32P distribution into 2,3-bisphosphoglyceric acid from human erythrocytes following molybdate-catalyzed hydrolysis

Normal human washed erythrocytes were incubated in autologous plasma with 32P for varying periods of time. 2,3-Bisphosphoglyceric acid the major organic phosphate ester of red cells, was first isolated and purified by anion-exchange chromatography; subsequently, the C-2Pi was selectively hydrolyzed by a molybdate-catalyzed reaction. The C-2-bound Pi of 2,3-P2-glyceric acid was found to have a significantly higher specific activity than C-3 Pi. Intramolecular equilibration was not reached until the 180-min incubation. No evidence of overhydrolysis or internal randomization by the molybdate reaction was noted which was confirmed following hydrolysis of double-label 2,3-P2-glyceric acid 32P C-2, 33P C-3.