The Formation of 2,3-Diphosphoglycerate in Rabbit Erythrocytes: The Existence of a Diphosphoglycerate Mutase

The red blood cells of most mammalian species are distinguished by the presence of 2,3-diphosphoglyceric acid, which accounts for about half of their total acid-soluble P (1).The compound is stable in contrast to the labile 1,3-phosphoglyceric acid (2), which is considered an obligatory intermediate in the glycolytic cycle of all tissues.From studies with radioactive P it would appear that 2,3-diphosphoglycerate has a rapid turnover comparable to that of other phosphate esters of the red cells (3), an indication that it lies either in the main path of their phosphate cycle or exists in free equilibrium with an obligatory intermediate.The mechanism of its formation is unknown.Similar uncertainty obtains with regard to the steps in its decomposition.In intact cells at a plasma pH below 7.35, breakdown occurs which is linked with maintenance of adenosine triphosphate (ATP) (4).In the unfortified hemolysate the compound is almost inert ( 5), but is decomposed readily when adenylic acid is added (6).The present study is concerned with the mechanism of the formation of 2,3-phosphoglycerate (2,3-P-GA). MethodsMost of the experiments were performed with hemolysates of rabbit erythrocytes, prepared by the addition of 2 volumes of distilled water to 1 volume of cells twice washed in the cold with 0.9 per cent NaCl.Hemolysates of beef cells were prepared in the same manner.Phosphate determinations were performed by the method of Fiske and Subbarow (7).Acid hydrolysis of the acid-soluble P was carried out for 15 or 150 minutes in N sulfuric acid in a boiling water bath.Total phosphoglycerate was determined by the naphthoresorcinol method (8) adapted to the photoelectric calorimeter, and 3-phosphoglycerate by polarimetry (9). ResultsFormation of Stable P in Rabbit Hemolysates-The formation of dill% cultly hydrolyzable P was studied in a system consisting of a hemolysate of 507