Properties of the metallochromic dyes Arsenazo III, Antipyrylazo III and Azo1 in frog skeletal muscle fibres at rest.

Abstract

Intact single twitch fibres from frog muscle were isolated and mounted in a normal Ringer solution (16.degree. C) on an optical bench apparatus for measuring fibre absorbance as a function of the wave-length and polarization of the incident light. Fibre absorbance was measured in resting fibres both in the absence and in the presence of one of three metallochromic dyes: Arsenazo III, Antipyrylazo III and Azol. In the absence of dye, the fibre intrinsic absorbance, Ai (.lambda.), measured as a function of wave-length, .lambda., was well described by the equation: Ai(.lambda.) = Ai(.lambda.long) [.lambda.long/.lambda.)X, where .lambda.long is a reference wave-length selected to lie beyond the absorbance band of the dyes and X is the exponential index. For wave-lengths between 480 and 810 nm, the average value of X was 1.1 for 0 deg polarized light (electric vector parallel to the fibre axis) and 1.3 for 90 deg polarized light (electric vector perpendicular to the fibre axis). The intrinsic absorbance at 0 deg, Ai,0 (.lambda.), was somewhat larger than the intrinsic absorbance at 90 deg, Ai,90 (.lambda.); for example, on average (n = 6), Ai,0 (810 nm) was 0.22, whereas Ai,90 (810 nm) was 0.016. Following dye injection, dye-related absorbance was estimated from the measured total fibre absorbance by subtracting the component attributable to the intrinsic absorbance; additionally, for comparison with in vitro calibrations as a function of wave-length, myoplasmic dye absorbance was corrected for the steady change in dye concentration with time that was attributable to dye diffusion. In fibres injected with either Arsenazo III or Antipyrylazo III, the dye-related absorbance measured with 0 deg light, A0 (.lambda.), was found to be significantly greater than that measured with 90 deg light, A90 (.lambda.), indicating the presence of a resting ''dichroic'' signal, A0 (.lambda.)-A90 (.lambda.), attributable to bound and oriented dye molecules. Over-all, the results strongly suggust that accurate calibrations of [Mg2+] or [Ca2+] in cytoplasm, either at rest or during activity, by means of metallochromic dyes cannot be carried out in a reliable fashion unless the properties of bound dye molecules are taken into account. An Appendix is included which analyses the in vitro spectral properties of Antipyrylazo III as a function of dye concentration and of free [Mg2+]. The dye chemistry was found to be more complicated than had been indicated by previous reports. The major complications observed in the absence of Ca2+ appear to be due to (i) the formation of metal-free dimers (dissociation constant estimated to be 4.0 mM), and (ii) the formation of a second type of Mg2+-dye complex, possibly a 1 Mg2+:2 dye complex, that produces an absorbance increase at 720 nm, the wave-length most commonly used to monitor changes in the Ca2+-dye complex.