ANALYSIS OF THE ION-BINDING SITES OF CALMODULIN BY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

The binding of Ca2+ and Mg2+ to four calmodulins (SynCaM 1, SynCaM 8, SynCaM 12A, and SynCaM 18A) has been studied by ESI-MS. The mass spect ra were recorded by dissolving the apoproteins in methanol/water (20/8 0, v/v) containing 1 mM CaCl2 or 1 mM MgCl2 and the pH adjusted to 6.0 with ammonia. The carrier solvent was methanol/water (20/80, v/v). In the case of Ca2+ complexation, ESI-MS reveals the presence of three k inds of sites: the first of high affinity corresponding to those deter mined using flow and equilibrium dialysis techniques and two others wi th lower affinities. These results clearly confirm the conclusion of M iles et al. [Miles, M., Comte, M., Schaer, J. J., & Cox, J. A. (1989) J. Inorg. Biochem. 36, 11-25] that there should exist between four and six auxiliary sites for Ca2+. Concerning the complexation of magnesiu m, the four proteins are able to bind two Mg2+ almost certainly on aux iliary cationic sites.