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On the use of luciferase to measure ATP Print E-mail

On the use of luciferase to measure ATP.
(extracted from the letter of Dr. Chinopoulos to MITOCHONDRIA newslist, with insignificant modifications).

“The luciferase assay is NOT useless, but …”

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     The luminometric method for detecting chemiluminescence upon ATP-dependent oxidation of luciferin, catalyzed by firefly luciferase is primarily employed in a form of an end-point assay. The biological material had to be lysed and the ATP released (there are major issues with that, as discussed later). This approach can in principle be used for a continuous on-line assaying of ATP [1], although with several drawbacks, such as:

  • • The lack of constant proportionality between ATP concentration and luminescence, caused by product inhibition of the luciferase reaction by oxyluciferin renders this method at best semi-quantitative.
  • • For reliable estimation of the kinetics of ATP formation, the endogenous ATP concentration prior to the assay has to be determined.
  • • The use of a low ionic strength medium and room temperature are required by the method, which restricts experiments to non-physiological conditions.
  • • The necessity to maintain [O2] below 50 micromolar in order to avoid significant drifts in luminescence, adds extra effort to any application of this method. Nevertheless, some of the disadvantages of this method have been overcome by various modifications [2].

    Problems with measuring ATP from lysates.

    First, it is possible that results obtained after lysis of the biological material are not relevant because of the time used for the separation process and possible inter-conversions of adenine nucleotides even in the presence of inhibitors [3-6].

    Second, a great part of adenine nucleotides is bound to proteins [7], a notion supported by the fact that tissues (in this ref isolated mitochondria) retain more than 50% of their total adenine nucleotide content after permeabilization by toluene [8]. Because of this potential binding of adenine nucleotides to intramitochondrial proteins [9-12] the relationship between the measured total ATP/ADP ratio to free intramitochondrial ATP/ADP ratio is difficult, if not impossible to estimate.

   One additional problem. You may want to measure ADP as well in your lysates, and a common way to do this is by converting it to ATP by pyruvate kinase and phosphoenolpyruvate (PEP). PEP is almost invariably contaminated with ATP and ADP, so that brings us back to the problem of not knowing the initial endogenous ATP (and ADP) concentration. Conclusion: The luciferase assay is NOT useless, but please consider all of the above in the interpretation of your results, should you choose to employ it.

Reference List

1. Lemasters, J. J. and Hackenbrock, C. R. (1979) Methods Enzymol. 56, 530-544
2. Wibom, R., Lundin, A., and Hultman, E. (1990) Scand. J. Clin. Lab Invest 50, 143-152
3. Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66-79
4. Brawand, F., Folly, G., and Walter, P. (1980) Biochim. Biophys. Acta 590, 285-289
5. Letko, G., Kuster, U., Duszynski, J., and Kunz, W. (1980) Biochim. Biophys. Acta 593, 196-203
6. Davis, E. J. and Lumeng, L. (1974) FEBS Lett. 48, 250-252
7. Lusty, C. J. (1978) Eur. J. Biochem. 85, 373-383
8. Matlib, M. A., Shannon, W. A., Jr., and Srere, P. A. (1977) Arch. Biochem. Biophys. 178, 396-407
9. Boyer, P. D. (2001) Biochemistry (Mosc. ) 66, 1058-1066
10. Senior, A. E., Nadanaciva, S., and Weber, J. (2000) J. Exp. Biol. 203, 35-40
11. Jault, J. M. and Allison, W. S. (1994) J. Biol. Chem. 269, 319-325
12. Harris, D. A., Rosing, J., van de Stadt, R. J., and Slater, E. C. (1973) Biochim. Biophys. Acta 314, 149-153

Last Updated ( Wednesday, 09 March 2011 )
 

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