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

Abstract

SUMMARY1. Intact single twitch fibres from frog muscle were isolated and mounted in a normal Ringer solution (16 '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.2. In the absence of dye, the fibre intrinsic absorbance, Ai(A), measured as a function of wave-length, A, was well described by the equation:Ai(A) = Ai(Aiong) (Aiong/A)X, where Along is a reference wave-length selected to lie beyond the absorbance band ofthe 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 S. M. BA YLOR AND OTHERSOn average, the lower limit estimated for the percentage of oriented dye was 2-8-3-0 00for Antipyrylazo III and 1-5-t180% 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. 5. For Arsenazo III, the wave-length dependence of the dichroic signal was not distinguishably different from the 'isotropic' signal, defined as (AO(A) + 2A90(A))/3, which represents the average spectrum of all the dye molecules independent of orientation. On the other hand, for Antipyrylazo III the dichroic spectrum appeared to be shifted significantly towards longer wave-lengths ('red-shifted') compared with the isotropic spectrum. From the magnitudes ofthe isotropic and dichroic absorbances at 650 nm compared with those measured in cuvette calibrations, it was estimated that roughly one-third of the Antipyrylazo III molecules in myoplasm might be bound. for Antipyrylazo III (n = 8), 0-21 x 10-6 cm2 S-1; for Arsenazo III (n = 3), 0-12 x 10-6 cm2 s-1; for Azol (n = 4), 0-10 X 10-6 cm2 s-1. These values are four to nine times smaller than estimates extrapolated from the data of Kushmerick & Podolsky (1969) 12. 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.

Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III.

“…The average value of the apparent diffusion constant D/(R + 1) was 0.259 X 10 -6 cm2/s (column 4). This value is similar to that obtained by Baylor et al (1986) for intact fibers at 16 0 C, 0 .21 X 10-6 cm2/s, and somewhat smaller than the value reported by Kovacs et al (1983a) for cut fibers at 6-9°C, 0.36 X 10-6 cm2/s.…”

“…Baylor et al (1986) also found that, in the absence of divalent cations, antipyrylazo III can form dimers with a dissociation constant, KDp, of 4 mM . The apparent dissociation constant of antipyrylazo III for Ca should be given by (1 + [Mg]/K MgAP + 2[Ap]/Kn,) 2Kc.APQ, in which [Ap] represents the concentration of free monomeric indicator .…”

“…3 in Maylie et al ., 1987b) was used to calculate, from A(550), the concentration of antipyrylazo III in the myoplasmic water ; the molar extinction coefficient, 11(550), was taken to be 2 .55 X 10" M -' cm-' (Rios and Schneider, 1981), and the optical path length, l, was taken to be 0.7 times fiber diameter (Baylor et al ., 1983a) . The value of 11(550) was considered constant, although Baylor et al (1986) have found a small concentration dependence in cuvette calibrations, which they attribute to dimer formation .…”

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III.

“…Fifth, the use of a constant scaling factor, -15 mM, to relate AA(590)/A(550) to A[Mg] may introduce an error. Bayior et al (1986) …”

Fura‐2 diffusion and its use as an indicator of transient free calcium changes in single striated muscle cells

“…[6]). Grynkiewicz et al [l] report a possible small component of fura-free acid (fura-2) binding to red cell membranes.…”