Estimation of parvalbumin Ca(2+)- and Mg(2+)-binding constants by global least-squares analysis of isothermal titration calorimetry data.

The use of competitive isothermal titration calorimetry (ITC) to measure high-affinity binding constants has been largely restricted to systems with a single binding site or multiple identical sites. This study demonstrates the extension of this approach to proteins with two nonequivalent EF-hand Ca(2+)-binding sites--rat beta parvalbumin and the S55D/E59D variant of rat alpha parvalbumin. The method involves simultaneous (global) least-squares analysis of titrations with Ca(2+), with Mg(2+), with Ca(2+) in the presence of Mg(2+), and with Ca(2+) or Mg(2+) in the presence of a competitive chelator (EDTA or EGTA). The Ca(2+) and Mg(2+) binding constants obtained for rat beta agree well with estimates obtained by flow dialysis. Although the Ca(2+) affinity of alpha S55D/E59D is too high to measure by flow dialysis, it was amenable to analysis using the ITC-based approach. The combined S55D and E59D mutations increase the Ca(2+) and Mg(2+) affinities of the mutated binding site by factors of 14 and 26, respectively. This behavior is consistent with that seen previously for the rat beta S55D variant.