Divalent ion-binding properties of the two avian beta-parvalbumins

Birds express three parvalbumins, one a isoform and two beta isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+- and Mg2+-binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes-buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 +/- 0.2 x 10(8) and 1.0 +/- 0.1 x 10(8) M-1. The corresponding values for CPV3-C72S are substantially lower, 4.5 +/- 0.5 x 10(7) and 2.4 +/- 0.2 x 10(7) M-1, a 1.9-kcal/mol difference in binding free energy. Thus, the P-parvalbumin lineage displays a spectrum of Ca2+-binding affinity, with ATH and the mammalian beta isoform at the high and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3-C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+-binding constants of 2.2 +/- 0.2 x 10(4) and 1.2 +/- 0.1 x 10(4) M-1, CPV3-C72S yields values of 5.0 0.8 x +/- 10(4) and 2.1 +/- 0.3 x 10(4) M-1.