Dissection of the radical reactions linked to fetal hemoglobin reveals enhanced pseudoperoxidase activity.
In the presence of excess hydrogen peroxide (H2O2), ferrous (Fe(+2)) human hemoglobin (Hb) (α2β2) 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 (β2α2) exhibits a different pseudoenzymatic activity than fetal Hb (γ2α2) due to the switch of γ to β subunits. Rapid mixing of the ferric forms of both proteins with excess H2O2 resulted in biphasic kinetic time courses that can be assigned to γ/β and α, respectively. Although there was a 1.5 fold increase in the fast reacting γ /β subunits the slower reacting phases (attributed to α 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 γ/β Cys93, to be more abundantly present in HbA than HbF, whereas higher levels of nitrated β 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.
Pure and Applied Biochemistry, Department of Chemistry, Lund University Lund, Sweden.
Khuanpiroon
K
Ratanasopa
Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration Silver Spring, MD, USA.
Michael Brad
MB
Strader
Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration Silver Spring, MD, USA.
Abdu I
AI
Alayash
Pure and Applied Biochemistry, Department of Chemistry, Lund University Lund, Sweden.
Leif
L
Bulow
fetal blood; hemoglobins; kinetics; peroxidases; quantitative mass spectrometry; spectrophotometry