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Background: Small Ca 2+ -binding protein parvalbumin possesses two strong Ca 2+ /Mg 2+ - 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 Ca 2+ association/dissociation., Objective: Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca 2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca 2+ -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 Ca 2+ dissociation from cod parvalbumin after fast mixing of Ca 2+ -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 [Ca 2+ ]/[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 Ca 2+ to cod parvalbumin is sequential., Conclusion: The results of the present spectral study showed that the binding of Ca 2+ 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 ., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)