Quantitative Analysis of α-Ketoglutarate

The reaction mixture (RM) contained 5 mM α-KGM, 5 mM DTT, 100 mM Tris-HCl buffer (pH 8.5), and an enzyme source. The final RM volume was adjusted with H₂O to 50 µL. Note that when the assays were conducted with purified ω-amidase, the blank contained a complete RM lacking the enzyme. For assays of crude tissue/cell homogenates, the blank contained a complete RM plus a homogenate and 200 mM of glycylglycine. After a 5–30 min incubation at 37 °C, the reaction was terminated by the addition of 20 µL of 5 mM 2,4-dinitrophenylhydrazine in 2 M HCl. After a further incubation for 5 min at 37 °C, 130 µL of 1 M NaOH was added and the absorbance was read at 430 nm within 5 min. The ε_430nm of α-ketoglutarate*2,4-dinitrophenylhydrazone under these conditions was 16,000 M⁻¹·cm⁻¹.