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.

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