A “Stripping” Ligand Tactic for Use with the Kinetic Locking-on Strategy: Its Use in the Resolution and Bioaffinity Chromatographic Purification of NAD-Dependent Dehydrogenases

The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD⁺-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD⁺-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously “dribbled” off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5′-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD⁺-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart -lactate dehydrogenase (-LDH, EC 1.1.1.27).