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 kinases¹, 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.

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.

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.

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.

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.

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.

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.