Development of a Tetramethyl-p-Phenylenediamine spectrophotometric assay for quantitating cytochrome oxidase activity in bacterial whole cells

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1977

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Physiologically diverse aerobic bacteria possess terminal oxidases in their respiratory electron transport chains that are capable of oxidizing the basic dye N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). This reaction has long been known in microbiology as the 'oxidase' test, whereby TMPD is oxidized to a colored semiquinone free radical, Wurster's blue, that imparts a purple color to the cells, as well as to the surrounding medium. Many taxonomic schemes have routinely employed the qualitative TMPD oxidase reaction because of its simplicity and usefulness, and today it is suspected that the TMPD oxidase reaction measures cytochrome c oxidase activity that is possessed, in varying degrees of complexity, by aerobic bacteria. Quantitative determinations of cytochrome oxidase activity have been confined, with rare exception, to the manometric or oxygraphic measurement of oxygen consumption during the oxidation of TMPD, or some other reduced artificial electron donor. In theory, the formation of the purple color of Wurster's blue, the major end-product of TMPD oxidation, allows for the possibility of quantitating cytochrome oxidase activity using a spectrophotometric assay. This would be performed by adding reduced TMPD to a precisely diluted bacterial cell suspension, and then measuring the rate of increase in color formation that occurs with the subsequent oxidation of the TMPD. This reaction would be monitored spectrophotometrically, the absorbance changes being measured at a specific wavelength. The development of such a TMPD oxidase assay is described herein and its use oriented toward measuring oxidase activities in turbidometrically standardized bacterial whole cell suspensions prepared from colonies grown under identical nutritional and cultural conditions on agar plates. TMPD, initially maintained in a reduced state by the presence of small amounts of ascorbate, was added to cuvettes containing aliquots of different bacterial suspensions, and the rate of TMPD oxidation was monitored by measuring the increase in absorbance that occurred at 610 nm over a given time interval. Micromoles of TMPD oxidized per unit time per unit dry weight of cells could be calculated from the known extinction coefficient for TMPD at 610 nm. The results obtained with this spectrophotometric procedure correlated well with the results obtained with the qualitative Kovacs oxidase test, but no quantitative relationship was apparent with the manometric QO[subscript 2] values previously reported for the TMPD oxidase activities for the same organisms. However, the simplicity and precision of the colorimetric assay developed herein make it a potentially practical alternative to the manometric procedure, and thereby further enhance the use of TMPD oxidation as a taxonomic tool for analyses using bacterial whole cells.

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