Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

“…Four test enzymes were used to evaluate NAD + derivatives I-III: YADH, L-LDH, bovine liver GDH, and L-PheDH. These enzymes were selected because it was reported that hydroxylamine, oxalate, and glutarate promoted biospeciWc adsorption of YADH, L-LDH, and GDH, respectively, to S 6 -and 8Ј-azo-linked NAD + [1]. It is also important to note that although Sporosarcina sp.…”

“…The type of inhibition in all cases was competitive. a Taken from [1]. The assay conditions for YADH were 100 mM sodium pyrophosphate, pH 8.8, 30°C, 0.08 mM NAD + .…”

“…This strategy uses soluble analogs (KBEC ligands) of the target enzymes' speciWc substrate to increase the selectivity of immobilized/cross-linked cofactors for the target enzyme and to push the rate of dissociation of the enzymes toward zero [1,2]. Four test enzymes were used to evaluate NAD + derivatives I-III: YADH, L-LDH, bovine liver GDH, and L-PheDH.…”

“…The NAD + derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospeciWc interference, reversible biospeciWc adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.  2004 Elsevier Inc. All rights reserved.Kinetic-based enzyme capture (KBEC) 1 strategies based on immobilized cofactor derivatives have considerable potential for protein puriWcation applications and for protein-detecting arrays/proteomic studies [1,2]. There are, however, a number of factors that require further development and reWnement if this technology is to reach its full potential.…”

“…Kinetic-based enzyme capture (KBEC) 1 strategies based on immobilized cofactor derivatives have considerable potential for protein puriWcation applications and for protein-detecting arrays/proteomic studies [1,2]. There are, however, a number of factors that require further development and reWnement if this technology is to reach its full potential.…”