Detection of two classes of Ca2+ binding sites in troponin C with circular dichroism and tyrosine fluorescence

We have observed a biphasic increase in [theta]222 and the tyrosine fluorescence of troponin C (TnC) with increasing [Ca2+]. Approximately 62% of the total helical change and 81% of the total increase in tyrosine fluorescence occurred with Ca2+ binding to a class of sites with a KCa of approximately 2.7 X 10(7) M-1. The remaining spectral changes occurred with Ca2+ binding to a class of sites with KCa of approximately 3.1 X 10(5) M-1. We interpret these biphasic responses in terms of distinct structural changes occurring in TnC with Ca2+ binding to the high affinity (KCa approximately 2.1 X 10(7) M-1) Ca2+-Mg2+ sites and to the low affinity (KCa approximately 3.2 X 10(5) M-1) Ca2+-specific regulatory sites.