Carbohydrate Intermediates and Related Cofactors in the Human Erythrocyte

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Abstract
Ion-exchange chromatography was used for the separation of certain carbohydrate intermediates and adenosine phosphates. With a Dowex-1-Cl column (0.8 sq. cm. x 10 cm.), flow rate 3 ml./ min., the following elution pattern was obtained with increasing molarities of HCl: adenine at 0.001 [image] HCl, adenosine-5-phosphate (AMP) at 0.002, mixed hexosemonophosphates, ribose-5-phosphate (RP) and inorganic phosphate (IP) at 0.003, 3-monophosphoglycerate (MPG) at 0.01, adenosine diphosphate (ADP) at 0.02, fructose diphosphate (FDP) at 0.04 and mixed adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) at 0.06. In order to separate its individual C parts the 0.06 mixture was hydrolyzed for 30 min. in 0.2 [image] HCl and rechromatographed. Adenine was recovered at 0.001, RP and IP at 0.003 and the acid resistant DPG at 0.06. The method was applied to the identification of the pool of carbohydrate intermediates and related cofactors in the human erythrocyte. A flow diagram is given covering approximately 90% of the trichloroacetic acid soluble P components of the red cell. The following micromole quantities of compounds were found in 100 ml. of blood: AMP1.1, ADP 11.6, ATP 45.2, glucose monophosphates 2, fructose monophosphate 1, MPG 3.0, DPG 157, FDP 10.5, IP 21.0, total P 597. There were 3 unidentified P peaks: 14 [mu][image] in 0.04, 10 [mu][image] in 0.06 and 19 [mu][image] in 1.0. The procedure has merit for the detection of unknowns and for following the movement of C14 and P32 in metabolism studies.