FURTHER-STUDIES ON THE BIOAFFINITY CHROMATOGRAPHY OF NAD(-DEPENDENT DEHYDROGENASES USING THE LOCKING-ON EFFECT())

Previous studies have capitalised on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly eff icient purifications and mechanistic studies of enzymes. The most dire ct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD(+) is the leading substrate. S uch immobilised specific substrates are, however, often difficult or i mpossible to synthesise. The locking-on strategy reverses the tactic b y using the more accessible immobilised leading substrate, immobilised NAD(+), as adsorbent with soluble analogues of the enzyme-specific li gands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to eff ect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrate d using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), a lcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described usi ng the tactic.