In this series, we have been breaking down one of our most popular educational pieces, "HRP Redox Reaction Driven TMB Color Development."
This post will conclude our series. Please see part one, two, three, or four if you missed them previously.
ImmunoChemistry Technologies gratefully acknowledges the significant contributions made by one of its founders, Brian W. Lee, Ph.D in the creation of this educational piece.
A Generalized Summary of Key Subject Matter
This review summary of the interactive chemical relationships existing between HRP and its H2O2 and TMB substrates, represents our attempt to provide a little deeper insight into the chemical dynamics occurring within routine HRP mediated TMB color development processes. We conclude this technical review effort by summarizing key subject matter points from each discussion topic section. The intent here is to distill down the technical text content into a minimal number of key fact statements from each of the discussion topic sections.
Desirable Performance Attributes of HRP
- Excellent enzyme stability properties
- High catalytic substrate turnover rates
- Ease of conjugation to other protein/hapten-like molecules. Contains a low number (4) of chemically accessible and reactive primary amines allowing for controllable conjugation reactions
- Relatively low molecular weight enzyme presenting minimal steric hindrance problems
- High (RZ-value) purity HRP enzyme less expensive on per mg basis than other EIA associated enzymes e.g. Alkaline Phosphatase.
- HRP can be used for both colorimetric and chemiluminescent signal generation assay formats.
HRP enzyme structural features
- Commercial HRP products consist of the isozyme-C (HRP-C) isotype. This prevalence is likely due to HRP-C being the most abundant isotype form (accounting for roughly 50% of the > 40 identified total peroxidase isozymes).
- HRP is a globular glycoprotein having an α-helical folding structure with a small section of β-sheet. 18%-22% of its molecular weight is contributed by its carbohydrate structure.
- These glycan structures are composed of N-acetylglucosamine, mannose, xylose, and fucose carbohydrates.
- HRP-C consists of two domains. Between these two domains is located a hydrophobic pocket in which lies the heme prosthetic group responsible for the enzyme’s redox activities.
- Glycan (carbohydrate) structures contribute to some of the high stability and aqueous solubility properties of HRP. These features include increased heat stability, increased resistance to free radical formation resulting from the enzymes redox duties, enhanced aqueous solubility properties, and increased structural integrity properties that lend themselves to a more robust enzyme stability in hostile environments.
Chromogenic Substrates for HRP
HRP happens to fall into the category of enzymes having lower specificity requirements for compatible electron donor substrates. This lack of substrate specificity characteristic lead to the synthesis and use of many different electron donor type dye substrates over the years. The goal was to create more sensitive substrates with enough colored oxidized product stability to enable their use in various diagnostics formats.
- Early HRP substrates displayed varying degrees of signal detection sensitivity.
- Many of these early substrates were chemical derivatives of the benzidine molecule which made them likely to have mutagenic and carcinogenic properties.
- Tetramethylbenzidine (TMB) was synthesized to be a sensitive and non-carcinogenic replacement for these older HRP substrate formulations.
- Contemporary TMB 1-Component HRP substrate products were created to provide a safe, sensitive, and oxidation product stable chromogenic signal generation mechanism for use in a range of EIA type assay formats.
Chemical Basis for Oxidizing Properties of Hydrogen Peroxide
- Hydrogen peroxide (H2O2) serves as the oxidizing HRP substrate that initiates the HRP redox cycle.
- Existing as a covalently linked two hydroxyl chemical structure where the oxygen molecules are linked by a weak (low bond energy) single O-O peroxide or peroxo covalent bond, the presence of this weak O-O peroxide linkage enables it to act as a potent two-electron electrophile oxidizing agent.
Generic HRP enzyme redox cycle overview
Our 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:
- Resting state enzyme + H2O2 à Cpd-I + H2O
- Cpd-I + AH2 à Cpd-II + AH
- Cpd-II + AH2 à resting state + AH
- Overall reaction can be condensed down to: 2AH2 + H2O2 à 2H2O + 2AH
A molecule of TMB may be substituted for the AH2 reactant.
Resting state enzyme contains heme in the Fe3+ oxidation state
Chemical Oxidation Steps Leading to Cpd-I Formation
H2O2 oxidation of heme prosthetic group can be defined by a three-reaction sequence.
- Heme + H2O2 à Heme-H2O2 = hydroperoxo-ferric complex
- Heme-H2O2 à Cpd-0
- Cpd-0 à Cpd-I + H2O along with conference of a 2-electron oxidation equivalence to the Cpd-I intermediate
Chemical Reduction Steps Returning Heme to Its Native Fe (III) Form
Conversion of high oxidation state (Fe(IV)=O) Cpd-I back to its native resting state Fe(III) form can be defined by a two reaction sequence.
- Cpd-I + AH2 à Cpd-II + AH
- Cpd-II + AH2 à the ferric resting state enzyme + AH + H2O
- Chemical basis for TMB oxidation to colored product
Unoxidized, colorless, native TMB starting material representing the diamine form of the dye (absorbance max at 285 nm) is oxidized by HRP associated Cpd-I intermediate oxidation state heme form. This oxidation event results in the formation of two new one electron TMB oxidation state forms.
- TMB radical cation
- Charge transfer complex (CTC) consisting of a colorless diamine form electron donor and a yellow colored diimine form electron acceptor
The diamine/diimine CTC is in rapid chemical equilibrium with a cation radical. Further oxidation of the one electron loss oxidation state CTC form into the two electron-loss oxidation state leads to the formation of the yellow colored diimine form. Within this dynamic reaction scheme there exists three different reaction components each having a different molar absorbance max.
- Colorless TMB (diamine form) starting material with molar absorbance max of 285 nm
- Blue-green colored CTC form with molar absorbance max of 652 nm
- Yellow colored diimine form with molar absorbance max at 450 nm
Exposing TMB to a large excess of H2O2 over TMB leads to the complete conversion of all the TMB (diamine form) into the two-electron-loss oxidation state yellow colored diimine form.
Experimental Basis for TMB Signal Enhancement Following Acid Stop of Color Reaction
Acid stoppage protocols within routine TMB color development processes reveal an enhancement of the absorbance signal going from the blue-green colored TMB oxidation product to the yellow colored TMB oxidation product. This occurs as the result of the following acid environment driven chemical processes:
- Complete oxidation, via acid conversion, of the TMB substrate leads to the complete elimination of the blue green A652 CTC peak with a concurrent increase in the yellow A450 diimine peak. The A450 yellow diimine absorbance signal was observed by one group to increase 3.2X over that present before the complete acid conversion step.
- Reduction of the pH environment from 5.5 to 1.0 of a completely oxidized (all diimine form) TMB solution lead to a 1.4X increase in the diimine oxidation product molar absorptivity constant.
- The 3.2 fold increase in the A450 signal described above, represents the culmination of the 1.4X increase in the yellow diimine molar extinction coefficient (ε450) plus the acid environment induced shift in TMB oxidation product equilibrium away from the CTC oxidation product formation to the formation of the yellow A450 diimine oxidation product.
We hope that this technical review was helpful in furthering the understanding of the functional mechanism (and history behind) one of the most widely accepted colorimetric EIA signal generation approaches in use today.
- 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 220.127.116.11). 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