Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran |
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Authors: | Ishida Akihiko Yoshikawa Takahiro Nakazawa Takatoshi Kamidate Tamio |
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Institution: | 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 |
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Abstract: | 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. |
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Keywords: | firefly bioluminescence ATP DEAE-dextran ATP extractant Escherichia coli |
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