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 corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(beta-aminoethyl)-N-N'-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca(++); the association constants of the high affinity site and low affinity site are 4.3 x 10(5) M(-1) and 9 x 10(3) M(-1), respectively. The advantages and limitations of this methodology are discussed.