A Kinetic Locking-On Strategy for Bioaffinity Purification: Further Studies with Alcohol Dehydrogenase

• Published in: Protein expression and purification 1999-02, Vol.15 (1), p.127-145
• Main Authors: O'Flaherty, Martina, McMahon, Mary, Mulcahy, Patricia
• Format: Article
• Language: English
• Subjects: affinity chromatography; alcohol dehydrogenase; Alcohol Dehydrogenase - isolation & purification; Alcohol Dehydrogenase - metabolism; Animals; Chromatography, Affinity - methods; Gram-Positive Asporogenous Rods, Irregular - enzymology; horse liver; Horses; immobilized NAD(P); immobilized pyrazole; Indicators and Reagents; kinetic locking-on strategy; kinetic mechanism; Kinetics; Liver - enzymology; NAD; NADP; Pyrazoles; Saccharomyces cerevisiae - enzymology; Thermoanaerobium brockii; yeast
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1998.0995

The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8′-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+derivatives. These studies were carried out using alcohol dehydrogenases fromSaccharomyces cerevisiae(YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), andThermoanaerobium brockii(TBADH, EC 1.1.1.2). The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference. The S6-linked immobilized NAD+derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy. This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the “test” enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase fromSaccharomyces cerevisiaewas purified to electrophoretic homogeneity in a single affinity chromatographic step.