It was found that the binding of PtCl42−
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 PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to
sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard
approach are characterized by n1 = 30
with average binding constant K1=1.0×107M-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 PtCl42−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 n2=26
with average binding constant K2=2.1×106M-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 PtCl42− binding, the G-actin turns to an open and loose structure in a
discontinuous mode.
