Electrochemical behavior of hemin binding with human centrin 3.

The electrochemical responses of human centrin 3 (HsCen3) binding with hemin were studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using glassy carbon electrodes (GCEs). In CV, the formal potential (E(0')) of hemin with the addition of HsCen3 shifted from -0.51 to -0.36V (versus saturated calomel electrode, SCE), indicating that a new species of hemin-HsCen3 had formed. Upon binding with HsCen3, the redox current of hemin in CV and DPV decreased significantly. Based on their titration curves, the association constant of HsCen3 with hemin was obtained with a logK of approximately 4, which was consistent with that obtained from spectroscopy. Combining UV-Vis, fluorescence emission, and electrochemical methods, His100 located on the α-helix between the two domains of HsCen3 was identified as the ligand binding residue of hemin. The protein binding-induced change in electrochemical signal was thus used to construct the diffusion coefficient (D=1.43×10(-7)cm(2)/s), the charge-transfer coefficient (α=0.49), and electron transfer standard rate constant (ks=2.54×10(-2)s(-1)) in the presence or absence of HsCen3. The electrochemical investigation of hemin bound with HsCen3 may provide useful data for understanding the biological processes of calcium-binding protein.