Hemoglobin E (β26Glu→Lys) Exhibits Altered Nitrite Reactivity.

Abstract 2564

Poster Board II-541

Hemoglobin (Hb) EE individuals exhibit a mild, chronic anemia while HbE/β thalassemia individuals show a range of clinical manifestations, including high morbidity, and death, often resulting from cardiac dysfunction. Yet, HbE (β26 Glu→Lys), known to be more unstable than HbA in vitro, remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms. Considering the above and that pediatric HbE/β thalassemia patients exhibit endothelial dysfunction with oxidative stress, we consider the possibility that HbE is not as effective as HbA with respect to generating bioactive forms of nitric oxide (NO) that can limit endothelial dysfunction. Since reactions of Hb with nitrite are central to their capacity to generate bioactive NO, we compare HbA and HbE with respect to the reaction of nitrite with both the deoxy and oxy derivatives. Additionally, as a means of comparing redox potentials, we use L-cysteine as a biologically relevant reductant to compare HbA and HbE with respect to reduction of the aquomet derivative to the deoxy derivative. The redox potential has been shown to be a major determinant of nitrite reactivity for Hb. Reactions of purified oxy HbE and deoxy HbE at neutral pH in the presence of excess nitrite (∼4:1 nitrite: tetramer) compared to oxy and deoxy HbA are monitored by visible absorption spectroscopy. Since deoxy Hb is a nitrite reductase [deoxy Hb + nitrite → met Hb + NO], the reaction rate was assessed by the loss of the characteristic deoxy Hb absorption spectrum. The initial rate of reaction was significantly slower for deoxy HbE than deoxy HbA. Oxy Hb reacts with nitrite to form met Hb [simplified as, oxy Hb + nitrite → met (Fe⁺³)Hb + nitrate] and is followed by formation of the characteristic met Hb absorption spectrum. Under these in vitro conditions, the oxy HbE reaction with nitrite is complete at ∼120 secs and faster than oxy HbA (∼180 secs), while deoxy HbE shows a reaction with nitrite that is slower to reach completion (deoxy HbE∼1400 secs) compared to deoxy HbA (∼1000 secs). Consistent with known Hb-nitrite reactivity, the reaction with nitrite for both oxy HbE and oxy HbA is faster than their respective deoxy Hb reaction with nitrite. Met HbE is more rapidly reduced by L-cysteine to form deoxy HbE compared to HbA. The nitrite reaction rates and the redox ordering are consistent with HbE populations having a greater propensity to remain in the T state compared to comparable samples of HbA. In brief, under these conditions, all results are consistent with HbE being shifted more towards the T-state in the R↔T equilibrium compared to HbA. Structural evidence in support of an allosteric alteration in HbE is seen in the high resolution deoxy and oxy HbE crystallographic structures compared to HbA structures grown under the same crystal forming conditions [Protein Data Bank entries 1YVT, 1YVQ, 3DUT; Hirsch et al. (2008) Blood 112(11):540a]. Our findings will be discussed from at least three perspectives: an altered nitrite reactivity for HbE and its physiological relevance; support for the findings of Kavanaugh et al. (2005, Biochemistry) and others that the α1β1 interface undergoes significant changes in the Hb allosteric transition; and the allosteric transition involves a Hb T-state continuum.