HRP Redox Reaction Driven TMB Color Development, Part Five

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.

  1. Heme + H2O2 à Heme-H2O2 = hydroperoxo-ferric complex
  2. Heme-H2O2 à Cpd-0
  3. 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.

  1. Cpd-I + AH2 à Cpd-II + AH
  2. Cpd-II + AH2 à the ferric resting state enzyme + AH + H2O
  3. 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.

  1. TMB radical cation
  2. 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.

  1. Colorless TMB (diamine form) starting material with molar absorbance max of 285 nm
  2. Blue-green colored CTC form with molar absorbance max of 652 nm
  3. 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:

  1. 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.
  2. 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.
  3. 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.


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