Symposium 1997: Abstract for A Lundin

Symposium 1997: Abstracts

Kinetic Properties of ATP Reagents as Influenced by Luciferase Level

Arne Lundin
BioThema, Strandvägen 36, S-130 54 Dalarö, Sweden

The luminometric ATP assay has a broad range of applications and a single ATP reagent cannot be not ideally suited for all purposes. With stable light emitting reagents the sample and the reagent may be mixed outside the luminometer and measured for any convenient period of time. Stable light emission requires that the luciferase activity is constant throughout the measurement, i. e. affected neither by product inhibition nor by inactivation by ATP extractants. If the luciferase level is too high the percentage of ATP consumed per minute will be significant resulting in a decay of the light. The peak light and the sensitivity are proportional to the luciferase activity. The present paper clarifies the kinetics of ATP reagents at different luciferase levels and should be useful in selecting reagents for various applications.

We have developed a range of three ATP reagents with increasing luciferase levels. All three reagents have the same optimised concentration of highly purified D-luciferin. They also contain stabilisers to give a constant ratio between light intensity and ATP concentration throughout the measurement. At low ATP concentrations the luciferase reaction follows first-order kinetics, i. e. (d[ATP] /dt) / [ATP] =k (the unit is min^-1 or %/min). Under the conditions used the rate constant k equals the decay rate of the light. This rate can be calculated from any two light intensity values, I₁ and I₂ measured at times t₁ and t₂, as k=(ln I₁ -ln I₂)/(t₂ - t₁). The k value is independent of ATP concentration and is a useful measure of luciferase activity. The peak light emission, I₀ , can be extrapolated from k and any light intensity value, I_t , as I₀= I_t /e ^-kt. The total integrated light, I_tot , emitted from peak to extinction can be calculated as I_tot= I₀/k. While I₀ is affected by various inhibitors and activators, I_tot is only affected by the quantum yield of the luciferase reaction. Thus I_tot is a valuable new parameter in ATP assays.

Analytical properties and application areas for the three ATP reagents are described in Survey Update 5 [Stanley PE, J Biolumin Chemilumin 12 (2)]. The k values are 0.5, 10 and 235 %/min. Thus with the same amount of ATP the peak light emissions are related as 1:20:470. Only with the most active reagent it is necessary to use automatic luminometers with reagent dispensers. The luciferase reaction consumes 90% of the ATP in 460, 23 and 1 min, resp.. Thus the two most active reagents rapidly consume contaminating ATP to give reagent blanks that are essentially zero. The three reagents have detection limits from 10^-15 to 10^-18 mol. The amount of ATP in a normal bacterial cell is around 2x10^-18 mol. Thus ATP technology can now be used for sterility testing.