Probing different conformational states of bovine alpha-lactalbumin: fluorescence studies with 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate].
The binding of the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate] (bis-ANS) to bovine alpha-lactalbumin (alpha-LA) was investigated. A strong dependence of the Kd value with the bound calcium stoichiometry was found, with Kd values ranging from 6.2 +/- 0.4 to 64.6 +/- 5.9 microM for apo-alpha-LA and 1:1 Ca(II)-alpha-LA, respectively. A 350-fold enhancement of the bis-ANS emission was observed in the protein-bis-ANS complex, along with an approximately 33-nm blue shift. Both appeared to be related to the hydrophobicity of the binding site and were independent of the Ca(II) ion content. From the difference in bis-ANS affinity between apo-alpha-LA and Ca(II)-alpha-LA, we demonstrated that Zn(II) and Al(III) were able to "lock" the protein into a new "apo-like" conformation, which was similar to, but not identical with, the apo conformation. The protein could be interconverted between all three conformations in a Mn(II) titration. The first Mn(II) shifted the apoprotein to the Ca(II) conformation; at higher Mn(II) levels, binding to the second site shifted the protein toward the apo-like conformation. The same behavior was observed with calcium in large excess. The evidence supported a model for the mutually nonexclusive binding of metals both to site I ("calcium site") and to site II ("zinc site") simultaneously. The results suggest that alpha-lactalbumin possesses a hydrophobic surface that becomes somewhat less accessible upon 1:1 calcium binding in the absence of metals that also bind to the zinc site.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D002413:Cations, Divalent; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D007768:Lactalbumin; D004492:Edetic Acid