Phenazine ethosulfate as a preferred electron acceptor to phenazine methosulfate in dye-linked enzyme assays.

Rapid nonenzymatic reduction of 2,6-dichlorophenolindophenol by N-methyl phenazonium methosulfate has been observed in aqueous solution and has been found to increase with increasing pH and ionic strength. The instability of N-methyl phenazonium methosulfate in aqueous solution has been explored in terms of change of absorption spectrum and formation of free radicals as evidenced by EPR spectroscopy. N-Ethyl phenazonium ethosulfate has been found to be much more stable than the methyl analog and did not reduce dichlorophenolindophenol nonenzymatically. The implications of these findings with respect to use of these dyes as artificial electron acceptors are discussed and the recommendation made that, wherever possible, use of N-methyl phenazonium methosulfate be discontinued in favor of use of the N-ethyl analog.