I'm testing the activity of horseradish peroxidase (HRP) under different conditions by measuring the change in concentration of hydrogen peroxide substrate after a given amount of time. To stop the enzyme activity, I add H2SO4 to drop the pH and denature the enzyme.

This was convenient because the following titration of peroxide using potassium permanganate calls for acidic conditions (according to many protocols I've seen). Acidic conditions are required to completely reduce Mn7+ to colorless Mn2+. Under neutral and basic conditions, Mn7+ will only be reduced to Mn4+ in the form of MnO2, a brown, water-insoluble precipitate.

However, my concern is that adding sulfuric acid to hydrogen peroxide will create a piranha solution, a strong oxidizing agent typically used to remove organic contaminants. With the HRP also in solution, I'm afraid the potential piranha solution will oxidize the HRP leading to some powerful exothermic reaction/explosion.

For reference, I'm performing all this with 40 µL HRP (1.0 mg/mL in phosphate buffer) combined with 500 µL of hydrogen peroxide. How much and what concentration of sulfuric acid can/should (if at all) should I add? Is there a better way to go about this whole thing?

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    $\begingroup$ Man, you're using highly dilute solutions - I can tell even though you ramble about Mn, but don't mention the concentrations. "Piranha solution" is 30 % H2O2 + 98 % H2SO4 and that makes all kinds of differences. $\endgroup$ – Mithoron Jul 16 at 16:34

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