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The distribution of proto-, meso-, and deuteroheme on hemoglobin apoprotein has been studied in mixtures containing heme-free and heme-bound hemoglobin chains. The difference between cy and /3 chains in their affinity for hemin, responsible for the formation of the intermediate compound ICrI, is absent in the case of deuterohemin. Binding of deuterohemin by globin appears to be strongly cooperative since no intermediate can be detected in the reconstitution process. For the case of protohemin, varying the pH from 6 to 9 did not influence the difference in affinity between cy and / 3 chains. The addition of cyanide strengthens the binding of the prosthetic

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (~-glycerate-2,3-P~) on the oxygen equilibrium of normal and SH(p93)-modified human hemoglobin. At sufficiently high D-glycerate-2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for ~-glycerate-2,3-P~ between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of ~-glycerate-2,3-P~ on the hemoglobin molecule.In the hemoglobins modified in position p93, the difference in affinity between deoxy and oxy hemoglobin for ~-glycerate-2,3-P~ decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for ~-glycerate-2,3-P~. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates.In spite of the very large number of investigations dealing with the effect of organic phosphates on the oxygen affinity of hemoglobin, some aspects of its interaction with organic phosphates still remain unclear.Thus, while in deoxy hemoglobin one high-affinity binding site has been identified by X-ray analysis at the entrance of the central cavity between the 8 chains [1,2], in oxyhemoglobin, the number and identity of the organic phosphate binding sites is still questioned [3 -61. Furthermore, a recent investigation [7 -91 of the interaction of organic phosphates with partially oxygenated molecular species has shown some interesting aspects of the phenomenon, indicating non-uniformity of anion release with respect to the oxygenation step. However, the general mechanism involved in the strong effect of D-glycerate 2,3-bisphosphate (~-glycerate-2,3-Pz) on both the oxygen affinity [lo-121 and the Bohr effect of hemoglobin [3,13] implies (a) that the affinity of the deoxy form for the ~-glycerate-2,3-P2 molecule is higher than that of the ligand-bound derivative [lo-12,14,15]; (b) that at constant ~-glycerate-2,3-P~ concentration, the amount of D-glycerate 2,3-P2 bound to hemoglobin changes with pH [4,16,17].In the course of an investigation on the functional properties of hemoglobin A modified at p93 with various sulphydryl reagents [18,19], it was necessary to reinvestigate the effect of ~-glycerate-2,3-P~ on the 0 2 affinity of normal human hemoglobin as a function of pH. The present paper reports data both for HbA and HbA modified by reaction of SH (893) with disulfides or with iodoacetamide. The results show that the total effect of ~-glycerate-2,3-Pz is independent of pH for normal hemoglobin, but not for the modified hemoglobin. Thus, at sufficiently high D-glycerate 2,3-P2 concen- _ _ _trations, the Bohr effect is unmodified in normal human hemoglobin, but is significantly decreased in hemoglobins modified at the SH of 893. MATERIALS AND METHODSFresh blood samples were obtai...

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