The kinetic locking-on strategy for bioaffinity purification: Further studies with bovine liver glutamate dehydrogenase

The locking-on strategy uses soluble analogues of the enzymes specific subs trate to produce biospecific adsorption of individual NAD(P)(+)-dependent d ehydrogenases on immobilized NAD(P)(+) derivatives, which is so selective t hat a single enzyme activity can be purified from crude cellular extracts i n a single chromatographic step with yields approaching 100%. However, atte mpts to further develop and apply this strategy to the biospecific chromato graphic purification of a range of NAD(P)(+)-dependent dehydrogenases revea led some anomalous chromatographic behavior and certain unexplained phenome non. Much of this can be attributed to nonbiospecific interference effects. Identification and elimination of this interference is discussed in the pr esent study focusing on bovine liver glutamate dehydrogenase (GDH; EC 1.4.1 .3) as the "test" enzyme. Results further confirm the potential of the lock ing-on strategy for the rapid purification of NAD(P)(+)-dependent dehydroge nases and provide further insight into the parameters which should be consi dered during the development of a truly biospecific affinity chromatographi c system based on the locking-on strategy. The kinetic mechanism of bovine liver GDH has been the topic of much controversy with some reports advocati ng a sequential ordered mechanism of substrate binding and others reporting a sequential random mechanism. Since the kinetic locking-on strategy is de pendent on the target NAD(P)(+)-dependent dehydrogenase having an ordered s equential mechanism of substrate binding, the bioaffinity chromatographic b ehavior of bovine liver GDH using the locking-on tactic suggests that this enzyme has an ordered sequential mechanism of substrate binding under a var iety of experimental conditions when NAD(+) is used as cofactor. (C) 1999 A cademic Press.