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 2O”C, the p50 values of Hb Al,,, Hb Alaz, Hb Al,, and Hb A, were 14,9.8,6.3, and 7.4 mm Hg, respectively, compared to 7.8 mm Hg for the major component, Hb Ao. The A log p&A pH (pH 7 to pH 8) was -0.5 for Hb A1 .1, Hb Alaz, and Hb A1, as compared with -0.65 for Hb Ale and Hb Ao; therefore, a significant alkaline Bohr effect exist 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”-nitriloethanol (bis-Tris)/ HCl buffer, pH 7, and 0.1 M NaCl at 20°C) as was that of Hb A1, (pEO = 5.1) and Hb Ale (PM = 4.5) while the affinities of Hb Al=, and Hb Al,, remained low (~50 = 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 Ao. The ratios ofpbO in the presence and absence of 500 pM inositol hexaphosphate were as follows: Hb Al., = 1; Hb Al,, = 1.5; Hb A1, = 2.2; Hb A1, = 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, (y&3N-g’ucose)2 and LQ.(p-glucose-B-P) 2 were similar to Hb A1 e and Hb ALz, 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 Ao. Two of these components, Hb Ala1, and Hb Al,,, are low affinity hemoglobins similar to certain rare human hemoglobin variants but present in every human hemolysate. Hb Al,,, and Hb Al< (1). These hemoglobins were first described by Allen et al. who obtained a good chromatographic separation of Hb Air from Hb Ala and Hb AI, as well as from the major component Hb A,I (2). Subsequent structural analyses of Hb AIM have shown that a glucose moiety is covalently attached to the NH, terminus of the p chain by a ketoamine linkage (3-6). Until recently no information on the structure of Hb Ala and Hb AI, was available. We have developed a chromatographic procedure using Bio-Rex 70 resin which allows not only complete separation of Hb Al* , Hb Al,, and Hb A,‘ but also resolution of Hb Ala into two components, designated Hb AIM, and Hb Ala,. All four of these components have been shown to contain ,0 chain modifications. Carbohydrate determinations on these purified components revealed that all four are glycosylated. Hb AI*, and Hb AIaT contain 2 and 1 mol of phosphate//3 chain, respectively. The finding that some minor components are modified by sugar phosphates was not unexpected since these compounds are known to form stable adducts with hemoglobin (7, 8). Presumably, the phosphorylated sugars serve as affinity labels with their phosphate group(s) forming electrostatic bonds at the 2,3-diphosphoglycerate binding site, their aldehyde (or ketone) group being thus properly positioned to form a covalent linkage with the NH2 termini of the ,L? chains (7). Hemoglobins altered covalently in this manner would be expected to have functional properties different from those of Hb Ao. We have examined the functional properties of the glycosylated minor components and have compared them with those of Hb A,, as well as synthetic glycohemoglobins. Here we present the effect of pH as well as organic phosphate on both equilibrium and kinetic parameters of these hemoglobins.