Transfer of human α- to β-hemoglobin via its chaperone protein: Evidence for a new state

Abstract

The α-hemoglobin-stabilizing protein (AHSP), a small protein of 102 amino acids, is synthesized in red blood cell precursors. It binds specifically to α-hemoglobin (α-Hb) subunits acting as a chaperone protein, preventing the formation of α-hemoglobin-cytotoxic precipitates. We have engineered recombinant AHSP in a pGEX vector to study the functional consequence of interaction between AHSP and α-Hb. By in vitro binding assays, we have isolated the complexes glutathione S-transferase-AHSP-α-Hb and AHSP-α-Hb. The latter assembles as a heterodimer based on size-exclusion chromatography. These complexes exhibited monophasic CO binding kinetics, as observed for isolated α- and β-subunits of hemoglobin. However, the rate of CO (or oxygen) binding to α-hemoglobin bound to its chaperone is three times slower than that observed for isolated α-hemoglobin, demonstrating a form that is intermediate to the R- and T-hemoglobin states. The physiologically relevant replacement of the chaperone by β-hemoglobin chains could be detected by both ligand binding kinetics and tryptophan fluorescence quenching.