Enhanced Firefly Bioluminescence Assay of ATP in the Presence of ATP Extractants by Using Diethylaminoethyl-Dextran

✍ Scribed by Akihiko Ishida; Takahiro Yoshikawa; Takatoshi Nakazawa; Tamio Kamidate

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 69 KB
• Volume: 305
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5680

✦ Synopsis

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13-to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml ؊1 of E. coli.