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Analysis of a calcium ion binding system composed of two different sites.

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca(++) binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca(++) binding sites and without such solute were titrated with Ca(++) using Mx as an indicator of free Ca(++) concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each