Karp M T, Raunio R P, Lövgren T N
Anal Biochem. 1983 Jan;128(1):175-80. doi: 10.1016/0003-2697(83)90359-7.
A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD+ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD+ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 X 10(-14) mol NADH or NAD+ per assay.
已开发出一种在室温下从组织样本中提取吡啶核苷酸的新方法，当使用70%缓冲乙醇溶液作为萃取剂时，该方法能够同时提取氧化型和还原型核苷酸。NAD⁺和NADH的提取效率分别为91%和102%。提取方法之后是对两种核苷酸的联合生物发光测定。一种细菌生物发光系统，其中包括荧光素酶和低水平的NADH特异性氧化还原酶，用于产生与组织提取物样本中NADH量直接成比例的恒定光强度。在测定NADH之后，通过添加乙醇脱氢酶将提取物中存在的NAD⁺酶促转化为NADH，然后记录光水平的第二次增加。此处介绍的生物发光测定的灵敏度为每次测定5×10⁻¹⁴摩尔NADH或NAD⁺。
