with ANS and TNS reflect this site multiplicity, i.e., it was not possible to analyze such curves with a single K diss. The apparent dissociation constants for binding of ANS and TNS to native bovine α-lactalbumin, as determined by an ultracentrifugal technique, ca. 950 and 900 μ m, respectively, indicate that such binding is considerably weaker than previously supposed. The A conformer (metal ion-free form) of α-lactalbumin binds ANS and TNS more tightly than the N (native) form of the protein with marked fluorescence enhancement. The A conformer has two dye binding sites with lifetimes for ANS and TNS comparable with those seen with native protein.