Effect of 2,3-diphosphoglycerate on O2-dissociation kinetics of hemoglobin and glycosylated hemoglobin using the stopped flow technique and an improved in vitro method for hemoglobin glycosylation.

Studies under equilibrium conditions have shown that the oxygen affinity of hemoglobin (Hb) is lowered by 2,3-diphosphoglycerate (2,3-DPG), the physiological allosteric effector in erythrocytes, and enhanced by glycosylation of Hb. The kinetics of oxygen release, as a function of 2,3-DPG and the degree of glycosylation have been determined using the stopped flow method and a new in vitro glycosylation procedure allowing adequate amounts of functionally intact hemoglobin to be obtained. The rate constant k of O2-dissociation in glycosylated Hb (8% HbA1c) was approximately 10% lower than in native Hb (4% HbA1c). The addition of 2,3-DPG in concentrations up to 20 mmol/l resulted in a progressive increase of k from 61.5 +/- 3.3 s-1 to 65.3 ae 4.1 s-1 for native Hb and from 56.8 +/- 5.2 s-1 to 59.4 +/- 4.1 s-1 for glycosylated Hb. We conclude that (a) the degree of glycosylation similar to that found in diabetic patients is responsible for a significant decrease of the oxygen dissociation velocity and (b) 2,3-DPG concentration similar to those occurring in vivo have only a weak effect on the oxygen dissociation velocity.