| Abstract: | 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(β-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 × 105 M-1 and 9 × 103 M-1, respectively. The advantages and limitations of this methodology are discussed. |
