HRP Redox Reaction Driven TMB Color Development, Part Two

In this series, we will break 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 two. See part one if you missed it previously and look for follow up posts in the near future to complete the series.


To further expand on this TMB substrate optimization discussion, efforts were made to modify TMB substrate formulations to maximize oxidized TMB (colored product) detection sensitivity. A major emphasis was also put toward being able to provide an absorbance-stable acid stopped, yellow-colored TMB-oxidation-product. This process would involve multiple TMB formulation adjustments. Providing a TMB substrate product with an extended time stable A450 absorbance property is an absolute imperative when running staggered multiple ELISA plate assays or when using an automated ELISA system. These TMB performance optimization efforts have also led to our product’s excellent long-term storage stability (> 2 years at 2 - 8°C). ICT offers several TMB 1-Step type product formulations (H2O2 and TMB provided within a common solution) that help you target your optimal ELISA detection sensitivity range. These include the TMB Supersensitive 1-Component HRP substrate formulation (SUBS, Catalog # 6275), the TMB 1-Component HRP substrate formulation (SUBT Catalog # 6276), and the TMB Slow-Kinetic 1-Component HRP substrate formulation (SUBK Catalog # 6277). All ICT brand HRP and Alkaline Phosphatase, ELISA and insoluble precipitate forming membrane substrate products, provide the end-user with a long-term storage stable, sensitive, safe, and user-friendly research tool.


Part 2: Hydrogen peroxide (H2O2) as oxidant for HRP redox cycle initiation

Initiation of an HRP enzyme-mediated color development process for signal generation is dependent upon the presence of H2O2 as the oxidizing agent to initiate the redox process (Figure 2). H2O2 consists of two OH molecules with the two oxygen molecules covalently bonded via a single O-O bond (Figures 2 and 3) [22]. Intracellular reduction of H2O2 via Fe2+ à Fe3+ (Fenton reaction) leads to the production of toxic reactive OH- + .OH radicals that are capable of reacting with other key intracellular components such as DNA, proteins or membrane lipids [23]. Its ability to perform as an oxidizing agent is attributed to the relatively low bond energy of the peroxide (peroxo) O-O covalent linkage. The energy requirement for disruption of this peroxide (O-O) bond was calculated to be ~ 45 kcal/mol compared to bond disruption energies for C-O and N-O linkages at 84 kcal/mol and 53 kcal/mol respectively [24]. This low O-O bond energy factor enables H2O2 to act as a potent two-electron electrophilic oxidizing agent as evidenced by its high (1.77 V) redox potential [25]. H2O2 migrates into a large cavity on the heme-distal side region that forms a small 3A by 3A cylindrical pocket where it binds to and reacts with the heme redox site [26]. A more detailed description of the H2O2 mediated oxidation of the HRP will be presented in the next section.


Figure 2. Ball and stick model of the hydrogen peroxide molecule responsible for initiating the HRP redox cycle with TMB.


Figure 3. Chemical structure of hydrogen peroxide (H2O2) highlighting the critical oxidation potentiating peroxide (peroxo) oxygen-oxygen linkage. The low bond energy of the O-O linkage (depicted in red) allows H2O2 to act as an efficient two-electron oxidizing electrophile.

Look for Part 3 of this series to be posted soon.


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