McNeil C J, Spoors J A, Cocco D, Cooper J M, Bannister J V
Anal Chem. 1989 Jan 1;61(1):25-9. doi: 10.1021/ac00176a006.
The use in amperometric enzyme assays of a highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), from the thermophilic organism Thermus aquaticus is described. The enzyme catalyses the oxidation of reduced nicotinamide adenine dinucleotide with concomitant two-electron reduction of dioxygen to hydrogen peroxide. In addition the enzyme used a substituted ferrocene as an alternative mediator of electron transfer. Hydrogen peroxide was detected at +650 mV vs Ag/AgCl at a platinum electrode. The current produced by oxidation of hydrogen peroxide was directly proportional to NADH concentration. The enzyme was used in solution to reoxidize enzymatically generated NADH and served as a basis for amperometric enzyme amplification systems for immunoassay as well as for the detection of substrate concentration for oxidoreductase enzymes. In the presence of alcohol dehydrogenase a rapid production of current occurred upon addition of ethanol over a clinically significant range. Thermus aquaticus NADH oxidase appears to be ideally suited for future exploitation in amperometric sensors for oxidoreductase substrates, offering a number of advantages over previously reported methods.
本文描述了来自嗜热栖热菌的一种高度稳定、对pH不敏感的黄素酶——还原型烟酰胺腺嘌呤二核苷酸氧化酶（NADH氧化酶）在安培酶分析中的应用。该酶催化还原型烟酰胺腺嘌呤二核苷酸的氧化，同时将二价氧双电子还原为过氧化氢。此外，该酶使用取代二茂铁作为电子转移的替代媒介。在铂电极上相对于Ag/AgCl在+650 mV处检测到过氧化氢。过氧化氢氧化产生的电流与NADH浓度成正比。该酶用于溶液中以酶促方式重新氧化产生的NADH，并作为免疫分析安培酶放大系统以及氧化还原酶底物浓度检测的基础。在存在乙醇脱氢酶的情况下，加入乙醇后在临床上有意义的范围内会迅速产生电流。嗜热栖热菌NADH氧化酶似乎非常适合未来用于氧化还原酶底物的安培传感器，与先前报道的方法相比具有许多优势。
