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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.

SUBMITTER: Mosbach K

PROVIDER: S-EPMC1178759 | biostudies-other | 1972 May

REPOSITORIES: biostudies-other

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Project description:1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.

Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

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

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