Lead ion effect on creatine kinase: equilibrium and kinetic studies of inactivation and conformational changes

Abstract

The effects of lead ions on creatine kinase (CK) were studied by measuring activity changes, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate (ANS)-binding fluorescence along with size-exclusion chromatography (SEC). Below 5 mM Pb 2+ concentration, there was nearly no change of the enzyme activity and a slight change of the ANS-binding fluorescence. The CK activity decreased significantly from 10 to 25 mM Pb 2+ concentrations. No residual activity was observed above 25 mM Pb 2+. The kinetic time courses of inactivity and unfolding were all mono-phase courses with the inactivation rate constants being greater than the unfolding rate constants for the same Pb 2+ concentration. The changes in fluorescence maximum and fluorescence intensity were relatively slow for 40–80 mM Pb 2+ as well as in the initial stage for less than 5 mM Pb 2+, showing that two transition states exist for Pb 2+ induced equilibrium-unfolding curves. The intrinsic fluorescence spectra and ANS-binding fluorescence measurements showed that even for high Pb 2+ concentrations, CK did not fully unfold. Additionally, the SEC results showed that the enzyme molecule still existed in an inactive dimeric state at 20 and 40 mM Pb 2+ solutions. All the results indicated the presence of at least one stable unfolding equilibrium intermediate of CK during Pb 2+ unfolding.