A comparison of biospecific affinity chromatographic methodologies for the purification of NAD+-dependent dehydrogenases: studies with bovine L-lactate dehydrogenase

Oakey, L.M.rtung, A.M.mahon, M.O.-Flaherty, M.M.lcahy, P.

Process biochemistry 35(3-4): 349-357

1999


ISSN/ISBN: 1359-5113
DOI: 10.1016/s0032-9592(99)00078-3
Accession: 014845674

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Abstract
The general potential of biospecific affinity chromatography in enzyme isolation and purification is well recognised. In the present study, three distinct approaches were assessed for the rapid, one-chromatographic-step affinity purification of l-lactate dehydrogenase (l-LDH, EC 1.1.1.27) from bovine heart: (i) the specific ligand approach using an immobilised oxamate derivative; (ii) the general ligand approach (immobilised NAD+ derivatives) in conjunction with a kinetic ‘locking-on’ strategy; (iii) the general ligand approach in conjunction with the kinetic locking-on strategy and an auxiliary tactic (the ‘stripping’ ligand tactic). Purification tables were constructed for each of the purification protocols and all purified enzyme samples were assessed for homogeneity using SDS–PAGE. Results confirm that the more widely applicable general ligand approach, when used in conjunction with both the locking-on strategy and stripping ligand tactic, has the potential to purify NAD+-dependent dehydrogenases to homogeneity in a single bioaffinity chromatographic step.