The Role of Alpha Hemoglobin Stabilizing Protein (AHSP) in the Formation of Hemoglobin A.

Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitro and in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α₂β₂) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:

1. Does bis-histidyl ferric α Hb-AHSP form more readily under physiologic conditions?

2. Can ferrous and ferric α Hb-AHSP complexes form functional tetrameric HbA upon reaction with β Hb?

The conversion of ferrous to ferric α Hb-AHSP is temperature-dependent with complete conversion occurring in 40 minutes at 37°C, as opposed to 5 hours at 22°C. Furthermore, the conversion is accelerated by partial oxygen saturation, increasing the likelihood that ferric-AHSP Hb will form in vivo. Using cellulose acetate gel electrophoresis, we demonstrated that β Hb displaces AHSP to form tetrameric HbA from both α Hb-AHSP complexes. The resulting tetramer from ferrous α Hb-AHSP is similar to HbA, both spectroscopically and in terms of oxygen binding. In contrast, the Hb tetramer formed from ferric α Hb-AHSP and β Hb is similar to HbY (αFeIII₂βFeII₂) spectroscopically. However, the α globin heme iron within this tetrameric complex appears to be occluded, as the rate of cyanide binding is reduced. Furthermore, it is resistant to reduction by sodium dithionite or methemoglobin reductase, suggesting that the α Hb retains its bis-histidyl nature. Together, these studies show that both α Hb-AHSP complexes are likely to form under physiological conditions. Upon interaction with β Hb, ferrous α Hb-AHSP forms functional Hb A tetramer. In contrast, ferric α Hb-AHSP is more chemically inert, but may not be able to form viable HbA.