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Accurate and rapid measurement of the oxygen concentration in biological samples, particularly when these oxygen concentrations are low, has been the goal of many research workers. By far the most successful method has been the use of oxygen electrodes (for review see Silver, 1984). However it is limited by the stability of the electrode surface and by instabilities in the oxygen diffusion barrier (because it measures the rate of diffusion of oxygen to the cathode). An alternate method, which measures the oxygen dependent quenching of the fluorescence of pyrene butyric acid (Vaughan and Weber, 1970; Knopp and Longmuir, 1972; Opitz and Lubbers, 1984), has been much less useful because the fluorescence intensity is affected by many experimental parameters other than oxygen concentration.

Oxygen dependence of the lifetime of the excited triplet state of phosphorescent molecules can be used to measure the oxygen concentration in aqueous media. These measurements are insensitive to much of the optical interference that limits the usefulness of measurements based on the oxygen dependent quenching of luminescence intensity. The measurements also extend to significantly lower oxygen concentrations than are normally attainable using oxygen electrodes. The phosphorescence lifetimes can be accurately measured from a few microseconds to seconds, permitting a wide dynamic range of oxygen concentration measurements. With currently available probes, for example, it is possible to make continuous measurement of oxygen concentrations from 10−4 M to 10−8 M in a single experiment.

There are very few proteins whose structure and properties have been more extensively studied than those of cytochrome c. The reason for this continual interest in cytochrome c lies in the particular characteristics of the molecule which make it a convenient object for studies by chemists, physicists, and biologists. The first of these characteristics is the indisputable importance of cytochrome c in the mitochondrial respiratory chain as a “mediator” of electron transfer between the reductase and the oxidase. Second, is the fact that cytochrome c can be easily dissociated from the membrane and isolated in a pure, crystalline state, suitable for physical-chemical studies (peripheral membrane protein in the nomenclature of Singer and Nicolson, 1972). Third, isolated cytochrome c reacts with other isolated enzymes or enzyme complexes and with artificial electron donors and acceptors, and thus, provides useful model systems. And last, cytochrome c once extracted from the mitochondrial membrane can be reincorporated into the membrane with full reconstitution of its catalytic activity; thus functional properties of the molecule can be well defined.

The measurement of oxygen in intact tissues has proven to be full of technical difficulties. For example, the direct determination of oxygen in tissue using microelectrodes is subject to error in the low range of oxygen pressures (< 1 uM) and is restricted to fixed sites of evaluation (Silver, 1984). Moreover, insertion of the electrode damages the tissue and disturbs the local milieu. Indirect qualitative measurements using surface fluorescence of NADH indicate the presence of anoxia in tissue (Barlow and Chance, 1976) but lacks internal standardization (Koretsky et al., 1987). Spectrophotometric measurements of hemoglobin (Kekonen et al, 1987) and myoglobin (Fabel and Lubbers, 1965; Tamura et al., 1978) are useful for oxygen pressures near their P50 but are subject to optical interference from changes in light scattering and from absorbtion by other chromophores. Alternatively, cryomicrospectrophotometry of myoglobin (Gayeski and Honig, 1986) can provide an accurate method for calculating intracellular oxygen pressure in some tissues. Because this method requires freezing of the tissue, it is impractical for monitoring changes in oxygenation during any length of time.