General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

Abstract

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.

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

“…Barry and O'Carra [4] and Mosbach et al [5] investigated such an approach using direct carbodiimide-promoted coupling to carboxylated Sepharose derivatives but did not report on the maintenance of cofactor activity postimmobilization or Wnal accessible immobilized cofactor concentrations. The assessment of these derivatives for biospeciWc binding was further complicated by the use of 6-aminohexanoic acid as the spacer arm compound (with the ligand being attached through the carboxylate function of the spacer).…”

“…Immobilized NAD(P) + derivatives synthesized by carbodiimide-promoted attachment to a carboxyterminating spacer arm Direct coupling of NAD(P) + to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling was based on methods originally described by Barry and O'Carra [4] and Mosbach et al [5] but with important modiWcations. DADPA was coupled to CNBr-activated Sepharose 4B and treated with glutaric anhydride to produce a carboxylate-terminating immobilized spacer arm.…”

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

“…O'Carra (2) and Mosbach (25) briefly investigated such an approach in the 1970s using direct carbodiimide-promoted coupling to carboxylatedSepharose derivatives, concluding that the attachment is predominantly through the ribosyl hydroxyl groups and/or the pyrophosphate grouping of the cofactors. But the matrices produced during these studies were not well characterized with respect to maintenance of cofactor activity postimmobilization or final accessible immobilized cofactor concentrations (2,25). The assessment of these derivatives for biospecific binding was further complicated by the almost universal use of 6-aminohexanoic acid as the spacer arm compound (the ligand being attached through the carboxylate function of the spacer).…”

“…Much of this effort was directed at NAD(P) ϩ -dependent dehydrogenases because of the important roles they play in biological processes and their potential biotechnological applications (e.g., [37][38][39][40][41][42][43]. At this time, two approaches to small ligand bioaffinity chromatography of NAD(P) ϩ -dependent dehydrogenases were advocated: (i) a general ligand approach employing immobilized derivatives of the common cofactor substrate (23)(24)(25) and (ii) a specific ligand approach employing immobilized derivatives of the enzymes-specific substrate (28,29,31,34). Arguments in favor of the general ligand approach were based upon the premise that a single immobilized cofactor derivative would exhibit bioaffinity for a great number of enzymes and that subsequent biospecific elution would then provide for the isolation of individual enzymes (23)(24)(25).…”

“…At this time, two approaches to small ligand bioaffinity chromatography of NAD(P) ϩ -dependent dehydrogenases were advocated: (i) a general ligand approach employing immobilized derivatives of the common cofactor substrate (23)(24)(25) and (ii) a specific ligand approach employing immobilized derivatives of the enzymes-specific substrate (28,29,31,34). Arguments in favor of the general ligand approach were based upon the premise that a single immobilized cofactor derivative would exhibit bioaffinity for a great number of enzymes and that subsequent biospecific elution would then provide for the isolation of individual enzymes (23)(24)(25). The fundamental argument against the general ligand approach was that what was gained in general applicability with immobilized cofactor derivatives was lost in selectivity (29,31).…”