The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies

Abstract

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9-3, increasing the H30+ concentration resulted in quenching of the fluorescence of fragment 1. Reversible pH-titration curves were obtained which appeared to consist of 2 regions. From pH 4-6.5 a broad titration curve was obtained, whereas from pH 6.5-9 a more pronounced titration behavior was evidenced by a group or groups on fragment 1 with an apparent pKa of .apprx. 7.5. The apparent association constant for Ca2+ and fragment 1 showed a sharp pH-dependence in the region between pH7-8 with tighter Ca2+ binding at higher pH values. A pKa of .apprx. 7.5 could be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. H3O+ and Ca2+ resulted in blue-shifts in the wavelengths of fragment-1 emission. These results were interpreted in terms of H+- and Ca2+-induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.