A microspectrophotometric study on the physicochemical properties of antipyrylazo III injected into rat myoballs for measuring free magnesium ion.

The accurate measurement of the intracellular concentration of free magnesium ions ([Mg2+]i) is essential for evaluating the role of Mg2+ in cellular functions. Among the specific (compared to fluorescent indicators) metallochromic dyes, antipyrylazo III (APIII) appears to be most suitable for measuring such concentrations in vertebrate cells according to in vitro studies. In this work, the intracellular physicochemical properties of APIII as a Mg2+ indicator were investigated in the cultured rat myoball by means of a microspectrophotometric technique, in order to obtain an accurate measurement of [Mg2+]i. A set of intracellular APIII-Mg2+ calibration spectra was established after permeabilization of the cell membrane with saponin. In comparison with recordings obtained in K+ solutions, the APIII absorption spectra recorded on a myoball exhibited a red shift and an overall change in absorbance, similar to that observed in a protein (bovine serum albumin: BSA) solution. The apparent dissociation constant of APIII for Mg2+ in the myoplasm was found to be 3.16 mM, significantly higher than the 1.86 mM measured in K+ solutions at the same pH (7.35). A stoichiometric ratio of 1:1 was found, however, both in solution and in the myoplasm. In addition, the injected APIII significantly affected the [Mg2+]i of myoballs. The [Mg2+]i was found to be 0.9+/-0.18 mM in 85 myoballs, on the basis of the intracellular calibration spectra obtained at the same myoplasmic APIII concentration ( approximately 2.5 mM), and after correction for the perturbing effect of the dye. It is concluded that an intracellular calibration and recording of whole spectrum are essential for proper interpretations of intracellular dye signals.