An optical stopped-flow and 1H and 113Cd nuclear magnetic resonance study of the kinetics and stoichiometry of the interaction of the lanthanide Yb3+ with carp parvalbumin

The rate constants for the dissociation of the lanthanide Yb3+ from the CD and EF calcium-binding sites of carp parvalbumin (isoelectric point, 4.25) have been measured using optical stopped-flow and 1H nuclear magnetic resonance (NMR) methods. The off-rate constants for Yb3+ are 1.5 × 10−1 and 1.3 × 10−3 s−1 respectively, at pH 6.6 and 23 °C. The relative displacement of Ca2+ from the two sites by Yb3+ was determined from the observed amplitude of the fast and slow kinetic phases. Yb3+-shifted 1H-NMR spectra of parvalbumin are presented as a function of pH, concentration, and H2O:D2O ratio to relate the NMR results to the kinetic and optical results. The displacement of Cd2+ from parvalbumin by Yb3+ was studied using 113Cd NMR. All of the results show the sequential displacement of Ca2+ from the CD and EF sites of parvalbumin by Yb3+. Some results are also presented for Tb3+ and Gd3+.