A sensitive bacterial luminescence probe for O 2 in biochemical systems

Abstract

1. 1. An oxygen assay method applicable at very low oxygen concentrations has been developed using bioluminescence as an oxygen indicator. The method can be applied with accuracy to the oxygen concentration range between 10 −6 and 10 −8 M. 2. 2. The reliability of the method was verified by determining the oxygenation state of horse heart myoglobin as a function of oxygen concentration, and comparing this curve with that obtained polarographically. The luminescence method gave a half-oxygenation concentration of 2.2·10 −6 M (1.1 mm Hg) with a Hill constant n = 1.0, while the polarographic method gave 2·3·10 −6 M (1.2 mm Hg) O 2 with n = 1.0. With a series of heme-substituted myoglobins with half-oxygenation concentrations ranging from 2.2·10 −6 (1.1 mm Hg) to 4·10 −7 M (0.2 mm Hg) O 2, the luminescence method yielded curves with n = 1.0 in all cases. 3. 3. The oxygenation state of both yeast hemoglobin and Ascaris perienteric fluid hemoglobin were continuously measured as a function of oxygen concentration from the fully oxygenated to the fully deoxygenated states. Both hemoglobins showed a half-oxygenation value of (0.01 mm Hg) 2 · 10 −8 M which is the lowest ever to have been measured. 4. 4. The method was applied to the measurement of the redox change in mitochondrial cytochrome c. The half-reduction of cytochrome c was observed at an oxygen concentration of 7·10 −8 M. 5. 5. The oxygenation state of yeast hemoglobin was compared with the redox state of cytochrome oxidase in yeast cells as a function of oxygen concentration. Half-oxygenation of hemoglobin in yeast cells is observed at an oxygen concentration of 2·10 −8M, which is identical to that observed in purified yeast hemoglobin and is lower than the oxygen concentration producing a 50% reduction of cytochrome a 3(3·10 −8 M) and cytochrome a (2·10 −7 M) in yeast cells.