Practice question

Horseradish peroxidase in presence of hydrogen peroxide oxidizes 3,3',5,5'-tetramethylbenzidine through single electron transfer steps. Initial oxidation yields a cation radical forming charge transfer complex that appears blue with absorption maxima near 650 nm. This intermediate is stable enough for kinetic reading without stopping. Addition of sulfuric acid halts reaction by denaturing enzyme and further oxidizes TMB to yellow diimine absorbing at 450 nm. Purple or brown precipitates belong to other systems like BCIP-NBT or DAB. Thus native peroxidase-TMB interaction generates distinctive blue, widely accepted for ELISA endpoint quantification.