Interference by cytochromes in the measurement of calcium with murexide

The calcium-binding dye murexide has become a widely-used tool for measuring changes in the free Ca*+ concentration in biological systems. The method was introduced in this context in [ 1,2] and the present status of the measurements, together with certain problems encountered in the use of the dye, was the subject of the review [3]. Murexide was first used to study mitochondrial Ca2+ accumulation in [4] where calciumdependent colour changes of the dye in suspensions of rat liver mitochondria were observed with a dual wavelength spectrophotometer. The wavelength pair 540-5 10 nm was employed for the murexide measurements [4]. Here, we show that, if this wavelength pair is used with mitochondrial suspensions, the contribution to the difference signal from changes in the redox poise of the cytochromes is incompletely eliminated. Since the original mitochondrial experiments [4], optical measurements have been made with murexide, of the free calcium concentration in mitochondrial suspensions (e.g., [S-8] and the reviews in [9,10]). In [5-lo] the wavelength pair 540-507 nm has generally been used. Our measurements show that although 540 nm is almost isosbestic for the cytochrome system during the aerobic to anaerobic transition, 5 10 nm (which is almost isosbestic for the calciuminduced spectral change of murexide) is not adequately isosbestic for this redox change in the cytochrome system. A dual wavelength instrument was employed in [4] which used interference filters of comparatively