The phenolphthalein used in this test has been modified from its conventional form, in that it has been reduced by two electrons and is pre-dissolved in alkaline solution. This is typically achieved by boiling an alkaline solution of phenolphthalein with powdered zinc, which reduces the phenolphthalein into phenolphthalin. Upon reduction, the very intense pink color of the cationic form of phenolphthalein fades to a faint yellow color. It is this form of phenolphthalein that is present in Kastle–Meyer test kits. In order to generate the intense pink color indicative of a positive test, the reduced phenolphthalein must be oxidized back to its normal, colored form.
In the relevant reaction, hydrogen peroxide reacts with the hemoglobin in the blood. Phenolphthalein does not directly participate in this process; instead, it acts as an external source of electrons. In its reaction with hydrogen peroxide, the heme center of hemoglobin behaves as a peroxidase, reducing the peroxide to water. This activity depletes hemoglobin of electrons that are, in turn, re-supplied by the phenolphthalein. Donating electrons to hemoglobin converts the phenolphthalin back into the intensely colored phenolphthalein. As long as the enzyme survives, the reaction of heme with peroxide is catalytic, making this test very sensitive to small quantities of blood present on the test swab. The hemoglobin-catalyzed reduction of peroxide that occurs is shown in the reaction below. The two electrons are supplied by phenolphthalein:
: HOOH + 2 e + 2 H → 2 HO
The consumption of protons during the course of the reaction has the effect of raising the pH of the solution, but the amount of base produced is negligible compared to the amount of base already present in the reagent mixture.