The present invention relates ~o a process and a reagent to be used in determining the ATP-concentration in, f~r exampl~, ATP-converting systems. More particularly the invention relates to a technique in which said system is brought into contact with a bioluminescence reagent includ-ing D-luciferin, luciferase and magnesium ions or certain other metal ions whereby a reaction takes place in which ATP and D-luciferin are bound to the luciferase and light is emitted and where the intensity of the e~itted light is measured, said intensity being a measure of the ATP-concentration.
According to the invention addit:ives to an Al'P dependent biolumine-scence reagent result in light emission which during the complete measuring time is in proportion to ATP-concent.ration. As the bioluminescence reagent itself consumes negligible amounts to ATP, samp].es with a constant ATP concen-tration will give rise to a constant light emission which facilitates the use of the reagent for ATP determination. Furthermore, a reagent wi~h the above cited properties can be added to other ATP converting systems providing a simple method for monitoring changes in the ATP concentration by means of a continuous measurement of the light intensity. ATP converting systems refers to combinations of enzymes and possibly a substrate which upon reaction give rise to a binding or consumption of ATP. The analytical use of the reagent consists in the dete~mination of ATP and substances and enzymes taking part in ATP converting reactions in the fields of clinical chemistry and clinical microbiology and in biochemical and biological research ~especially bioenergetics~.
In figures which illustrate embodiments of the invention~
Figure 1 is a schematically simplified and partly hypothetical representation of the sequence of luciferase catalyzed reactions, and Pigure 2 is a graph illustrating the effect of various embodi-ments of the invention on emitted light intensity over time.
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- ~ , ~ TP dependent bioluminecense reagents, where ATP is the commonly used abbreviatiorl fo~ adenosinetriphosphate, are known per se, said reagents being based on an enzyme, luciferase, and one of the two substrates, namely D-luciferin plus magnesium ions plus certairl other metal ions to which the other substrate, ATP, has to be complexed in order to react. When ATP is brought into contact with the reagent~ the reaction or rather th~ sequence Of luciferase catalyzed reactions which in a schematically simplified and partly hypothetical form are shown in Figure 1 will take place. The reac~
tions 1-3 have been studied in detail for luciferase from the common American firefly Photinus pyralis and these reactions represent the prior art.
Several survey articles have been published in recent years, see DeLuca, M.
(1976), "Advance in Enzymology"., CA. Meister.ed.) Vol. 44~ 37-68, John Wiley ~, Sons, New York and McElroy~ W.D., Seliger, H.H. and DeLuca, M.
(1974), "The Physiology of Insecta", 2nd ed (M. Rockstein, ed.~ Vol. 11, 411-460, Academic Press, New York.
In reaction 1 luciferase (E), ATP and D-luc;ferin ~LH2) react to form free pyrophosphate (PPi) and enzyme bound luciferyladenylat~. This reaction is not limiting for the rate of the complete reaction. The enzyme-luciferyladenylate complex obtained is subject to two processes limiting the speed of the initially emitted light, namely a conformation change and an abstraction of a proton from luciferyladenylate (see DeLuca, M. and McElroy, W.D., ~1974), Biochemistry, 13, 921-925). In reaction number 2, luciferyladenylate is oxidized with oxygen to produce AMP (adenosine mono-phosphate) and excited oxyluciferin ~P*), which both remain enzyme bound, and carbon dioxide. Oxyluciferin is in reaction 3 transfor~ed into its ground state while emitting a photon. The energy for emitting the pho~on has been obtained from the oxidation of luciferin and not fro~ splitting of the pyrophosph~te bound in ATP.
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The ~nzyme-oxyluciferin-AMP-comp]ex generated in reaction 3 is stable and can be isolated by gel filtration iE the reaction is performed in the presence of pyrophosphatase (Gates, B.J., and DeLuca, M. (1975~
Arch., Biochem, Biophys., 169, 616-621). tn the absence of pyrophosphatase an enzyme-oxyluciferin-complex is isolated without AMP, having the same activity as a free enzyme (see the last mentioned article of Gates and DeLuca).
As a result of the stability of the enzyme-oxyLuci~erin-AMP-complex, the mixtwre of luciferase, D-luciferin and ATP gives rise to a light flash rather than a constant light emission. The maximum light inten-sity will be reached within one second and thereafter declines to a steady-state level, whilst the regeneration of free enzyme is obtained with essentially the same speed as the light emission. The st~ady-state level is, however, not completely constant mainly because of the fact that the free products are inhibiting the reactions.
The determination R ATP has routinely been made in the follow-ing manner. The sa~ple containing an unknown ATP-concentration is mixed with the bioluminescent reagent which con~ains luciferase~ D-luciferin and magnesium ions. In order to ensure maximum reliability of the analysis the mixing should suitably take place with a given reaction rate and in a mea-suring position so that the initial parts of the light characteristics can be registered ~see Lundin, A. and Thore, A. (1975) Anal. Biochem., 66, 47-63~. -When the measure of the light intensity of the experimental sample has been registered, the analysis is repeated with a sample containing a known ATP-concentration and with a blank without ATP. It is from these three measure-mentS that the unknown ATP-concentration has been calculated. If the experimental sample has contained substances which could interfere with the analysis an internal standard technique has been used, i.e. the sample has - . , , . . - .
been analysed with Qnd without addition of a known ATP-concentration.
In 1952 it was shown that ATP dependent bioluminescence systems are available not only for determining the ATP, but also in principle for determining any substance which takes part in ATP converting reactions ~Strehler, B.L. and Totter, J.R. (1952) Arch. Biochem. Biophys. 40, 28-41).
The possibility of adding a bioluminescent reagent to an ATP conver~ing system in order to continuously follow the ATP-concentration by measuring the light intensity has, however, obiained very little practical signifi-cance. This is due partly to the fact that the actlvity of the luciferase 1~ during the reaction declines as a result of product inhibition as described above, and partly because of the fact that the luciferase reagents have been contaminated with ATP converting systems. Interest in khe method was, however, heightened when it became possible, by means of using purified luciferase and with a synthetic preparation of the luciferin, to obtain throughout a reaction time of several minutes a negligible decrease of emitted light intensity as well as of ATP-concentration (Lundin, A. and Thore A. ~1975) Anal. Biochem. 66, 47-63). Suitable conditions for the ana-lysis have been investigated and the method has proven useable for ATP con-centrations up to 10 M (Lundin, A., Rickardsson, A. and Thore, A., (1977) 2Q Anal. Biochem. 75, 611-620). However in spite of repeated experiments a reagent with the above cited properties could be prepared only in very few cases.
It has however been possible to shov the analytical use of a reagent with the above cited properties for kinetic determinations of substrates and enzymes, for end-point determination of substrates~ to moni-tor photophosphorylation and to follow lytic reactions ~Lundin, A., Rickardsson, A., and Thore, A. ~1~76), Anal. Biochem. 75, 611-620; Lundin A., Rickardsson, A., and Thore~ A. (1977)~ "Proceedings of ~he 2nd Bi-Annual ATP
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9~,~3 M~thodology Symposium, SAI Technology Company~ San Diego; Lundin, A., lhore, A.
and Baltscheffsky, M. ~1977), F~BS Lett. 79, 73-76; Lundin, A. (1~78), "Methods in Enzymology CM. DeLuca, ed.) Vol. 57, Academic Press, New York; Lundin, A.
and Baltscheffsky, M. ~1978) "Methods in Enzymology ~M. DeLuca, ed.) Vol. 57, Academic Press, New York; Lundin A., and Styrelius, I., (1978) Clin. Chem. Acta and Thore A., and Eriksson, A.C. (1977), FOA-report).
As a result of the difficulties in pr~paring in a reproducible way a reagent with the above cited properties, the above cited applications have not obtained any use outside the laboratory at which the technique was developed.
According to the present invention, there is provided a process for the determination of ATP-concentrations which comprises reacting the sample to be assayed with a bioluminescence reagent including D-luciferin, luciferase, metal ions, and one or more competitive inhibitors of the reaction in the form of D-luciferin analogs, whereby ATP and D-luciferin are bound to the luciferase and light is emitted, and measuring the intensity of the emitted light, which intensity is a measure of the ATP~concentration.
The present invention also provides a bioluminescence reagent for use in the determination of ATP-concentrations which reagent includes D-luciferin, lucierase, metal ions, and one or more competitive inhibitors in the form of D-luciferin analog~s~.
Before the use of the present invention the stability of the light level varied in different reagents from a decline of a few per cent ;
per minute to a decline to half the initial light intensity after one minute. According to the present invention it has, however, been shown that an addition of D-luciferin analogs make it possible to prepare in a reproducible way a reagent with the desired propertiesJ i.e. with a s~able light level. The effect o the D-luciferin analogs on the stability of the ;.~ : .
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light level has not previously been observed and it would be predicted, in analytical applications, to avoid said analogs as they have been shown to inhibit the light reaction competitively with the D-luciferin ~DeJIburg, J.L.) By a D-luciferin analog is meant with respect this invention, substances which inhibit the previously described lucif~rase reaction, this inhibition being competitive with respect to D-luciferin. The specific D~
luciferin analogs giving the desired result are easily found by adding them in inhibiting concentrations to the reagent and measuring the stability of the light level after addition of ATP.
Although the invention is not limited to any specific theory as concerns the reaction mechanics upon the addition of a D-luciferin analog, said addition could mean that a smaller part of the total luciferase present will exist as an inactive en7yme-product-complex. As the free enzyme concentration decreases upon formation of an enzyme-product-complex, the enzyme-luciferin analog complex is probably dissociated with correspond-ing formation of free enzyme as is shown in the reaction 5 in Figure 1 where I represents the D-luciferin analog. By using the D-luciferin analog9 i.e. the competitive inhibitor, the luciferase amount can be increased without any corresponding increase of the reaction rate. This is true irrespective of whether the D-luciferin analog reacts with ATP according to reaction 1 or is forming an enzyme inhibiting complex according to ~;~
reaction 5. The essential prerequisite is solely that the reactions be reversible and faster than reactions 2-4 ~reaction 4 will be discussed in detail below~.
To the extent that free AMP and free oxyluciferin are formed in the reaction it could also be of interest to study whether this would affect product inhibition. If for eample lQ 6 M ATP would lead to the generation `
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of 10 6 M AMP and 10 M oxyluciferin the inhibition of AMP would, as Ki for AMP is 2.4 x 10 M ~see Lee, R.T.) Denburg, J.L. and McElroy, W.D.
(1970) Arch. Biochem. Biohpys, 141, 38~52) affect ~he luciferase activity by less than 0.5 %. On the other hand the forming of oxyluciferin, the Ki value of which is 2.3 x 10 M ~Goto, T., Kuoota, 1., Suzuki, N. and Kishi,Y.
~1973) "Chemiluminescence and Bioluminescencc" (M.J. Cormier, D.M. Hercules, and J. Lee, eds.) pages 325-335, Plenum Press, New York) would decrease the luciferase activity significantly. Consequently it may undar certain con-ditions be important to counteract the effect of the generation of free 10 oxyluciferin. The addition of D-luciferin analog makes the initial inhibi-tion o the luciferase so great that the additional inhibition from the oxyluciferin, which is continuously formed during the reaction, will be negligible. The addition of inhibiting concentrations of a D-luciferin analog will thus stabilize the light level.
In the analysis the concentration of D-luciferin should be saturating i.e. so high that a small change of the concentration will not affect the reaction rate. This is essential as small changes in volume would otherwise affect the reaction rate. Furthermore, components of biological samples could affect the D-luciferin concentration available for the luciferase reaction and thus inhibit that reaction. A satisfactory accuracy of the analysis could thus only be achieved by using saturating concentrations of D-luciferin. By adding a D-luciferin-analog to D-luciferin an apparent saturation can be achieved at a low concentra~ion of D-luciferin without affecting the accuracy of the analysis in any way other than by a reduction of the sensitivity. This is an additional advantage of the inven-tion, since D-luciferin is an expensive substance which only is exceptional cases can be added in saturating concentrations.
The reduced luciferase activity o~tained by the addition of an -: . , : . . : ~ .: :
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analog can be compensated for by an increase of the luciferase concentration.
Thus, the luciferin-luciferase ratio can be optimized with respect to, for example, cost for reagent production, without affecting the sensitivity of the analysis. The impo~tance of the invention in this respect is easily realized from the fact that the world market for only one of the developed applications is about 5 millions analyses per year. Thus a decrease of the cost for the reagent by less than one cent per analysis will imply con-siderable reductions in cost which means that the present invention also in this respect involves a very essential contribution to the technique in the field.
In summary, it could thus be concluded that the use according to the invention of the luciferin-analog makes it possible to optimize the luciferin/luciferase ratio with respect to, for example, production cost of the reagent and also for increase in stability of the light level. A
suitable concentration of the D-luciferin analog is one which gives an in-hibition of the luciferase reaction, and thus of light intensity, by at least 25 % since at a lower degree o~ inhibition, the effect on the stability of the light level and the required concentration of the D-luciferin ~or saturation will be too small to be of economical analytical importance. A
specifically preferred range is 50-90 %. An inhibition of the intensity of more than 90 % makes among other things the demands on the light regis-tration equipment unnecessarily large and also makes it necessary to increase the amount of the luciferase so much that the analysis will be un-economical. ~urthermore, an analytical interference can easily be obtained when using high amounts of luciferase, In accordance ~ith a preferred embodiment of the invention the previously mentioned competitive inhibitor is added to the reagent together with pyrophosphate. It is kno~n per se that concentrations of pyrophosphate " ~ , sufficient to inhibit thc lwciierase reaction counteract the decline of the light intensity ~IcElroy, W.D., Hastings, J.W., Coulombre, J. and Sonnenfeld, V. ~1953~, Arch. Biochem. Biophys. 46 399-416). This has, however, never previously been used to improve the assay conditions in the determination of ATP.
According to the present invention it has surprisingly appeared that a combined use of pyrophosphate in a much lower concentration than it has been used before, preferably at most in a concentration of la 4 M and specifically not more than 10 M, along wil:h a competitive inhibitor in the concentration defined above results in the obtalning of a very stable light-level. Through this combination the same stability of light~level can be achieved as would require a considerably higher degree of inhibition of the luciferase to achieve by use of either pyrophosphate or D-luciferin analog independently~ At a competitive inhibition with a D-luciferin analog of around 5Q % and a concentration of pyrophosphate of 10 6 M it has been possible to achieve a decline of the light intensity curve which is below 3 percent and at an inhibition of around 75 % and a concentration of pyro-phosphate of 10 6 M it has been possible to achieve decline of the light intensity curve in the order of 1 %. Such a stable light intensity will of course imply that much less expensive and simpler light registration equip-ment can be used and above all it gives very good prerequisites for continu-ous analysis of ATP converting reactions.
Although the reaction in this respect is not limited in any specific theory concerning the reaction mechanics it is possible that pyro- ;
phosphate reacts with AMP in the enzyme-oxyluciferin-AMP complex according to reaction 4 of Figure 1. The formation of ATP from pyrophosphate and AMP
requires energy. This energy is probably obtained from the transformation of the enzyme back to i.ts original conformation from the conformation which _ 9 _ . .
was obtained before ~he oxidation reaction (reaction 2 of Pigure 1~. The conformation change would explain why product inhibition is non-competitive tLemasters, J.J. and Hacknebrock, C.R. ~1977) Biochemistry 16, 44s-447 whereas oxyluciferi~ is a competitive inhibitor (see the above cited application by Goto et al., 1973). The reaotion with pyrophosphate would thus change a strong non-competitive inhibition (see the above cited publi cation by Goto et al., 1973) to a weaker compctitive inhibition. The effect of tle competitive inhibition on the light level could according to the invention be counteracted by means of adding a D-luciferin-analog tsee above). According to reaction 4 of Pigure 1 the luciferase reaction in the presence of pyrophosphate does not give any net consumption of ATP.
This would contribute to the stabilisation of the light level.
In addition to taking part in splitting enzyme from the enzyme-oxyluciferin- AMP-complex, pyrophosphate pressnt in inhibiting concentra-tions could cont~ibute by driving the reaction 1 of Figure 1 backwards.
By adding pyrophosphate the total enzyme concentration could thus be in-creased without a corresponding increase of the light intensity. A smaller part of the total enzyme concentration will therebybe present as an inactive enzyme-product-complex. Inhibiting pyrophosphate concentrations would thus contribute to the stabilisation of the light level.
According to another preferred embodiment of the invention, co-enzyme A is added to the bioluminescent reagent either in combination with the competitive inhibitor only or in combination ~ith the competitive in-hibitor and pyrophosphate. The addition of co-enzyme A has also been shown to stabilize the light-level. The fact that one could obtain an increase of the light intensity by adding only co-enzyme A is known per se as for example by the publication of Airth, R.L., Rhodes, W.C. and McElroy, ~ D.
~1~58), Biochem. Biophys. Acta, 27 519-~ but the use of co~enzyme A for -- 10 -- ., . .. , ;.
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improving the analysis conditions when det~rmining Al'P has not previously been sugg~sted, The reaction mechanics for co-enzyme A is thus probably that the compound reacts with oxyluciferin in the enzylne-oxyluciferin-AMP-complex according to reaction 4 in Figure 1 ~where co-enzyme A is denoted CoA~.
The reaction between co-enzyme A and oxyluciferin changes the non-competitive product inhibition of the ~nzyme-oxyluciferin -AMP-complex to a weak com-petitive inhibition by AMP, which is negligible under normal analytical conditions.
Thus, co-cnzyme A will perform its effect without inhibiting the luciferase reaction. This is in contrast to D-luciferin analogs and in certain cases also to pyrophosphate. The inhibition obtained with D-luciferin analo~s and pyrophosphate will however in many analytical applica-tions not be of importance since generally the sensitivity of the luciferase reactiOn will be satisfactory even after the inhibition. If this is not the case the concentration of the luciferase may be increased provided that the luciferase preparation does not contain too high proportions of contaminants which interfere with the analysis. In certain applications of the present invention it could therefore be of special interest to use a highly purified luciferase preparation. Different methods for purifying luciferase are known per se and anyone of these methods could be used in most cases. A pre-ferred embodiment of the inv0ntion, when the demands for purification are very high, would be the use of a reagent which contains luciferase purified by means of isoelectric focusing. Luciferase could furthermore be protected from unspecific activation by means of chosing the correct reaction conditions and through addition of protecting substances such as bovine serum albuminJ
thiol compounds and/or EDTA ~ethylenediamine tetraacetic acid~.
The use o~ pyrophosphate and co-enzyme A may be limited by ~he .
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' . : ,. . . ' . ': ' existence in certain biological samples o~ enzyme systems which degrade these substances. In these cases the effect o such enzymes could be prevented hy adding an inhibitor which does not affect the luciferase ,ql ~anq~h ~ s reaction. For example pyrophosphatase activity could be inhibited by m&~
g~n or fluoride ions. Considering the synthetic nature of the luciferin analogs these would presumably not be subject to an enzymatic degradation in biological samples.
When producing a bioluminescence reagent with the desired pro-perties, the choice of substances or the combination of substances within the group of D-luciferin-analogs, pyrophosphate and co-enzyme A has to be determined by the application. This is due to the fact that the demands of different applications vary with respect to sensitivity, sample composition, existing interfering reactions, the price level of the reagent, storing stability etc.. Considering that the present invention has made it possible to make the choice ~rom within a defined group of substances, it would not be difficult for the man skilled in the art to find a suitable reagent composition for each single application.
In addition to the above mentioned advantages and the application of the present invention it may be added that continuous measurment of ATP
converting reactions with the improved bioluminescence reagent according to the invention has a sensitivity which normally is several powers of ten higher than the corresponding spectrophotometric method. The analytical procedure is however very similar to the procedure for coupled spectrophoto-metrical analysis based on e.g. NAD /NADH-conversion. Also ~hen determining ATP in non-ATP-converting systems, i.e. in samples with a constant ATP-con-centration, a reagent with the above cited properties has obvious advantages.
Since the light is constant no demands are put on the velocity of mixing reagent and samples. The mixing does not have to take place in the measuring . , .
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., , ~ . ~ , position and the light measurement may continue durirlg any desired period of time. Thereby the sensitivity as well as the reproducibility will be in-creased. Also when determining ATP in cellular systcms the reagent according to the invention is advantageous since it makes it possible to measure the concentration of extracellular and intracel:lular ATP in the same sample. The concentration of extracellular ATP is first measured whereafter some lytic reagent, which does not affect the luciferase system, is added and the light increase corresponding to the concentration of intracellular ATP is measured.
Although the various components of the bioluminescent reagent according to the invention have for simplicity's sake been described as part of the reagent, it is of course possible to add them separately. Thus, one Or several of the components can be added together with the buffer required for achieving the desired pH value.
An embodiment of the invention will now be further described by means of the following non-limiting example.
This example, which refers to Figure 2, illustrates how D-luci-ferin analogs (in this case L-luciferin) and pyrophosphate could be used together so that a reagent producing a stable light level is achieved having a reason-able degree of inhibition. The luciferase used in th0 example has been puri-fied by means of isoelectric focusing. In Figure 2 the light intensity is shown as a function of time after adding a final concentration of 10 6 M ATP
to the reaction mixture. In all cases the reaction mixture ~final volum~ 1 ml~ consists of luciferase, D-luciferin ~100/ug/ml), magnesiumacetate (lOmM), bovine serum albumin ~Q.l %), EDTA C2mM), and 0.1 M tris (hydroxymethyl~
aminomethane buffer adjusted to pH 7.75 by using acetic acid.
Figure 2 A shows the light curve obtained without any further additives. A reagent with a declining level according to Figure 2 A is not , :
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sui~able for continuous measurement on ATP-conver~ing systems. Figure 2B shows the effect of the addition of L-luciferin ~lO pg/ml) resulting in an inhibition of about 70%. The decline of the curve is accepkable but the initial peakmakes the reagent unacceptable at high ATP-concentrations for continuous measurements in ATP~convcrting systems. When using higher concentrations of the additive and thus a higher d~gree of inhibition one will however obtain straight light curves and suitable reagents. In ~igure 2C there is shown the effect of pyrophosphate (10 6 M). The decline o the light curve is still too large and there is a small initial peak. At higher and more inhibiting concentrations of the additive the light curve will be straighter. ~igure 2D shous the effect of L-luciferin ~10 ~g/ml~
and pyrophosphate ClQ ~). The decline as well as the initial peak has in ~his case been almost completely eliminated. This reagent is extremely suitable for analytical purposes. Only the reagents which contain L-luciferin (2B and 2D) apparent saturated with respect to luciferin (D+L, see above) will consequently give the maximum analytical accuracy.
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