HRP Redox Reaction Driven TMB Color Development, Part Four

In this series, we are breaking down one of our most popular educational pieces, "HRP Redox Reaction Driven TMB Color Development."

ImmunoChemistry Technologies gratefully acknowledges the significant contributions made by one of its founders, Brian W. Lee, Ph.D in the creation of this white paper.

This post will cover part four. See part one, part two, and part three if you missed them previously. And look for part five to be posted soon!


Over a span of almost four decades, HRP has proven quite useful in its ability to reliably conduct the critical redox activities that are required of any chromogenic substrate-dependent signal generation processes. Its low substrate specificity requirements, high substrate turnover rates, and robust stability properties made it the type of enzyme that would make it conducive to its incorporation into many EIA diagnostic testing formats.

Chemical Basis for TMB Oxidation to Colored Product

In 1982, Josephy devised and published one of the first reaction schemes defining the TMB oxidation product intermediates that occur as the result of the HRP oxidation process [15]. Using electron spin resonance and optical spectroscopy methods, they identified the presence of a TMB semiquinone-imine cation free radical and the associated Charge Transfer Complex (CTC). They envisaged the molecular reaction scheme shown in Figure 5. Within this TMB oxidation scheme there exists two chemically distinct, one electron TMB oxidation products. These consist of; 1) a TMB radical cation and 2) the CTC which is made up of a diamine form electron donor and a diimine electron acceptor. This diamine/diimine charge transfer complex is in rapid chemical equilibrium with the cation radical. Within this dynamic reaction scheme there exists three different reaction component entities; each with its own separate molar absorbance max properties. These reaction component entities consist of the native TMB starting material (diamine form) having an absorbance max of 285 nm followed by the CTC having an absorbance max of 652 nm and finally the two-electron loss/complete oxidation state diimine form with an absorbance max of 450 nm.

Figure 5. 3,3’,5,5’-Tetramethylbenzidine (TMB) oxidation product intermediates formed following horseradish peroxidase (HRP) oxidation of the native diamine form of TMB substrate solution. HRP removal of a single electron from the native diamine TMB form leads to the creation of two new intermediate oxidation-state products. These consist of a colorless TMB Cation radical that is in rapid chemical equilibrium with a second blue-green colored intermediate oxidation state entity identified as the Charge Transfer Complex (CTC). The CTC is composed of two different TMB molecules; a colorless diamine form (electron donor) and a yellow colored diimine form (electron acceptor). Following a second HRP facilitated one electron oxidation event, the green TMB product is converted to the yellow colored diimine oxidation product. The CTC has an absorbance max at 652 nm while the diimine oxidation state form displays a maximum absorbance at 450 nm. These two one-electron loss and two-electron loss oxidation state TMB products account for the pre-acid stopped blue-green and acid stopped yellow colored TMB solutions.

The Josephy group also made the experimental observation that if you use a large enough molar excess of H2O2 over the TMB (diamine form) starting material, it was possible to convert all the TMB (diamine form) into the two-electron-loss oxidation state diimine form. By combining this fact with the observation that each of the individual TMB, CTC, and diimine oxidation state derivatives possess unique molar absorptivity properties, the Josephy group was able to determine the molar absorptivity constants for both the CTC and diimine oxidation state forms. They assigned a ε450 = 5.9 x 104 M-1 cm-1 to the diimine form and a ε652 = 3.9 x 104 M-1 cm-1 to the CTC form. These two molar absorptivity determinations have been accepted as fact by all future investigators and commercial distributors of TMB substrate products.

Chemical Basis for Acid Amplification of TMB Absorbance Signal

It is customary in most routine ELISA protocols using TMB as the chromogenic substrate to stop the TMB color development process by the addition of some formulation iteration of acidic stopping solution. The intent here is to create a low enough pH environment from addition of the acid stop solution to shut down the HRP redox activity. In some scenarios (typically involving larger numbers of ELISA plates being tested) it may not be feasible to read each plate immediately after a fixed predetermined color development time frame. It is therefore necessary to stop the catalytic HRP substrate oxidation process to allow completed ELISA plates to be read as soon as time permits.

It was noted early on that a partial oxidation of TMB lead to the production of a blue-green reaction product and complete oxidation of TMB lead to a yellow reaction product [15, 31]. Bally and Gribnau sought to provide an explanation for the significant A450 absorbance increases typically observed upon acid stoppage of HRP-mediated TMB oxidation reactions. In one experiment, using UV/VIS (200 nm – 700 nm) spectral scanning methods for monitoring the oxidation product accumulations, they evaluated an HRP-mediated TMB oxidation reaction that proceeded until the A650 absorbance values plateaued. At this point, the reaction pH of 5.5 was lowered to pH 1.0 with 2N H2SO4. Upon superimposing the pH 1.0 absorbance spectrum over the pH 5.5 absorbance spectrum, the oxidation state product composition within each pH environment was clearly revealed (Figure 6). Within the pH 5.5 reaction environment, one could clearly identify both an A450 diimine absorbance peak as well as an A650 Charge-Transfer-Complex (CTC) absorbance peak. Upon acid conversion of the pH 5.5 reaction environment to a pH 1.0 reaction environment, the A650 CTC absorbance peak was eliminated. In contrast, the A450 diimine absorbance peak was visibly enhanced by what the authors estimated to be 3.2-fold multiplier.

Figure 6. Acid induced shift in the chemical equilibrium governing the formation of TMB oxidation products. Acidification of TMB oxidation product reaction conditions going from slightly acidic (pH 5.5) dotted line to highly acidic (pH 1.0) solid line, lead to the complete dissociation of the blue-green colored Charge Transfer Complex (CTC) with a corresponding increase in the yellow colored diimine oxidation product. By shifting the TMB oxidation product equilibrium away from formation of the green colored CTC oxidation product and toward exclusive formation of the yellow colored diimine product, the A450 absorbance signal was observed to increase 3.2-fold over that associated with the pH 5.5 A450 absorbance signal. Figure image from Bally, R.W. and T.C. Gribnau. Some aspects of the chromogen 3,3′,5,5′-tetramethylbenzidine as hydrogen donor in a horseradish peroxidase assay. J Clin Chem Clin Biochem, 1989. 27(10): p 791-6.

In a different experimental setting, the authors sought to create an HRP-mediated TMB oxidation environment containing a large excess of HRP as well as equal molar concentrations of H2O2 and TMB. Their goal in this case was to achieve a rapid and complete conversion of TMB into its A450 absorbing diimine form. As was the case in the previous reaction scheme, this initial reaction was performed at pH 5.5. Once again UV/VIS (200 nm – 700 nm) spectral scanning methods were utilized to monitor the time point at which the A450 diimine absorbance peak was maximized. At this time point the pH 5.5 reaction conditions were reduced to pH 1.0 by the addition of 2N H2SO4 and another spectral scan was performed to visualize in this case a second single A450 diimine oxidation product. Upon superimposing the pH 1.0 absorbance spectrum over the pH 5.5 absorbance spectrum, the change in molar extinction coefficients between the two pH conditions was revealed (Figure 7). Since the same A450 diimine oxidation product is common to both the pH 5.5 and pH 1.0 reaction environments, any differences in the A450 diimine absorbance values is the direct result of the different pH conditions in which the two spectral scans were taken (Figure 7). As the net result, it was determined that the acidified (pH 1.0) conditions were directly responsible for increasing the molar absorptivity constants by a factor of 1.4.

Figure 7. Reduction of the reaction pH conditions from 5.5 to 1.0 of the completely oxidized diimine form lead to a 1.4X increase in the diimine oxidation product’s molar absorptivity constant. Figure image from Bally, R.W. and T.C. Gribnau. Some aspects of the chromogen 3,3′,5,5′-tetramethylbenzidine as hydrogen donor in a horseradish peroxidase assay. J Clin Chem Clin Biochem, 1989. 27(10): p 791-6.

Summation of Acid Induced TMB Absorbance Enhancement Study

Lowering the pH from 5.5 (dotted line) to 1.0 (solid line) leads to complete disassociation of the blue-green CTC oxidation product. This action corresponds with an immediate shift in the reaction equilibrium to instead favor formation of exclusively the completely oxidized yellow diimine product. This event is validated by a 3.2X concurrent increase in the A450 absorbance peak associated with formation of additional quantities of the diimine oxidation state form (Figure 6).

It was also found that lowering the pH of completely oxidized (yellow colored) diimine oxidation product from 5.5 (dotted line) to 1.0 (solid line) conferred a 1.4X increase in the yellow diimine product’s molar extinction coefficient (Figure 7).

The final yellow diimine form A450 absorbance increase represents the culmination of the acid induced 1.4X increase in the yellow diimine molar extinction coefficient (ε450) plus the acid environment shift in the TMB oxidation product equilibrium leading to the dissipation of the CTC oxidation product in favor of the formation of a corresponding amount of the yellow diimine product. The summation of these two events was used to justify the 3.2X increase in acid induced A450 absorbance as depicted in Figure 6.


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