Estimation of parvalbumin Ca2+- and Mg2+-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 Ca2+-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 Ca2+, with Mg2+, with Ca2+ in the presence of Mg2+, and with Ca2+ or Mg2+ in the presence of a competitive chelator (EDTA or EGTA). The Ca2+ and Mg2+ binding constants obtained for rat P agree well with estimates obtained by flow dialysis. Although the Ca2+ 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 Ca2+ and Mg2+ 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. (C) 2003 Elsevier Science (USA). All rights reserved.