Effect of Hydrogen Peroxide on Methemoglobin

Abstract

Barnard and Gowen 1 reported that the addition of hydrogen peroxide to methemoglobin solutions resulted in the formation of oxyhemoglobin. This change was considered to be in harmony with that deduced from the relative redox potentials of the hydrogen peroxide-oxygen system and the methemoglobin-oxyhemoglobin system.

Keilin and Hartree 2 subsequently showed that the addition of hydrogen peroxide to methemoglobin solutions led to the formation of a substance which they characterize as a methemoglobin peroxide compound.

Because of the discrepancy between the results of Barnard and Gowen and those of Keilin and Hartree the question was reinvesti-gated. It was found that the observations of the latter investigators could be confirmed when the methemoglobin used was prepared by the oxidation of hemoglobin with ferricyanide. However when methemoglobin is prepared by the oxidation of hemoglobin with quinhydrone the addition to it of hydrogen peroxide results in the formation of oxyhemoglobin. That this latter compound was actually oxyhemoglobin and not a substance with similar absorption bands was demonstrated by treatment with carbon monoxide when CO-hemoglobin resulted.

In a typical experiment, a sample of a 0.5 millimolar oxyhemoglobin solution (molarity in this case refers to iron molarity) is divided into two portions. Each of these portions is placed in the chamber of a double spectrophotometer vessel at the collimator of a Hillger spectrophotometer. The telescope slit of the spectrophotometer is opened to its maximum extent. The photometer is adjusted so that these two solutions cast spectra of corresponding intensities, one above the other—the absorption bands are of course identical since they emanate from identical solutions.

To the contents of the upper chamber, a small quantity of powdered quinhydrone is added, sufficient to change the upper spectrum to that of methemoglobin (about 3 milligrams suffices for the usual contents of one chamber).