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 en zymes' specific substrate to promote selective adsorption of individual NAD (+)-dependent dehydrogenases on their complementary immobilized cofactor de rivative. Application of this strategy to the purification of NAD(+)-depend ent dehydrogenases from crude extracts has proven that it can yield bioaffi nity systems capable of producing one-chromatographic-step purifications wi th yields approaching 100%. However, in some cases the purified enzyme prep aration was found to be contaminated with Other proteins weakly bound to th e immobilized cofactor derivative through binary complex formation and/or n onspecific interactions, which continuously "dribbled" off the matrix durin g 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 subst rate analogue (locking-on ligand) is clear from the results presented in th is report. The general effectiveness of this auxiliary tactic has been asse ssed using model studies and through incorporation into an actual purificat ion from a crude cellular extract. The results confirm the usefulness of th e stripping-ligand tactic for the resolution and purification of NAD(+)-dep endent dehydrogenases when using the locking-on strategy. These studies hav e 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 mitocho ndrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (L-LDH, EC 1.1.1.27). (C) 1999 Academic Press.