Purification of Glutamate Dehydrogenase in Bovine (Bos taurus) Liver

  • Malika Prasad Ganguli Trent University
Keywords: Glutamate Dehydrogenase, Enzyme, Protein, Purification, Isolation, Bovine, Liver


Glutamate dehydrogenase (GDH) is found in the mitochondrial matrix and catalyzes the reaction of L-glutamate to alpha-ketoglutarate, reducing NAD+ to NADH in the process. Alpha-ketoglutarate is processed through the citric acid cycle and the electron transport chain, respectively, to produce the high-energy molecules NADH, FADH2, GTP, and ATP. The purification of GDH has not been thoroughly explored in recent literature, thus driving the attempt in successfully isolating a kinetically active GDH from bovine (Bos taurus) liver. 50 grams of bovine liver was homogenized in buffer and centrifuged to remove cell debris. Ammonium sulfate fractionations (ASF) removed unwanted proteins, and two samples were saved: 50% ASF and 70% ASF. Each sample underwent ion exchange (IEX) and size exclusion chromatography (SEC). Kinetic assays were used to analyze total catalytic activity in which the reverse reaction was monitored, where NADH consumption was measured at 340nm. In the 70% ASF sample, 17.00mg of total protein remained after SEC and showed the greatest amount of NADH consumption, with an enzyme activity of 1.44 x 10-10 U and a specific GDH activity of 8.47 x 10-12 U/mg. The 50% ASF sample showed less NADH activity, with an enzyme activity of 1.05 x 10-8 U, a specific GDH activity of 3.5 x 10-10 U/mg, and a total protein mass of 30.00 mg. Quantifiable GDH identification on SDS-PAGE showed a dark band at the expected protein location, ~60kDa. Overall, the purification of GDH was successful, though controlling for reaction temperature, buffer pH, addition of stabilizing agents, and other suggestions have been listed to improve the quality of GDH isolate.