Proton nuclear magnetic resonance determination of the sequential ytterbium replacement of calcium in carp parvalbumin.

The substitution of the paramagnetic lanthanide ion ytterbium for the calcium ions bound to the CD and EF sites of carp parvalbumin results in a series of 1H NMR resonances which are shifted far outside the envelope of the 1H NMR spectrum of the diamagnetic form of the protein. Titration of Ca2+-saturated parvalbumin with ytterbium (YB3+) demonstrate that Yb3+ sequentially replaces the two bound calcium ions of the protein. Analysis of the 1H NMR data yields the relative affinities of the two sites (CD and EF) for ytterbium with respect to calcium. The dissociation constants for ytterbium are then calculated to be KYb3+CD equals (4-7) x 10(-10) M and KYb3+EF equals (2-6) x 10(-10) M from the known dissociation constants for calcium [Haiech, J., Derancourt, J., Pechere, J.-F., & Demaille, J. G. (1979) Biochemistry 18, 2752-2758]. The approximate equality of these constants is verified by Yb3+ titrations of apoparvalbumin.