Horseradish Peroxidase (HRP) is a commonly used enzyme in many applications. HRP is used in conjunction with various colorimetric or fluorometric probes to detect hydrogen peroxide. In these applications, H2O2 is generated either as a primary or secondary signal, which in turn is used by HRP to oxidize a probe that can be detected colorimetrically. This assay is widely used in immunological assays, molecular-based medical diagnostic testing, and biotechnology applications. However, there are numerous isoforms of HRP and numerous probes to select from. To guide their work, a systematic investigation of these isoforms and probes would be useful to those using and designing HRP assays. The biochemical activity of HRP isoforms I, II, ViA, X, and XII was each measured against the most common colorimetric probes: Amplex Red, ABTS, TMB, OPD, and DAB. Kinetic parameters of each HRP isoform for both H2O2 and probe were determined. Further, each probe was characterized with respect to temperature optimum, pH optimum, metal ion reactivity, oxygen sensitivity, and reducing agent inhibition. All HRP isoforms exhibit the same biochemical properties with each other for a given probe. However, different probes do have different pH optimums, metal tolerance, reducing agent tolerance, and different sensitivity and dynamic range capabilities. The Kmapp values for colorimetric probes varies generally within the sub-mM range while prbe turnover numbers vary up to an order of magnitude. Amplex Red showed the greatest sensitivity with detection limits for hydrogen peroxide in the low µM concentration range. Additionally, certain divalent metal cations have an inhibitory effect with some probes. Finally, kinetic isotope effects observed with HRP provide some mechanism insight into the oxidation of the various colorimetric probes. This information is of value for those who construct peroxide detection assays for their chosen application. While there is a wide range in price and availability for the various HRP isoforms, we recommend researchers choose based on factors other than catalytic performance as we see no significance difference between isoforms. However, the choice in probe may depend upon the application as pH, temperature, metal ion concentration, and sensitivity needs may dictate usage.

Kinetic equivalence of HRP isoforms for colorimetric probes and mechanistic insight to guide enzyme usage in applications

ACS Spring Meeting 2021

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