Murexide for determination of free and protein-bound calcium in model systems [Milk]

The determination with murexide of free and protein-bound calcium in model systems of known composition, ionic strength, and pH was investigated. The spectra of calcium murexide in the presence of varying amounts of calcium ions indicated that the absorption maximum fo calcium murexide complex occurs at 480 nm while that of murexide ion is at 520 nm. The absorbance at 509 nm is independent of calcium ion concentration and, therefore, could be used to measure the total dye. The spectra are pH dependent but constant in the range 6.5 to 7.0. The apparent dissociation constant of calcium murexide is dependent upon ionic environment, ionic strength, and free calcium ion concentration. The relationship between the apparent dissociation constant and free calcium concentration was established. Whole casein had no effect on the absorption spectra of calcium murexide and no affinity for calcium murexide complex or murexide ion. Beta-casein, at the concentrations employed, did not influence the dissociation fo calcium murexide. At pH 7.0, ionic strength .1, and 2 C, Beta-casein bound calcium as if there were 8.65 binding sites per molecule, each of pK 2.23, corresponding to an intrinsic association constant of 168.9 liters per mole.