Tsuji A, Maeda M, Arakawa H
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
J Biolumin Chemilumin. 1989 Jul;4(1):454-62. doi: 10.1002/bio.1170040160.
Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 X 10(-14) to 5 X 10(-12) mol/assay. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.
生物发光法广泛用于辅助因子NADH和ATP的测定。尽管生物发光法灵敏度很高，但所使用的酶不稳定且昂贵。因此，化学发光法在临床常规检测中具有重要价值。我们开发了一种使用硫酸1-甲氧基-5-甲基吩嗪鎓甲酯（1-MPMS）/异鲁米诺（IL）/微过氧化物酶（m-POD）体系测定NADH的化学发光法。为了提高该方法的灵敏度，将酶循环系统与NADH的化学发光检测相结合。使用乙醇脱氢酶和苹果酸脱氢酶作为循环酶。标准曲线在3×10⁻¹⁴至5×10⁻¹²mol/次检测范围内获得。NADH的检测限为30fmol/次检测，与使用细菌荧光素酶的生物发光法相当。已经开发了两种用于测定ATP的化学发光法。方法1是使用己糖激酶/G6PDH和1-PMS/IL/m-POD的体系，方法2是基于己糖激酶/丙酮酸激酶对ATP进行酶循环反应的体系。方法2比方法1灵敏1000倍。ATP的检测限为10fmol/次检测。
