Abstract:

Calmodulin(CaM) is a highly conserved Ca²⁺ binding protein ubiquitously found in animals and plants, which is involved in a large variety of cellular functions. The presence of many other metal ions in the physiological and nonphysiological environment such as heavy metal ions suggests that CaM might be binding other metal ions than Ca²⁺, which might influnce CaM′s function. It is important to investigate the general metal ion binding properties of CaM. Based on high sensitivity of square wave polarographic signal of Pb²⁺, the direct binding reaction of Pb²⁺ to CaM was studied by square wave polarography(SWP). The complexing specie, Pb²⁺-CaM, was detected for the first time by SWP in the Pb²⁺-CaM system, and electrochemical reaction characterization was done by cyclic voltammetry. Two reduction peaks were detected in SWP polarograms obtained at different concentration ratios of Pb²⁺ to CaM at pH=6.5, indicating that two electroactive species of Pb²⁺ exist, the reduction peak potentials of two species are in the range of -0.44—-0.47 V and -0.73—-0.77 V vs. SCE, respectively. The peak with a maximum at ca. -0.44—-0.47 V is corresponding to the reduction of free Pb²⁺ under our experimental conditions and the peak with a maximum at ca. -0.73—-0.77 V, clearly more negative than that for the reduction of free Pb²⁺, allows us to interpret it as due to the reduction of Pb²⁺ complexed by CaM. Moreover, prior to the addition 10 times of Pb²⁺, two peak currents increase gradually with the increasing of the Pb²⁺ concentration. At higher metal ion concentration(10—16 times), the peak currents of free Pb²⁺ increased linearly with a higher value of the slope, while the peak currents of the complexing specie, Pb²⁺-CaM, reached maximal and constant. The polarographic titration curves of the two species show that there are ten binding sites in CaM at pH=6.5. Furthermore, the reduction species of Pb²⁺-CaM system was confirmed by cyclic voltammetry with Controlled Growth Mercury Electrode(CGME). One couple of the free Pb²⁺ redox waves were observed clearly in the cyclic voltammogram, and only the reduction peak of the complexing specie of Pb²⁺-CaM system was detected. The results obtained in the paper show a direct evidence for the mechanism of the toxicity of Pb²⁺ by CaM mediating.

Key words: Calmodulin; Heavy metal ion Pb²⁺; Binding site; Square wave polarography; Cyclic voltammetry