In vitro calibration of the equilibrium reactions of the metallochromic indicator dye antipyrylazo III with calcium.

The equilibrium reactions of the metallochromic indicator dye Antipyrylazo III with calcium at physiological ionic strength have been investigated spectrophotometrically. Dye absorbance as a function of wavelength was measured at various total dye and calcium concentrations. Analysis of the absorbance spectra indicated that at pH 6.9 at least three calcium:dye complexes form, with 1:1, 1:2, and possibly 2:2 stoichiometries. The dissociation constant and the changes in dye extinction coefficients on formation of the 1:2 complex, the main complex which forms when Antipyrylazo III is used to study cytoplasmic calcium transients, have been characterized.