An experimental artifact in the use of chelating metal ion buffers. Binding of chelators to bovine alpha-lactalbumin.

The binding of EDTA and EGTA to bovine alpha-lactalbumin was shown to stabilize the native conformation relative to that characteristic of the metal-free protein (A conformer). Fluorescence titration of the metal-free protein with Ca2+ in the presence of EDTA was markedly influenced by micromolar concentrations of EDTA such that the apparent association constant of calcium binding would be significantly larger than obtained in the absence of chelator. These observations explain the discrepancy in values of K alpha reported by different investigators for binding near neutral pH: 2.75 X 10(6) M-1 (Kronman, M. J., Sinha, S. K., and Brew, K. (1981) J. Biol. Chem. 256, 8582-8586); 6.3 X 10(8) M-1 (Permyakov, E. A., Yarmolenko, V. V., Kalinichemko, L. P., Morozova, L. A., and Burstein, E. A. (1981) Biochem. Biophys. Res. Commun. 100, 191-197); and 4 X 10(9) M-1 (Murakami, K., Andree, P., and Berliner, L. J. (1982) Biochemistry 21, 5488-5494). The last two high values were obtained by fluorescence titration in the presence of EGTA, while the former lower one was determined by a gel filtration technique in the absence of chelators. The anomolous association constants for calcium binding and the alteration of a conformational equilibrium observed in the presence of chelators demonstrate the need for great care in the use of chelating metal ion buffers in the studying of metalloproteins.