Fluorescence Studies of the Calcium Binding to Whiting (Gadus merlangus) Parvalbumin

Abstract

The Ca binding by parvalbumin of whiting (Gadus merlangus) was studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changes proceed in 2 stages reflecting the existence of at least 3 forms which can be interpreted as the protein without Ca2+, with 1 Ca2+ and with 2 bound Ca2+ ions/molecule. The fluorescence of these forms was identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into 3 components corresponding to these spectral forms. The dependence of the relative concentration of the 3 forms on Ca2+ concentrations agree well with the 2-step binding of Ca2+ to parvalbumin: .**GRAPHIC**. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 .times. 108 M-1 and 6 .times. 106 M-1. This scheme and the K1 and K2 values are in a good agreement with the independent experimental data resulting from EGTA [ethylene glycol bis(2-amino ethyl ether)-N,N''-tetraacetic acid] titration of Ca2+-saturated parvalbumin and pH titration of parvalbumin in the presence of EGTA and Ca2+.