HRP Redox Reaction Driven TMB Color Development, Part Three

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 three. See part one and part two if you missed them previously. And look for part four to be posted soon!


HRP plays a central catalytic role in many enzyme immunoassay (EIA) signal generation schemes. Possession of a catalytically efficient, redox capable, enzyme-prosthetic group structure is a mandatory prerequisite for its centralized substrate processing role. In this section, we list the key reaction steps that are driving the HRP oxidation and reduction processes. A logical starting point for describing the interactive chemical relationships between HRP and its oxidizing agent (H2O2) and its reducing agent (TMB), would be to split this discussion topic into two subsections. To this end, we broke this HRP redox chemistry discussion into two parts; 1) redox cycle initiation involving H2O2 as the oxidizing agent and 2) the redox cycle completion reaction involving TMB as the electron donor reducing agent.

Part Three: HRP Enzyme Redox Cycle Driven by H2O2 and TMB Substrates

The first subsection summarizes the key H2O2 initiated heme oxidation reactions. The second subsection summarizes the key chemical interactions between HRP and TMB during the HRP reduction process. A generic enzyme redox reaction pathway defining both the oxidation and reduction steps within our HRP redox cycle, can be represented by four generic reaction sequence equations:

  1. Resting state enzyme + H2O2 à Cpd-I + H2O
  2. Cpd-I + AH2 à Cpd-II + AH
  3. Cpd-II + AH2 à resting state + AH
  4. Overall reaction à 2AH2 + H2O2 à 2H2O + 2AH

The AH2 in this HRP redox cycle discussion can be represented by the TMB substrate.

The resting state enzyme above would be represented by the HRP Fe3+ or Fe (III) heme form.

A simplified summary figure of this HRP redox cycle is illustrated in Figure 4 below.

Figure 4. Hydrogen peroxide (H2O2) driven initiation of the horseradish peroxidase (HRP) redox cycle via the creation of high oxidation state Fe(IV=O) Cpd-I. This two electron oxidation equivalents bearing heme-Cpd-I acts as the driving oxidative force behind the conversion of colorless TMB starting material into its blue-green one electron oxidation state product. Along the redox cycle pathway, Cpd-I is subsequently reduced via a molecule of TMB forming a second lower oxidation potential oxidizing heme intermediate in the form of heme-Cpd-II. Heme-Cpd-II is subsequently reduced via another molecule of TMB returning the heme to its Fe (III) resting state form.

We used TMB as the reducing substrate example in this discussion because it is the electron donor/chromogenic component in the H2O2 + HRP + TMB redox reaction cycle. It should be stated however that because of HRPs notoriously low specificity for compatible electron-donor-substrate candidates, it became possible over the years for the development of many chemical-structure-variable chromogenic substrates [27]. That said, TMB is just one of multiple chromogenic HRP-compatible electron donor candidate as seen in Table 1 above.

Initiation of the HRP Oxidation Process to Begin its Redox Cycle

Activation of the HRP redox cycle is made possible via a two-electron oxidation of HRP event that is initiated by its oxidizing substrate H2O2. Occurring as a two electron oxidation of the heme prosthetic group, native resting-oxidation-state [Fe3+ or Fe (III)] heme is converted into a high oxidation state [Fe+4 or Fe (IV=O)] oxoferryl species that is typically referred to as Cpd-I [1, 6]. This two-electron oxidation event curtesy of the H2O2 oxidizing substrate leads to the formation of the heme intermediate oxidation state Cpd-I. It is the oxidizing power of Cpd-I that serves as the driving force behind the conversion of colorless TMB substrate into the blue-green colored oxidation product used for signal generation in ELISA type EIA formats.

Chemical Reaction Steps on the Oxidation Side of the HRP Redox Cycle

  1. The generalized reaction of H2O2 with the native resting oxidation state heme can be defined by a three-reaction sequence.
  2. Heme + H2O2 à Heme-H2O2 = hydroperoxo-ferric complex
  3. Heme-H2O2à Cpd-0
  4. Cpd-0 à Cpd-I + H2O along with conference of a 2-electron oxidation equivalence to the Cpd-I intermediate.
    Initial binding of the neutral-charge-form of H2O2 to the resting state heme [Fe3+ or Fe (III)] occurs within the distal heme pocket as defined by Arg 38, Phe 41, and His42. This binding event leads to the formation of the hydroperoxo-ferric complex (heme-H2O2) [2, 28].
  5. Heme-H2O2 converts to Cpd-0 as the result of a His42 facilitated deprotonation of the H2O2 while still bound to the Fe (III).
  6. Cpd-I is then formed by the heterolytic cleavage of the peroxide (O-O) bond after back protonation via His42 of the distal OH group on Cpd-0. Cleavage of the peroxide bond results in simultaneous reduction of the H2O2 to H2O with concurrent 2 electron oxidation of Cpd-0 to create the Cpd-I intermediate [28, 29]. This reaction requires the joint participation of His42 and Arg38. Arg38s role in the heterolytic cleavage of H2O2 is to lower the pKa of the His42 as well as align the H2O2 within the reactive site. The role of His42 is to cleave the H2O2 (O-O) bond resulting in a 2-electron oxidation event that converts Cpd-0 to Cpd-I. Two electron equivalents are simultaneously transferred to the H2O2 oxidizing agent subsequently reducing it to H2O. This reaction requires a neutral to slightly basic pH environment to put the His42 in its neutral form and Arg38 in its cationic form [28, 29].
  7. Cpd-I exists as a high oxidation state [Fe4+ or Fe (IV)=O] oxoferryl containing heme intermediate bearing a two oxidizing equivalent of oxidizing potential [1, 2, 6]. The 2 positive charges are distributed between the oxoferryl center and a porphyrin ring-based cation radical. The oxidative capacity of Cpd-I becomes the driving force perpetuating the subsequent HRP dependent TMB oxidization process.

Chemical reaction steps on the reduction side of the HRP redox cycle

Completion of the HRP redox cycle is made possible via a two-electron reduction of HRP at the expense of its TMB substrate. Occurring as a two consecutive event single electron oxidation reaction, colorless TMB substrate is converted into its blue-green colored single-electron oxidation state product. When H2O2 oxidized-HRP contacts the colorless unoxidized TMB starting material, the TMB can act as an electron donor substrate converting oxidized HRP back into its reduced Fe (III) oxidation state native form. In so doing, this two-electron acquisition event (reduction) at the expense of TMB, completes the HRP enzyme’s internal redox cycle. Once HRP is converted back into its native Fe (III) oxidation state, it is free to once again participate in another redox cycle with the aid of its H2O2 and TMB respective oxidizing and reducing substrates.

Heme Facilitated TMB Oxidation Leading to HRP Reduction to Native Fe (III) Form

  1. The HRP Cpd-I [Fe4+ or Fe (IV=O)] reduction process converting heme back to its native (Fe3+) ferric oxidation state form can be defined by two reaction sequences.
  2. Cpd-I + AH2 à Cpd-II + AH
  3. Cpd-II + AH2 à the ferric resting state enzyme + AH + H2O

    In these two reaction sequences, AH2 represents a reducing (electron donor) substrate and AH, the oxidized radical form of the reducing substrate. In this discussion, TMB substitutes in for AH2, acting as the electron donor substrate [29, 30].

  4. Cpd-I is a high oxidation state oxidizing agent existing as a Fe4+ or Fe (IV=O) oxoferryl species containing heme intermediate. As a result of its two-electron oxidation by H2O2, it bears a two oxidizing equivalent oxidizing potential. As stated above, the 2 positive charges are distributed between the oxoferryl center and a porphyrin ring based cation radical [1].
  5. Cpd-I can be converted to Cpd-II which also exists as an oxoferryl species, by a one electron transfer step from an electron donor substrate which in this case is TMB.
  6. Cpd-II can be converted back into its Fe3+or Fe (III) (native ferric state) following a second one electron transfer step from another TMB electron donor substrate. In so doing, another H2O is liberated in the process [6, 26].

Look for part four to be published soon!


  • 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 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.
Elisa reagents