Summary:Enzyme immunoassays frequently incorporate the use of horseradish perosidase äs the enzyme label. This enzyme usually catalyses the oxidation of a ehromogen which can be quantified after termination of the enzyme reaction. A chromogen widely used for this purpose is S^'^S'-tetramethylbenzidine. The two electron oxidation of tetramethylbenzidine yields a component with an absorbance maximum at 450 nm. If the enzyme reaction is terminated by lowering of the pH (< 1.0), an additional increase of the absorbance at 450 nm is observed.It is shown that this additional increase is partly due to a l .4-fold increase in the molar lineic absorbance of oxidized tetramethylbenzidine, caused by the acidic pH, äs well äs a quantitative shift of the existing equilibrium between tetramethylbenzidine, oxidized tetramethylbenzidine and their charge-transfer complex. The total absorbance increase upon acidification of the reaction mixture depends therefore on the reaction conditions äs well äs the reaction coordinate.

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