Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase.

A method for the assay of 2-oxoglutarate decarboxylating enzymes based on determination of the reaction product [1-14C]succinate after precipitation of remaining 2-oxo[5-14C]glutarate with 2,4-dinitrophenyl hydrazine is reported. It is particularly useful for the study of the 2-oxoglutarate-coupled dioxygenase prolyl 4-hydroxylase (EC 1.14.11.2); it is superior to previously described assay methods of this enzyme with respect to simplicity of the procedure, speed, cost, and radiochemical safety. The results are highly reproducible, the standard deviation of repeated measurements being about 2% of the mean. The commercially available 2-oxo[5-14C]glutarate used in this study contained approximately 3% of radioactivity coeluting with succinate in HPLC and 1.5% of an unidentified radioactive compound as impurities, which contributed to the background.