EFFECT OF 2,3-DIPHOSPHOGLYCERATE ON O-2-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 affini ty 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 th e stopped flow method and a new in vitro glycosylation procedure allow ing adequate amounts of functionally intact hemoglobin to be obtained. The rate constant k of O-2-dissociation in glycosylated Hb (8% HbA(1c )) was approximately 10% lower than in native Hb (4% HbA(1c)). The add ition of 2,3-DPG in (-1) to 65.3 concentrations up to 20 mmol/l result ed in a progressive increase of k from 61.5 +/- 3.3 s(-1) to 65.35 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 glycosylat ion similar to that found in diabetic patients is responsible for a si gnificant decrease of the oxygen dissociation velocity and (b) 2,3-DPG concentration similar to those occuring in vivo have only a weak effe ct on the oxygen dissociation velocity.