HRP Redox Reaction Driven TMB Color Development, Part Four

Chemical Basis for TMB Oxidation to Colored Product

In 1982, Josephy et.al 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.

References

• Veitch, N.C., Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 2004. 65(3): p. 249-59.
• Azevedo, A.M., et al., Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol Annu Rev, 2003. 9: p. 199-247.
• Paul, K.G.a.S., T., Four Isoperoxidases from Horse Radish Root. Acta Chemica Scandinavica, 1970. 24(10): p. 3607-3617.
• Shannon, L.M., E. Kay, and J.Y. Lew, Peroxidase isozymes from horseradish roots. I. Isolation and physical properties. J Biol Chem, 1966. 241(9): p. 2166-72.
• Wilson, M.B., and Nakane, P.K., Recent Development in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. Immunofluorescence and Related Staining Techniques, ed. W. Knapp, Holubar, K., and Wick, G. 1978: Elsevier/North-Holland Biomedical Press.
• Ngo, T.T., Peroxidase in chemical and biochemical analysis. Analytical Letters, 2010. 43: p. 1572-1587.
• Welinder, K.G., Covalent structure of the glycoprotein horseradish peroxidase (EC 1.11.1.7). FEBS Lett, 1976. 72(1): p. 19-23.
• Welinder, K.G., Amino acid sequence studies of horseradish peroxidase. Amino and carboxyl termini, cyanogen bromide and tryptic fragments, the complete sequence, and some structural characteristics of horseradish peroxidase C. Eur J Biochem, 1979. 96(3): p. 483-502.
• Yang, B.Y., J.S. Gray, and R. Montgomery, The glycans of horseradish peroxidase. Carbohydr Res, 1996. 287(2): p. 203-12.
• lebedeva, O.V., and Ugarova, N.N., Mechanism of peroxidase-catalyzed oxidation. Sunstrate-substrate activation in horseradish peroxidase-catalyzed reactions. Russian Chemical Bulletin, 1996. 45(1): p. 25-32.
• Gajhede, M., et al., Crystal structure of horseradish peroxidase C at 2.15 A resolution. Nat Struct Biol, 1997. 4(12): p. 1032-8.
• Hosoda, H., et al., A comparison of chromogenic substrates for horseradish peroxidase as a label in steroid enzyme immunoassay. Chem Pharm Bull (Tokyo), 1986. 34(10): p. 4177-82.
• Porstmann, B., T. Porstmann, and E. Nugel, Comparison of chromogens for the determination of horseradish peroxidase as a marker in enzyme immunoassay. J Clin Chem Clin Biochem, 1981. 19(7): p. 435-9.
• Josephy, P.D., Oxidative activation of benzidine and its derivatives by peroxidases. Environ Health Perspect, 1985. 64: p. 171-8.
• Josephy, P.D., T. Eling, and R.P. Mason, The horseradish peroxidase-catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine. Free radical and charge-transfer complex intermediates. J Biol Chem, 1982. 257(7): p. 3669-75.
• Garner, R.C., Testing of some benzidine analogues for microsomal activation to bacterial mutagens. Cancer Lett, 1975. 1(1): p. 39-42.
• Holland, V.R., Saunders, B.C., Rose, F.L., and Walpole, A.L., A safer substitute for benzidine int he detection of blood. Tetrahedron, 1974. 30: p. 3299-3302.
• Bos, E.S., et al., 3,3′,5,5′ – Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J Immunoassay, 1981. 2(3-4): p. 187-204.
• Frey, A., et al., A stable and highly sensitive 3,3′,5,5′-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays. J Immunol Methods, 2000. 233(1-2): p. 47-56.
• Gerber, B., Block, E., Bahar, I., Cantarow, W., Coseo, M., Jones, W., Kovac, P., and Bruins, J., Enzyme immunoassay with two-parts solution of tetramethylbenzidine as chromogen. 1985, BTC Diagnostics Limited Partnership, Cambridge, Mass.: USA.
• Cattaneo, M.V. and J.H. Luong, A stable water-soluble tetramethylbenzidine-2-hydroxypropyl-beta-cyclodextrin inclusion complex and its applications in enzyme assays. Anal Biochem, 1994. 223(2): p. 313-20.
• Lo Conte, M.a.C., Kate S., The chemistry of thiol oxidation and detection. Oxidative Stress and Redox Regulation, ed. U.a.R. Jakob, Dana. 2013: Springer.
• Chen, S.X. and P. Schopfer, Hydroxyl-radical production in physiological reactions. A novel function of peroxidase. Eur J Biochem, 1999. 260(3): p. 726-35.
• Bach, R.D., Ayala, Philippe Y., and Schlegel, H. B., A Reassessment of the Bond Dissociation Energies of Peroxides. An ab Initio Study. J. Am. Chem. Soc., 1996. 118(50): p. 12758-12765.
• Finnegan, M., et al., Mode of action of hydrogen peroxide and other oxidizing agents: differences between liquid and gas forms. J Antimicrob Chemother, 2010. 65(10): p. 2108-15.
• Yoshida, T., et al., The catalytic mechanism of dye-decolorizing peroxidase DyP may require the swinging movement of an aspartic acid residue. FEBS J, 2011. 278(13): p. 2387-94.
• Conyers, S.M. and D.A. Kidwell, Chromogenic substrates for horseradish peroxidase. Anal Biochem, 1991. 192(1): p. 207-11.
• Tatoli, S., et al., The role of arginine 38 in horseradish peroxidase enzyme revisited: a computational investigation. Biophys Chem, 2009. 141(1): p. 87-93.
• Rodriguez-Lopez, J.N., et al., Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle. J Am Chem Soc, 2001. 123(48): p. 11838-47.
• Krainer, F.W. and A. Glieder, An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Appl Microbiol Biotechnol, 2015. 99(4): p. 1611-25.
• 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.