Investigation of the Molecular Basis of High Oxygen Affinity Hemoglobin Brigham (α₂^Aβ₂^{100 Pro→Leu}) In a Subject with Familial Erythrocytosis

Abstract 4806

A 40 year old female was seen at the NIH Clinical Center for polycythemia (Hb 16.3 g/dL) and recurrent pulmonary embolism. It was determined that she was a member of the original kindred with familial erythrocytosis due to the high oxygen affinity Hb Brigham first reported by Lokich et al (JCI, 1973;52:2060-7) where leucine replaces proline at amino acid 100 in the Hb beta globin chain. We sought to isolate and characterize the abnormal Hb in order to elucidate the mechanism by which the amino acid substitution causes a high affinity for oxygen. Fresh RBCs exhibited high oxygen affinity (P₅₀ = 23.6 mmHg; a Hill coefficient of n=2.02) compared to that of fresh normal RBCs (P₅₀ = 31.1 mmHg; n=2.1) in Hemox buffer, pH 7.4 at 37°C using the TCS automated Hemox analyzer. After 2, 3-diphosphoglycerate (DPG) depletion (24 hours incubation of RBCs at room temp) the RBCs exhibited a higher oxygen affinity, that decreased when inositol hexaphosphate (a 2, 3-DPG analogue) was electroporated into erythrocytes, indicating a normal allosteric response at the 2,3-DPG binding site. The Bohr effect (pH dependence of oxygen affinity) of stripped Hb Brigham was reduced by approximately 30% when compared to that of HbA₀. Hb Brigham was separated from Hb A in this subject using strong cationic HPLC and the molecular composition of each protein was verified by tryptic peptide mapping and mass spectrometry of purified peptides. The purified fractions; F1 (HbA₀) and FII (Hb Brigham) which represent approximately 60% and 40% of total Hb, respectively, were characterized using rapid mixing stopped-flow kinetics and compared to RBCs Brigham. The time course of the oxygen dissociation reaction from both (oxy) fractions in the presence of sodium dithionite were monophasic with apparent rate constants derived for FI (k_off = 38 s^-1) which is comparable to that of whole blood (k_off = 37.9 s^-1) and that of normal control HbAo (k_off = 39.6 s^-1). However, the rate constant for oxygen dissociation from FII was slightly reduced (k_off = 33.9 s^-1). To further characterize oxygen dissociation kinetics, carbon monoxide (CO) combination kinetics with the (deoxy) forms of each hemoglobin were also carried out. The kinetics of CO binding to deoxyHb were comparable between both purified fractions and whole blood (k_on =0.22-0.23 mM^-1 s^-1). Unlike FI or control Hb A₀, FII showed no change in its CO binding kinetics in the presence of increasing concentrations of IHP. We propose that the IHP-dependent difference in CO binding resulted from destabilization of the deoxy (tense) structure due to the β2 100 Pro→Leu substitution. Analysis of purified Hb Brigham, which was not previously possible, provides valuable insights into the contribution of this mutant to the overall oxygen affinity in this patient and may be useful in the design and evaluation of hemoglobin-based blood substitutes.