Preparation of ³²P-2, 3-Diphosphoglyceric Acid Labeled at a Single Position and Its Application to a Study on Diphosphoglycerate Phosphatase

Abstract

³²P-Diphosphoglyceric acid labeled at a single position was prepared, (1) Labeling at C-2. 3-Phosphoglyceric acid and ³²P-phosphoric acid were treated with dicyclohexylcarbodiimide in pyridine and the synthesized ³²P-2, 3-diphosphoglyceric acid isolated by column chromatography. (2) Labeling at C-3, ³²P-3-Phosphoglyceric acid labeled at C-3 was prepared by yeast fermentation and esterified with non-radioactive phosphoric acid by the same method as (1) .
Two kinds of the labeled diphosphate were subjected to hydrolysis by dphosphoglycerate phosphatase prepared from rabbit muscle. The results revealed that the liberated phosphate comes almost exclusively from C-2 position but not from C-3 position. When hemolysate was incubated with added inosine and³²P-phosphoric acid the radioactivity was incorporated into 2, 3-diphosphoglyceric acid, Diphosphoglycerate phosphatase was employed to determine the distribution of³² P. The isotope was shown to be evenly distributed between both of the phosphate groups. This can be explained by that both originate from a common inorganic phosphate pool.