Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators. I. General calculational method.

A calculational method is developed for spectrophotometric determination of stoichiometrics and individual equilibrium constants in the complexing of metal ions with metallochromic indicators. Implicit expressions are developed for the calculation of all parameters necessary to describe mixtures of 1 : 1, 1 : 2 and 2 : 1 metal-indicator complexes. The analysis of titration curves entails a series of one-variable best-fit determinations based on mass action and conservation laws; this reduction in the number of degrees of freedom in the curve-fitting procedure yields greater resolution of the complexing parameters than is allowed by conventional methods. Since a common application of metallochromic indicators is to the determination of metal-binding properties of biological molecules, accurate description of metal-indicator complexing is vital for investigation of the regulatory roles of metal ions in biological events.