RESUMO
The binding of Ca+ to whiting parvalbumin has been studied by intrinsic (tryptophan) protein fluorescence. It has been shown that parvalbumin can exist in three states, which are interpreted as the protein without Ca2+ (P), with one bound Ca2+ (PC) and with two bound Ca2+ (CPC). The spectra of the states have been identified and the protein fluorescence spectra at different Ca2+ concentration have been decomposed on the components corresponding to these states. Relative populations of the P, PC and CPC states have been plotted against Ca2+ concentration. On the basis of a scheme of the successive Ca2+ binding; P+Ck1 in equilibrium PC; PC+Ck2 in equilibrium CPC, corresponding theoretical dependences have been computed and fitted to the experimental ones. The fitted equilibrium constants K1 and K2, are 5.10(8) and 6.10(6)M-1, correspondingly. The schemes are in a good agreement with the experimental data on EGTA-titration of Ca2+-saturated parvalbumin and on pH-titration of parvalbumin in the presence of EGTA.