A kinetic locking-on strategy for bioaffinity purification: Further studies with alcohol dehydrogenase

The kinetic locking-on strategy improves the selectivity of protein purific ation procedures based on immobilized cofactor derivatives through use of e nzyme-specific substrate analogues in irrigants to promote biospecific adso rption. This paper describes the development and application of this strate gy to the one-chromatographic step affinity purification of NAD(P)(+)-depen dent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)(+), S-6- linked and N-6-linked immobilized NAD(+), and N-6-linked immobilized NADP() derivatives. These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1 .1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reve al 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 co factor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identificatio n and elimination of nonbiospecific interference. The SG-linked immobilized NAD(+) derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present s tudy for use with the locking-on strategy. This conclusion was based primar ily on the observations that this affinity adsorbent was stable, retained c ofactor activity with the ''test" enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunct ion with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step. (C) 1999 Academic Press.