Functional properties of the glycosylated minor components of human adult hemoglobin

The functional properties of four glycosylated minor components of human hemolysate have been examined. In 0.1 M potassium phosphate buffer, pH 7 and 20°C, the p₅₀ values of Hb A₁(a1), Hb A₁(a2), Hb A₁(b) and Hb A₁(c) were 14, 9.8, 6.3, and 7.4 mm Hg, respectively, compared to 7.8 mm Hg for the major component, Hb A₀. The Δ log p₅₀/Δ (pH 7 to pH 8) was -0.5 for Hb A₁(a1), Hb A₁(a2), and Hb A₁(b) as compared with -0.65 for Hb A₁(c) and Hb A₀; therefore, a significant alkaline Bohr effect exists for all these components. After being freed of phosphate, the ligand affinity of Hb A₀ was noticeably increased (p₅₀ = 4.3 mm Hg in 0.05 M 2,2'-bis(hydroxymethyl)-2,2',2''c-nitriloethanol (bis-Tris)/HCl buffer, pH 7, and 0.1 M NaCl at 20°C) as was that of Hb A₁(b) (P₅₀ = 5.1) and Hb A₁(c) (P₅₀ = 4.5) while the affinities of Hb A₁(a1) and Hb A₁(a2) remained low (P₅₀ = 14 and 8.0 mm Hg, respectively). Organic phosphate had a much weaker effect on the oxygenation of the four glycosylated hemoglobins compared to Hb A₀. The ratios of p₅₀ in the presence and absence of 500 μM inositol = were as follows: Hb A₁(a1) = 1; Hb A₁(a2) - 1.5; Hb A₁(b) = 2.2; Hb A₁(c) = 3.6; and Hb A₀ = 13. The time course of carbon monoxide binding in the presence and absence of inositol hexaphosphate reflected the equilibria findings. The equilibrium properties of two synthetic glycohemoglobins, α₂(β(N-glucose)₂ and α₂(β(N-glucose-6-P)₂ were similar to Hb A₁(c) and Hb A₁(a2), respectively. These functional studies show that the four native glycosylated minor components possess unique functional properties which differ from those of the major component, Hb A₀. Two of these components, Hb A₁(a1), and Hb A₁(a2), are low affinity hemoglobins similar to certain rare human hemoglobin variants but present in every human hemolysate.