Characterization of Hemoglobin Bassett (α94Asp→Ala), a variant with very low oxygen affinity

Abstract

Hemoglobin (Hb) Bassett, an abnormal Hb variant with a markedly reduced oxygen affinity, was discovered in a Caucasian (Anglo-Saxon) male child who experienced episodes of cyanosis. Cation-exchange and reversed-phase (RP) high-performance liquid chromatography (HPLC) showed that the patient has an abnormal Hb, with a mutation in the α-globin. Tryptic peptide digest of the abnormal α-globin with subsequent HPLC analysis revealed abnormal elution of the α-T11 peptide. Further studies with Edman sequencing and electrospray mass spectrometry of tryptic peptide α-T11, as well as structural analysis by X-ray crystallography revealed an Asp→Ala substitution at the α94 (G1) position, a match for Hb Bassett. Detailed functional studies showed that this Hb variant had a markedly reduced oxygen affinity (P50 at pH 7.0 = 22 mmHg; Hb A P50 = 10.5 mmHg), reduced Bohr effect (-0.26 compared to -0.54 in Hb A), and low subunit cooperativity (n = 1.4, compared to 2.6 in Hb A). X-ray crystallography results explain the probable effects of the structural modification on the oxygen-binding properties of this Hb variant. © 2004 Wiley-Liss, Inc.