Comparison of nitric oxide-induced oxidation of recombinant oxyhemoglobin subunits using a competition experiment

A low reaction rate with nitric oxide (NO) is one of the important characteristics of hemoglobin (Hb)-based oxygen carriers. The reaction rate between oxyHb and NO is usually measured by stopped-flow spectrophotometry. However, the reported rates vary due to the difficulty of accurately determining the NO concentration and the limit of the instrument dead time. To circumvent these problems, we developed an experiment using oxymyoglobin (oxyMb) to compete with oxyHb for NO that is released from an NO donor. Determination of the rate constants in the competition experiment no longer depends on accurate measurement of time or NO concentration, since this approach instead measures the ratio of rate constants for the reaction of oxyHb and oxyMb with NO. For recombinant mutant Hb α(L29F)β the rates for α(L29F) and β are approximately 15- and 1.6-fold smaller than for wild-type Hb. In conclusion, the competition experiment provides an alternative method for determination of relative reaction rates of recombinant Hb subunits with NO.