Every day we find new applications for the technology that is the utilization of firefly reaction

The activity of all ATP dependent enzymes, whether they form or degrade ATP, can be continuously monitored with the luminescent reaction of firefly luciferase. The light emission is directly proportional to the ATP concentration in the reaction mixture. This circumstance makes it possible to investigate inhibitors of these enzymes as well as their concentrations and their substrates, either kinetically or with endpoint assays.

When continuously monitoring ATP concentration as a function of enzyme activity, the ATP depletion related to luciferase should be negligible compared to that of the enzymatic reactions being monitored. It is possible to follow reactions with ATP concentrations ranging from 1 to 1,000,000 pmol/L. Furthermore, kinetic ATP depletion assays are best set up as 1st order reactions, plotting the natural logarithm of the measured light versus measurement time. The slope in these plots is the rate constant of the reaction. ATP forming reactions can be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test.

Examples of assays of enzymes:
protein kinases,¹ adenylate kinase², pyruvate kinase³, pyruvate phosphate dikinase, glycerol kinase^{4, 5}, creatine kinase isoenzymes ^{6, 7, 8, 9}, ATPases², ATP synthases¹⁰, aminoacyl tRNA synthetase.

Examples of assays of metabolites:
ADP, AMP, pyrophosphate.

1. Hoffner, S., Jimenez-Misas, C. and Lundin, A. (1999) Improved extraction and assay of mycobacterial ATP for rapid drug susceptibility testing. Luminescence 14, 255-261.

2. Lundin, A., Karnell Lundin, U. and Baltscheffsky, M. (1979) Adenylate kinase activity associated with coupling factor ATPase in Rhodospirillum rubrum. Acta Chem. Scand. B33, 608-609.

3. Lundin, A., Rickardsson, A. and Thore, A. (1977) Substrate and enzyme determinations by continuously monitoring the ATP level by a purified luciferase reagent. Proceedings: 2nd Bi-annual ATP Methodology Symposium, March 22-24, 1977 (G. Borun, Ed.), pp. 205-218, SAI Technology Company, A Division of Science Applications, Inc., San Diego, Cal.

4. Lundin, A., Arner, P. and Hellmér, J. (1989) A new linear plot for standard curves in kinetic substrate assays extended above the Michaelis-Menten constant: Application to a luminometric assay of glycerol. Anal. Biochem. 177, 125-131.

5. Hellmér, J., Arner, P. and Lundin, A. (1989) Automatic luminometric kinetic assay of glycerol for lipolysis studies. Anal. Biochem. 177, 132-137.

6. Lundin, A. and Styrélius, I. (1978) Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase. Clin. Chim. Acta 87, 199-209.

7. Lundin, A. (1978) Determination of creatine kinase isoenzymes in human serum by an immunological method using purified firefly luciferase. Methods in Enzymology, 57, 56-65.

8. Lindberg, K., Lundin, A., Nordlander, R., Nyquist, O and Styrélius, I. (1980) Detection of acute myocardial infarction by a new sensitive and rapid method for determination of creatine kinase B-subunit activity. Eur. Heart J. 1, 327.333.

9. Lundin, A. Jäderlund, B. and Lövgren, T. (1982) Optimized bioluminescence assay of creatine kinase and creatine kinase B-subunit activity. Clin. Chem. 28, 609-614.

10. Lundin, A., Karnell Lundin, U. and Baltscheffsky, M. (1979) Adenylate kinase activity associated with coupling factor ATPase in Rhodospirillum rubrum. Acta Chem. Scand. B33, 608-609.

11. Wibom, R., Lundin, A. and Hultman, E. (1990) A sensitive method for measuring ATP-formation in rat muscle mitochondria. Scand. J. Clin. Invest 50, 143-152.

Extracellular ATP can be continuously monitored simply by adding ATP Reagent SL (Stable Light) to cells undergoing lysis. The synthesis of ATP by isolated organelles like mitochondria, chloroplasts or chromatophores can be monitored in the same way. This methodology can be used in e.g diagnosis of inborn metabolic errors in mitochondrial ATP production, studies on photophosphorylation in chloroplasts and chromatophores, Studies on ATP release from cells, pyrosequencing etc.
Extracellular ATP can be continuously monitored in cell lysis, stimulated release of ATP, platelet aggregation, oxidative- and photophosphorylation. This monitoring can be performed while cells and organelles can remain in the medium during the measurement. The wide linear range from 10^-12 to 10^-6 mol/L provided excellent assay flexibility.

1. Wibom, R., Lundin, A., Hultman, E. (1990) A sensitive method for measuring ATP-formation in rat muscle mitochondria. Scand J Clin Lab Invest 50(2), 143-152.

2. Lundin A., Wibom, R., Söderlund, K., Hultman, E. (1991) Luminometric ATP monitoring of oxidative phosphorylation and energy metabolites in needle biopsies from skeletal muscle. In: Stanley P, Kricka L (eds) Bioluminescence and chemiluminescence: current status. Wiley, Chichester, pp 413-416.

3. Wibom, R., Hagenfeldt, L., von Döbeln, U. (2002) Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples. Anal. Biochem. 311,139-151.

4. Wibom, R., Hagenfeldt, L., von Döbeln, U. (2003) Erratum to "Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples". Anal. Biochem. 317, 288.

5. Lundin A, Thore A, Baltscheffsky M (1977) Sensitive measurement of flash induced photophosphorylation in bacterial chromatophores by firefly luciferase. FEBS Lett 79(1), 73-76.

6. Lundin A, Baltscheffsky M (1978) Measurement of photophosphorylation and ATPase using purified firefly luciferase. Methods in Enzymology 57, 50-56.

7. Lundin, A., Baltscheffsky, M., Höijer, B. (1979) Continuous monitoring of ATP in photophosphorylating systems by firefly luciferase. In: Schram E, Stanley P (eds) Proceedings: international symposium on analytical applications of bioluminescence and chemiluminescence. State Printing & Publishing, Inc, Westlake Village, Cal, pp 339-349.

8. Baltscheffsky, M., Lundin, A. (1979) Flash-induced increase of ATPase activity in Rhodospirillum rubrum chromatophores. In: Mukohaka Y, Packer L (eds) Cation flux across membranes. Academic Press, New York, pp 209-218.

9. Baltscheffsky, M., Lundin, A. (1982) Photophosphorylation after single turnover light flashes in chromatophores from Rhodospirillum rubrum. In: Kaplan N (ed) From cyclotrons to cytochromes. Academic Press, New York, pp 347-354

10. Lundin, A., Hasenson, M., Persson, J. and Pousette, Å. (1986) Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods in Enzymology 133, 27-42.

11. Kowal, J., Yegutkin, G. and Novak, I. (2015) ATP release, generation and hydrolysis in exocrine pancreatic duct cells. Purinergic Signalling 11, 533-550.

12. Orriss, I., Knight, G., Utting, J., Taylor, S., Burnstock, G., Arnett, T. (2009) Hypoxia stimulates vesicular ATP release from rat osteoblasts J. Cell. Physiol. 220, 155-162.

13. Sathanoori, R., Swärd, K., Olde, B., Erlinge, D. (2015) The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells. PLoS ONE 10(5), e0125111

14. Bagoly, Z., Sarkady, F. , Magyar, T. , Kappelmayer, J. , Pongrácz, E , Csiba, L. , Muszbek, L. (2013) Comparison of a New P2Y12 Receptor Specific Platelet Aggregation Test with Other Laboratory Methods in Stroke Patients on Clopidogrel Monotherapy. PLoS ONE 8(7), e69417.

15. Nordström, T., Ronaghi, M., Forsberg, L., de Faire, U., Morgenstern, R.. and Nyrén (2000) Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. Biotechnol. Appl. Biochem. 31, 107-112.

Enzymatic ATP depletion assays can be continuously monitored with Kinase RR Kit. The assays are set up as 1st order reactions and with an ATP concentration well below the Km (Michaelis-Menten constant) values of both luciferase and the reaction being studied. The logarithm of the light emission, i.e remaining ATP, versus time will then be a straight line. This can be used for HTS of compound libraries to find inhibitors of e.g. protein kinases measuring remaining ATP by the luciferase reaction.

The Kinase RR (Reaction Rate) kit can be used to monitor ATP depletion reactions by e.g., protein kinases, ATPases and aminoacyl tRNA transferases. The Kinase RR kit has the following features:

• No interference from luciferase inhibiting compounds or variations in ATP concentration
• All library compounds can be classified without follow-up testing.
• Very low ATP depletion by the luciferase reaction (<0.06 % per minute corresponding to t1/2=14 h).
• Microplates (96 or 384 wells) can be used.
• Z’ values as high as 0.96 have been obtained.

1. Lundin and Eriksson (2008) A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition, Assay Drug Dev Technol 6 (4), 531-541.

2. Auld, D. S. et al. (2008) Characterization of chemical libraries for luciferases inhibitory activity. J. Med. Chem. 51, 2372-2386.

3. Saint-Léger,A. and de Pouplana, L. (2017) A new set of assays for the discovery of aminoacyl-tRNA synthetase inhibitors. Methods 113, 34–45.

Biological residues contain the molecules ATP, ADP and AMP. When the cells in these residues die, their ATP is degraded to ADP, and finally to AMP. Measuring only ATP… can result in not detecting severe contamination. In contrast, measuring ATP+ADP+AMP (called A3) provides an estimate of the level of biological residues present. High levels of these residues indicate insufficient cleaning and may become a source of nutrients from which bacteria can rapidly grow to dangerous levels. Even if a surface is sterile after cleaning/disinfection, airborne bacteria may infect the biological residues. Additionally, these residues may contain allergens, which may cross-contaminate non-allergenic food stuff if processed in/on the same production line.

For portable estimation of biological residues, BioThema recommends using the unique ATP+ADP+AMP (A3) single use Kikkoman swab - LuciPac A3 (surfaces) or LuciPac A3 Water (liquids). These swabs are used in combination with the portable instrument - Lumitester Smart. Contrastingly, in the case of processed and sealed off food stuff, live bacteria, or microbes within these packages, are the main concern. Please read more about this application under “Quantification of bacteria”.

A3 is a measure of biological residues, not bacteria. Biological materials, e.g. various foods, contain different levels of A3 per gram. Consequently, the same A3 level from two different biological materials does not necessarily mean the same degree of contamination in terms of biological residue weight. However, in the case of contamination testing in production of a certain product, one can determine the amount of A3 per gram of said product*. It is then possible to determine the amount of biological residues on a surface or in a liquid in grams per dm2 or mL as the sample area/volume is known. Bacterial cells are much smaller than plant or animal cells. Consequently, the bacterial contribution to the total ATP or A3 in e.g. food is generally negligible. The same methodology can be used in verification of cleaning procedures and cleaning equipment. In this case, A3 per gram/mL is determined for a specific contaminant used when testing cleaning efficacy.

1. Bakke and Suzuki (2018) Journal of Food Protection, 81, 729-737

Rapid and sensitive quantification of bacteria has many applications, such as diagnosing urinary tract infection, QC of packed food stuff, antibiotic susceptibility testing etc. All living cells contain ATP (adenosine triphosphate) in a similar concentration, and bacteria are no exceptions. Because animal and plant cells are much larger than bacteria (about 100 to 10 000 times), ATP not originating from bacteria must either be removed or negligible in the application of interest. By selectively lysing animal and plant cells and simultaneously degrade their ATP prior to lysing the bacteria, it is possible to differentiate between bacterial ATP and other sources of ATP.

Laboratories equipped with either a luminometer or a plate reader with luminescence mode, have the possibility to perform bacteria quantifications with our kits. These assays can be calibrated by adding a known amount of certified liquid-stable ATP standard at the end of every measurement, either with automatic injectors or manually. As previously mentioned, all living cells contain ATP (adenosine triphosphate) but also its degradation products ADP (adenosine diphosphate) and AMP (adenosine monophosphate). However, in healthy cells, only a few percent of the total adenosine nucleotides are made up of ADP and AMP. ATP is formed in energy yielding processes like oxidative phosphorylation and depleted in energy requiring processes like synthesis of new intracellular components. Consequently, the intracellular ATP concentration must be regulated within narrow limits. Therefore, the amount of total intracellular ATP can be used as an estimate of the total intracellular bacterial volume of a sample. If you know the amount of intracellular bacterial ATP per cell and there is only one type of cells in the sample, you can calculate the number of cells in the sample. The bacterial species is sometimes known beforehand but can otherwise be determined with PCR or by conventional cultivation. The ATP per bacteria has been determined for many species in scientific publications. Although growth conditions will somewhat affect the ATP per cell, it is possible to make an estimate of the number of bacteria one has in a sample.

1. Lundin, A. (1982) Analytical applications of bioluminescence: The firefly system. In Clinical and Biochemical Luminescence (L. Kricka and T. Carter, Eds.), pp. 43-74, Marcel Dekker, Inc., New York.

2. Koutny et al. (2006) Acquired biodegradability of polyethylenes containing pro-oxidant additives. Polymer Degradation and Stability 91, 1495-1503

3. Ludwicka, A., Switalski, L., Lundin, A., Pulverer, G. and Wadström, T. (1985) Bioluminescent assay for measurement of bacterial attachment to polyethylene. J. Microbiol. Meth. 4, 169-177.

4. Hunter, D. and Lim, D. (2010) Rapid detection and identification of bacterial pathogens by using an ATP bioluminescence immunoassay. Journal of Food Protection, 73 (4), 739-746.

5. Lee, J., and Deininger, R. (2004) Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence. Luminescence 19, 31-36.

6. Hammes et al. (2010) Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments. Water Research 44, 3915-3923.

7. Pedersen, K. (2013) Metabolic activity of subterranean microbial communities in deep granitic groundwater supplemented with methane and h6. The ISME Journal 7, 839-849

8. Jensen, S., Hubrechts, P., Klein, B., Hasløv, K. (2008) Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine. Biologicals 36, 308-314.

9. Ho et al. (2008) Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine. Vaccine 26, 4754-4757

10. Lundin, A., Hallander, H., Kallner, A., Karnell Lundin, U., Österberg, E. (1984) Luminometric detection of bacteriuria in primary health care. In Analytical Applications of Bioluminescence and Chemiluminescence (L.Kricka, P. Stanley, G. Thorpe and T. Whitehead, Eds.), p. 71, Academic Press, New York.

11. Hallander, H., Kallner, A., Lundin, A. and Österberg, E. (1986) Evaluation of rapid methods for the detection of bacteriuria (screening) in primary health care. Acta Path. Microbiol. Immunol. Scand. Sect. B, 94, 39-49.

12. Gästrin, B., Gustafsson, R. and Lundin, A. (1989) Evaluation of a bioluminescence assay for detection of bacteriuria. Scand. J. Infect. Dis. 21, 409-414.

All living cells contain ATP, where it plays the role of energy currency between different cellular processes. When cells die due to induced with stress from e.g a cytotoxic substance or through apoptosis, ATP is normally degraded, and the cell viability can therefore be monitored. Cell proliferation, as a function of e.g good growth conditions or issues in regulation of apoptosis (which can lead to cancer), can also be monitored. Determination of intracellular ATP can also be used to estimate biomass. If the amount of ATP per cell is known, the cell number may also be estimated.

The intracellular ATP concentration is similar in different cell types. Bacterial cells contain 1-2 attomoles per cell while animal and plant cells contain 100 to 10 000 times as much ATP. Therefore, ATP from bacteria is most often negligible when measuring animal or plant cells but not the other way around. Extracellular ATP may be degraded by adding ATP consuming enzymes, such as ATP Eliminating Reagent, or by physical separation, e.g. centrifugation. All ATP assays should be calibrated by measuring the light before and after adding a known amount of ATP standard at the end of each test. This calibration obviates analytical interference from all sorts of variation of reagents and sample matrices and provides a result expressed in moles rather relative light units.

1. Nilsson, H. et al. (2015) Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate. Cell Death and Disease (2015) 6, e1585

It is often difficult or impossible to directly measure the activity of the regulatory sequence of a gene of interest. The solution is to attach a reporter gene coding for an easily measured substance to the regulatory sequence. The luc gene coding for firefly luciferase may be attached to the gene of interest. The expression of this gene may then be estimated from the level of luciferase expression measured as light. The luc gene coding for firefly luciferase is an ideal reporter as it is very easy to measure luciferase in extremely low amounts simply by measuring the emitted light.

There are two ways to measure:

• Lyse the cells and add optimal levels of D-luciferin and ATP. Use the BioThema Luciferase Assay Kit with a detection limit of 10^-19 mol luciferase, a dynamic range of 5 orders of magnitude and a decay rate of light from 1 to 4 percent per minute (t_1/2 70 respective 17 minutes) depending on the stability of the luciferase used.
• Add D-luciferin directly to the cells and D-luciferin will then interact with intracellular ATP and any luciferase expressed. Use the BioThema D-luciferin (free acid, sodium or potassium salt). In a test among 8 manufacturers the BioThema D-luciferin gave the highest activity.