Kinetics of Ca2+ Dissociation from Cod Parvalbumin Studied by Fluorescent Stopped-flow Method

Abstract

Background: Small Ca2+-binding protein parvalbumin possesses two strong Ca2+/Mg2+- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca2+ association/dissociation. Objective: Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca2+-induced changes in the protein, both in steady-state and kinetic mode. Methods: Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca2+ dissociation from cod parvalbumin after fast mixing of Ca2+-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). Results: The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca2+]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca2+ to cod parvalbumin is sequential. Conclusion: The results of the present spectral study showed that the binding of Ca2+ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s-1 and koff2 = 1.5 s-1.