Chemiluminescent assay of co-factors

✍ Scribed by Tsuji, Akio ;Maeda, Masako ;Arakawa, Hidetoshi

• Publisher: John Wiley and Sons
• Year: 1989
• Weight: 486 KB
• Volume: 4
• Category: Article
• ISSN: 0884-3996
• DOI: 10.1002/bio.1170040160

✦ Synopsis

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 I-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(lL)/microperoxidase(m-POD) system.

In order t o 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 to 5 x 10-12mol/assay. The detection limit of NADH was 30fmol/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 hexokinaseIG6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinasdpyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10fmoVassay.