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

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.

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.

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).

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.

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 k_off1 = 1.0 s^-1 and k_off2 = 1.5 s^-1.