On experimental artifacts in the use of metal ion chelators for the determination of the cation binding constants of alpha-lactalbumin. A reply.

The binding constant of Ca2+ to the strong cation site of bovine alpha-lactalbumin has been measured directly by monitoring the free calcium concentration by Quin 2 fluorescence. A dissociation constant of 1-4 nM was calculated, which confirms the strong calcium binding properties of this protein. In order to examine whether the metal ion chelators EDTA or EGTA affect the cation binding equilbria by binding to bovine alpha-lactalbumin, calcium binding equilibria were carefully measured under highly stabilized pH and temperature conditions. Within the concentration ranges required for competitive binding by these ligands (EDTA or EGTA) (less than 1-3 mM) these chelators produced no artifacts, in contradiction to the data of Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709).