Scientific Publications by FDA Staff

Front Physiol 2015 Feb 20;6:39

Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.

Ratanasopa K, Strader MB, Alayash AI, Bulow L

In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe(+2)) human hemoglobin (Hb) (alpha2beta2) undergoes a rapid conversion to a higher oxidation ferryl state (Fe(+4)) which rapidly autoreduces back to the ferric form (Fe(+3)) as H2O2 is consumed in the reaction. In the presence of additional H2O2 the ferric state can form both ferryl Hb and an associated protein radical in a pseudoperoxidative cycle that results in the loss of radicals and heme degradation. We examined whether adult HbA (beta2alpha2) exhibits a different pseudoenzymatic activity than fetal Hb (gamma2alpha2) due to the switch of gamma to beta subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to gamma/beta and alpha, respectively. Although there was a 1.5 fold increase in the fast reacting gamma /beta subunits the slower reacting phases (attributed to alpha subunits of both proteins) were essentially the same. However, the rate constant for the auto-reduction of ferryl back to ferric for both proteins was found to be 76% higher for HbF than HbA and in the presence of the mild reducing agent, ascorbate there was a 3-fold higher reduction rate in ferryl HbF as opposed to ferryl HbA. Using quantitative mass spectrometry in the presence of H2O2 we found oxidized gamma/beta Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated beta Tyr35 containing peptides were found in HbA samples treated with nitrite. The extraordinary stability of HbF reported here may explain the evolutionary advantage this protein may confer onto co-inherited hemoglobinopathies and can also be utilized in the engineering of oxidatively stable Hb-based oxygen carriers.