Abstract
Phenazine methosulphate (PMS) is used widely as an intermediate electron carrier for coupling the production of NADH or NADPH to the reduction of tetrazolium salts to coloured formazans. Nitro-blue tetrazolium (NBT) is the most popular tetrazolium salt for this purpose, for both spectrophotometric assay of dehydrogenases and the visualisation of dehydrogenases after electrophoresis or in tissue sections. It has been widely assumed (1) that oxygen does not interfere with this reaction, even though it is known that reduced PMS reacts very readily with oxygen (2–4). This assumption has been supported in a study by Altman (5). Sowerby and Ottaway (6) observed that oxygen reduced the yield of formazan in their assay for glutamine, but they failed to detect its consumption during the reaction and postulated other reasons for their nonquantitative results. Oxygen always appears to be ignored when this reaction is used to stain enzyme zones in histochemistry or after electrophoresis. We report here that atmospheric oxygen interferes seriously with this reaction under a wide range of conditions, especially at low rates of reduced pyridine nucleotide production.
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