Effect of Accessible Immobilized NAD+ Concentration on the Bioaffinity Chromatographic Behavior of NAD+-Dependent Dehydrogenases Using the Kinetic Locking-on Strategy

In preparation for studies aimed at establishing the relationship between immobilized NAD+ concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD+-dependent dehydrogenases to immobilized NAD+ derivatives (the “locking-on” strategy), two app...

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Bibliographic Details
Published in:Protein expression and purification 1999-07, Vol.16 (2), p.261-275
Main Authors: Mulcahy, Patricia, O'Flaherty, Martina, McMahon, Mary, Oakey, Laura
Format: Article
Language:English
Subjects:
Animals
bioaffinity chromatography
bovine heart [formula omitted]-lactate dehydrogenase
Cattle
Chromatography, Affinity - methods
Electrophoresis, Polyacrylamide Gel
immobilized NAD+ derivatives
Kinetics
L-Lactate Dehydrogenase - metabolism
Myocardium - enzymology
NAD - metabolism
yeast alcohol dehydrogenase
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Summary:In preparation for studies aimed at establishing the relationship between immobilized NAD+ concentration and the concentration of soluble locking-on ligand required to promote biospecific adsorption of NAD+-dependent dehydrogenases to immobilized NAD+ derivatives (the “locking-on” strategy), two approaches were evaluated for varying substitution levels: (i) suitable dilution of the affinity matrix with unsubstituted Sepharose 4B and (ii) direct coupling of the required ligand concentration to the inert matrix. The latter approach was found to be the preferable strategy for evaluation of the locking-on tactic because it produced a more homogeneous distribution of immobilized NAD+ concentration. Affinity chromatographic studies using S6-linked NAD+ derivatives synthesized to various substitution levels showed that the total accessible immobilized NAD+ concentration has a direct effect on the locking-on behavior of pyridine nucleotide-dependent dehydrogenases. The one-chromatographic-step bioaffinity purification of l-lactate dehydrogenase (L-LDH, EC 1.1.1.27) from bovine heart illustrates the potential of the locking-on strategy for protein purification applications.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1999.1050