A Spectroscopic Study on PtCl(4)((2−)) Binding to Rabbit Skeletal Muscle G-Actin

It was found that the binding of PtCl(4)(2−) to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl(4)(2−)/actin (R). In the first step, R less than 25, the PtCl(4)(2−) ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n(1) = 30 with average binding constant K(1)=1.0×10(7)M(-1). The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl(4)(2−)binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n(2)=26 with average binding constant K(2)=2.1×10(6)M(-1). Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl(4)((2−)) binding, the G-actin turns to an open and loose structure in a discontinuous mode.