Scientific Publications by FDA Staff

Redox Biol 2016 Aug;8:363-74

Oxidative instability of hemoglobin E (beta26 Glu-->Lys) is increased in the presence of free alpha subunits and reversed by alpha-hemoglobin stabilizing protein (AHSP): relevance to HbE/beta-thalassemia.

Strader MB, Kassa T, Meng F, Wood FB, Hirsch RE, Friedman JM, Alayash AI

When adding peroxide (H2O2), ß subunits of hemoglobin (Hb) bear the burden of oxidative changes due in part to the direct oxidation of its Cys93. The presence of unpaired a subunits within red cells and/or co-inheritance of another ß subunit mutant, HbE (ß26 Glu¿Lys) have been implicated in the pathogenesis and severity of ß thalassemia. We have found that although both HbA and HbE autoxidize at initially comparable rates, HbE loses heme at a rate almost 2 fold higher than HbA due to unfolding of the protein. Using mass spectrometry and the spin trap, DMPO, we were able to quantify irreversible oxidization of ßCys93 to reflect oxidative instability of ß subunits. In the presence of free a subunits and H2O2, both HbA and HbE showed ßCys93 oxidation which increased with higher H2O2 concentrations. In the presence of Alpha-hemoglobin stabilizing protein (AHSP), which stabilizes the a-subunit in a redox inactive hexacoordinate conformation (thus unable to undergo the redox ferric/ferryl transition), Cys93 oxidation was substantially reduced in both proteins. These experiments establish two important features that may have relevance to the mechanistic understanding of these two inherited hemoglobinopathies, i.e. HbE/ß thalassemia: First, a persistent ferryl/ferryl radical in HbE is more damaging to its own ß subunit (i.e., ßCys93) than HbA. Secondly, in the presence of excess free a-subunit and under the same oxidative conditions, these events are substantially increased for HbE compared to HbA, and may therefore create an oxidative milieu affecting the already unstable HbE.