O'Carra P, Griffin T, O'Flaherty M, Kelly N, Mulcahy P
Department of Biochemistry, University College, Galway, Ireland.
Biochim Biophys Acta. 1996 Oct 17;1297(2):235-43. doi: 10.1016/s0167-4838(96)00100-8.
Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct 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. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol 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 using the tactic.
以往的研究利用有序动力学机制设计生物特异性亲和色谱方法，用于酶的高效纯化和机理研究。最直接的策略是使用以下物质的固定化类似物，通常是酶特异性底物，例如，对于以NAD⁺为主要底物的乳酸脱氢酶，使用乳酸/丙酮酸。然而，这种固定化的特异性底物通常很难合成或无法合成。锁定策略则颠倒了这种策略，使用更容易获得的固定化主要底物——固定化NAD⁺作为吸附剂，酶特异性配体的可溶性类似物（例如，对于乳酸脱氢酶，使用乳酸）可显著增强生物特异性吸附，足以从一组酶（如NAD⁺特异性酶）中对一种酶进行吸附选择。使用乳酸脱氢酶（LDH，EC 1.1.1.27）、乙醇脱氢酶（ADH，EC 1.1.1.1）、谷氨酸脱氢酶（GDH，EC 1.4.1.3）和苹果酸脱氢酶（MDH，EC 1.1.1.37）进行模型研究，证明了这种方法的价值。文中描述了使用该策略从粗提物中高产率纯化牛肝GDH的过程。
