[75] Determination of pH Gradients in Intact Cyanobacteria by Electron Spin Resonance Spectroscopy

The most accepted methodology for the measurement of internal pH in cells, organelles, or membrane vesicles involves the determination of the distribution of weak acids or bases across the membrane. Nitroxides are relatively stable free radicals, which can be designed to be of a varied chemical nature, for instance, that of a weak acid or an amine. They are easily detected by room-temperature electron spin resonance (ESR) spectroscopy, and unlike radiolabeled probes their distribution data are available immediately on the introduction of the sample into the spectrometer. In addition, the method allows for fast and reliable measurements of the water volume of the cell, under the same conditions employed for the determination of internal pH. The ESR method has been shown to be effective and accurate in the determination of internal pH in various systems; it has, however, very rarely been applied to intact cells. The choice of a spin probe for determination of pH gradients in a particular experimental system should be dictated by several factors, the most important of which is probe stability. A set of three probes answering to these specifications is required for a complete pH assay: a neutral, acidic, and an amine nitroxide.