Thiol species interfere with H 2 O 2 detection, but adjustments can correct for the problem (A) Dithiothreitol suppresses half the fluorescence otherwise produced in an HRP/AR/H 2 O 2 reaction containing 0.5 μM H 2 O 2 . (B) Even at high levels of dithiothreitol (here, 100 μM), standard curves permit H 2 O 2 quantification. (C) Thiol species only slowly scavenge H 2 O 2 . Dithiothreitol (50 μM) and 50 μM H 2 O 2 were coincubated in RT KPi/DTPA buffer at the indicated pH values, and H 2 O 2 was periodically quantified. Data indicate a rate constant for H 2 O 2 reduction by the DTT thiolate anion of 15 M −1 s −1 , which equates to an H 2 O 2 half-life of 3 hours in 100 μM DTT.
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