Some aspects of the chromogen 3,3',5,5'-tetramethylbenzidine as hydrogen donor in a horseradish peroxidase assay

Enzyme immunoassays frequently incorporate the use of horseradish peroxidase as the enzyme label. This enzyme usually catalyses the oxidation of a chromogen which can be quantified after termination of the enzyme reaction. A chromogen widely used for this purpose is 3,3',5,5'-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 (less than 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 1.4-fold increase in the molar lineic absorbance of oxidized tetramethylbenzidine, caused by the acidic pH, as well as 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 as well as the reaction coordinate.