Experiments were carried out on intact, single skeletal muscle fibres from frog in order to estimate the apparent diffusion constant of myoglobin (denoted DAPP) in the myoplasm of living muscle cells. An optical technique was employed to measure myoglobin concentration along the fibre axis following injection of metmyoglobin (denoted metMb) at a point source. The concentration profiles were fitted by the one-dimensional diffusion equation to give estimates of DAPP. The method relied on the fact that myoglobin is normally absent from these frog fibres, thus permitting resolution of the myoglobin-related absorbance above the intrinsic absorbance of the fibre. 2. One complication in the method was that metMb became significantly reduced to oxymyoglobin (denoted MbO2) during the elapsed time before measurement of the concentration profile. The rate of reduction was evaluated by fitting myoglobin-related absorbance spectra, measured at different times following injection of metMb, with in vitro absorbance spectra of metMb and MbO2. Results from four experiments indicated that reduction could be described by a first-order, irreversible reaction having an average rate constant of 0.0164 min-1 (22 degrees C). The effect of reduction on the fitting of DAPP was taken into account. 3. DAPP was determined under three fibre conditions: (1) long sarcomere spacing (3.6-3.8 microns) at 16 degrees C, (2) long sarcomere spacing at 22 degrees C, and (3) normal sarcomere spacing (2.4-2.7 microns) at 22 degrees C. The average values for DAPP under these conditions were: (1) 0.12 (n = 5); (2) 0.17 (n = 5); and (3) 0.15 (n = 7) x 10(-6) cm2 s-1. The average value at 22 degrees C, 0.16 x 10(-6) cm2 s-1, is about 4 times smaller than values for myoglobin diffusivity at 20 degrees C commonly assumed in models of facilitated transport of oxygen by myoglobin. 4. In order to test the possibility that the unexpectedly low value of DAPP found in intact fibres might be due to the binding of myoglobin to relatively immobile sites in myoplasm, experiments were carried out in a cut-fibre preparation using a technique described by Maylie, Irving, Sizto & Chandler (1987 b) for determining the diffusion constants and degree of myoplasmic binding of absorbance dyes. Values for DAPP and the factor (denoted 1 + beta) by which the total myoglobin concentration exceeded the free myoglobin concentration were obtained by fitting the absorbance data by solutions of the one-dimensional diffusion equation.(ABSTRACT TRUNCATED AT 400 WORDS)
