A comparison of biospecific affinity chromatographic methodologies for thepurification of NAD(+)-dependent dehydrogenases: studies with bovine L-lactate dehydrogenase

The general potential of biospecific affinity chromatography in enzyme isol ation and purification is well recognised. In the present study, three dist inct approaches were assessed for the rapid, one-chromatographic-step affin ity purification of L-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovin e heart: (i) the specific ligand approach using an immobilised oxamate deri vative; (ii) the general ligand approach (immobilised NAD(+) derivatives) i n conjunction with a kinetic 'locking-on' strategy; (iii) the general ligan d approach in conjunction with the kinetic locking-on strategy and an auxil iary tactic (the 'stripping' ligand tactic). Purification tables were const ructed for each of the purification protocols and all purified enzyme sampl es were assessed for homogeneity using SDS-PAGE. Results confirm that the m ore widely applicable general ligand approach, when used in conjunction wit h both the locking-on strategy and stripping ligand tactic, has the potenti al to purify NAD(+)-dependent dehydrogenases to homogeneity in a single bio affinity chromatographic step. (C) 1999 Elsevier Science Ltd. All rights re served.