Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin.

Abstract

We have reported previously that organic phosphates have a profound effect On the reaction of hemoglobin with oxygen.' Organic polyphosphates such as 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) in the millimolar concentrations at which they occur in the red cell can decrease the oxygen affinity of hemoglobin about 30-fold. These compounds therefore greatly facilitate the unloading of oxygen from hemoglobin. Some of these findings have recently been confirmed by Chanutin and Curnish.2 The displacement of oxygen from hemoglobin by DPG strongly suggested that the affinity of this compound for the deoxy form of hemoglobin is greater than for the oxy form. Direct measurements of the binding of DPG by oxy-and deoxyhemoglobin, described below, confirm this assumption. It will be shown that the difference in affinity can account for the observed displacement of the oxygen dissociation curve in the presence of DPG.Preparation of Hemoglobin.-The method of Drabkin,I previously used by us and other workers, results in preparations which contain variable amounts of organic phosphate. A representative example of the DPG content at various stages of this procedure is shown in Table 1. It can be seen that the final preparation contains about 0.5 mole DPG per mole of hemoglobin. The presence of 0.6 atom of P per mole of human hemoglobin, also prepared by Drabkin's procedure, was already noted by Havinga.4 Since DPG is a highly charged molecule (almost four of the five acid groups are ionized at neutral pH), it can only be displaced from the protein in the presence of sufficient concentrations of electrolyte.The following procedure was finally adopted: Blood collected in neutral citrate (4 ml of 3.2% sodium citrate per 20 ml blood) was washed four times with ice-cold 0.9% sodium chloride, the cells were hemolyzed by shaking for 3 min with 1 vol of water and 0.4 vol of toluene followed by centrifugation. The clear hemoglobin solution was siphoned off and 5 ml (concentration approximately 15%) were placed on a 2.5 cm X 30-cm column of Sephadex G-25 equilibrated with 0.1 M sodium chloride. The protein was eluted with the same solvent at a rate of about 20 ml/hr.

Oxygenation Properties and 2, 3-Diphosphoglycerate of Human Adult and Fetal Blood

“…5, show clearly that the oxygen affinity increases parallel with the decrease of DPG content in both adult and cord blood and that the increase in oxygen affinity is less marked in the latter than in the former. A biphasic pattern in the dissociation curves as observed in free hemoglobin solution with a DPG/Hb molar ratio of less than unity (BENESCH et al, 1968), is not present in whole blood under comparable conditions (Fig. 6), and therefore a normal heme-heme interaction is always maintained (Fig.…”

The in-vivo oxyhaemoglobin dissociation curve at sea level and high altitude

“…Like CO 2 and H + , 2,3-DPG preferentially binds to, and stabilizes the reduced form of Hb, decreasing saturation for any given PO 2 (Benesch et al, 1968). 2,3-DPG also affects the position of the ODC via its effect on pH; increases in 2,3-DPG lower RBC pH, and a decrease in RBC pH has been shown to shift the ODC to the right (Bellingham et al, 1971).…”

“…The right shift may also have been due to the increase of [2,3-DPG] (Benesch et al, 1968). Morpurgo et al (1976) later conducted a study of Sherpas native to an altitude of 3800-3900 m. adapted to life at high altitude due to prolonged exposure over many more generations to the stress of hypoxic hypoxia, which would have increased the possibility of natural selection and evolution.…”

“…They discovered that this organic phosphate, a by-product of RBC metabolism, is an allosteric modulator of Hb that binds to its reduced form and decreases its affinity for O 2 (Benesch et al, 1968). Soon thereafter, Lenfant et al (1968) Once it was established that [2,3-DPG] increases upon ascent to high altitude, conducted an experiment to determine the relationship between pH change, [2, and shifts of the ODC at high altitude.…”

Purification of Bisphosphoglyceromutase, 2, 3-Bisphosphoglycerate Phosphatase and Phosphoglyceromutase from Human Erythrocytes. Three Enzyme Activities in One Protein

“…Since the binding of 2,3-bisphosphoglycerate to hemoglobin, which facilitates the release of oxygen to tissues, was found by Benesch and Benesch [3,4], much interest has centered on the regulatory mechanisms of the 2,3-bisphosphoglycerate metabolism in human red cells.…”