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 degrees 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 Azo1. 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. On average, the lower limit estimated for the percentage of oriented dye was 2.8-3.0% for Antipyrylazo III and 1.5-1.8% for Arsenazo III, the population differences between the two dyes being statistically significant. The actual percentage of bound and oriented dye molecules is likely to be considerably larger for both dyes. For Arsenazo III, the wave-length dependence of the dichroic signal was not distinguishably different from the 'isotropic' signal, defined as (A0(lambda) + 2A90(lambda))/3. which represents the average spectrum of all the dye molecules independent of orientation.(ABSTRACT TRUNCATED AT 400 WORDS)