TMB produces a soluble end product which is blue in color that is quantitated at 650nm. Upon addition of an equal volume of 0.5M/Liter HCl, the resulting yellow chromogen is quantitated at 450nm. The yellow product is stable at room temperature for at least 1 hour displaying less than a 1% loss in absorbance during this time interval. 1.56mM TMB is recommended for manual assays.
Benzidine and its derivatives are well documented electron donors for the horseradish peroxidase (HRP) /H2O2 oxidation/reduction system. These amines are known to be potent carcinogens.
The synthesis of TMB by Holland, et al., provided a reagent more sensitive than benzidine and much less hazardous due to ortho methylation.
Using H2O2 as substrate, the reaction of TMB with HRP proceeds through 2 phases. First, a charge transfer complex, the result of a 1-electron oxidation is formed. This is a blue product displaying an absorption maximum at 650nm. Further oxidation proceeds through a green color shift to the fully oxidized yellow diimine with an absorption maximum at 450nm.
The diimine is quantitatively formed only if the molar concentration of H2O2 is twice that of TMB. The molar extinction coefficient of the charge transfer complex is reported to be 3.9x10e4 mol-1 cm-1 and that of the diimine to be 5.9x10e4 mol-1 cm-1. The blue reaction product can be converted to the diimine non-enzymatically by the addition of acid providing an increase in sensitivity of at least 1.5.
1.56mM/Liter TMB is designed to measure low levels of HRP preventing the formation of the diimine allowing the user to determine enzyme activity or analyte concentration kinetically at 650nm or as an end point at 450nm after the addition of acid.