کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1173876 | 1491405 | 2012 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines: Versatile probes for enzyme kinetics Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines: Versatile probes for enzyme kinetics](/preview/png/1173876.png)
14C-labeled nicotinamide cofactors are widely employed in biomedical investigations, for example, to delineate metabolic pathways, to elucidate enzymatic mechanisms, and as substrates in kinetic isotope effect (KIE) experiments. The 14C label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors and disruptions in the availability of enzymes required for established syntheses have recently made the preparation of labeled nicotinamides such as [Ad-14C]NADPH unviable. Here, we report the syntheses and characterization of several alternatives: [carbonyl-14C]NADPH, 4R-[carbonyl-14C, 4-2H]NADPH, and [carbonyl-14C, 4-2H2]NADPH. The new procedures use [carbonyl-14C]nicotinamide as starting material, because it is significantly cheaper than other commercial 14C precursors of NADPH, and require only one commercially available enzyme to prepare NAD(P)+ and NAD(P)H. The proximity of carbonyl-14C to the reactive center raises the risk of an inopportune 14C isotope effect. This concern has been alleviated via competitive KIE measurements with Escherichia coli dihydrofolate reductase (EcDHFR) that use this specific carbonyl-14C NADPH. A combination of binding isotope effect and KIE measurements yielded no significant 12C/14C isotope effect at the amide carbonyl (KIE = 1.003 ± 0.004). The reported procedure provides a high-yield, high-purity, and cost-effective alternative to labeled nicotinamide cofactors synthesized by previously published routes.
Journal: Analytical Biochemistry - Volume 430, Issue 2, 15 November 2012, Pages 123–129