Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.
An assay for prolyl 4-hydroxylase (EC 1.14.11.2) is described which measures succinic acid produced during the decarboxylation of 2-oxoglutaric acid in the presence of poly(L-Pro-Gly-L-Pro). [1-14C]Succinic acid was separated from its precursor 2-oxo[5-14C]glutaric acid by using ion-exchange minicolumns. The contamination of succinic acid by 2-oxoglutaric acid was approx. 1%, and the recovery of succinic acid was 100%. Kinetic parameters of prolyl 4-hydroxylase measured by the assay showed good agreement with published values. Our experience indicates that the measurement of prolyl 4-hydroxylase by the production of succinic acid is especially suited to investigations involving large numbers of assays.
D002250:Carbon Radioisotopes; D002852:Chromatography, Ion Exchange / Q000379:methods; D007656:Ketoglutaric Acids / Q000032:analysis; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis*; D013386:Succinates / Q000032:analysis*; D019802:Succinic Acid
D002250:Carbon Radioisotopes; D007656:Ketoglutaric Acids; D013386:Succinates; D019802:Succinic Acid; D011393:Procollagen-Proline Dioxygenase