Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran.
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.
Ishida
Akihiko
A
Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku, Kita-13, Nisihi-8, Sapporo, 060-8628, Japan. ishida@dove-mc.hokudai.ac.jp
Yoshikawa
Takahiro
T
Nakazawa
Takatoshi
T
Kamidate
Tamio
T
D000255:Adenosine Triphosphate / Q000032:analysis*; D000818:Animals; D001517:Coleoptera / Q000201:enzymology / Q000378:metabolism; D003637:DEAE-Dextran; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D017830:Octoxynol / Q000378:metabolism; D014238:Trichloroacetic Acid / Q000378:metabolism
D014238:Trichloroacetic Acid; D000255:Adenosine Triphosphate; D017830:Octoxynol; D003637:DEAE-Dextran; D008156:Luciferases