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The present invention refers to a method and a reagent to be used in
determining the ATP-concentration in e.g. ATP-converting systems. More
particulaly the invention refers to a technique in which said system is
brought into contact with a bioluminiscence reagent based o 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 be
luciferase and light is emitted and where the intensity of the emitted
light is measured, said intensity being a measure of the ATP
concentration.
According to the invention additives to an ATP dependent bioluminiscence
reagent result in a light emission which during the complete measuring
time has the same proportionality factor to the ATP concentration. As the
bioluminiscence reagent itself consumes negligible amounts of ATP, samples
with a constant ATP concentration will give rise to a constant light
emission which facilitates the use of the reagent for ATP determination.
Furthermore, a reagent with the above cited properties can be added to
other ATP converting systems so as to in a simple manner make it possible
to monitor changes in the ATP concentration by means of a continuous
measurement of the light intensity. ATP converting systems could e.g. be
combinations of enzymes and possible a substrate which at a reaction give
rise to a binding or consumption of ATP. The analytical use of the reagent
comprises determining of ATP and substances and enzymes taking part in ATP
converting reactions within clinical chemistry and clinical microbiology
and within biochemical and biological research (especially bioenergetics).
ATP dependent bioluminicense reagents, where ATP is the commonly used
abbreviation for adenosinetriphosphate, are known per se, said reagents
being based on an enzyme, luciferase, and one of the two substrates,
namely D-luciferin and magnesium ions and certain other metal ions to
which the other substrate, ATP, has to be complex bound in order to react.
When ATP is brought in contact with the reagent, the reaction or rather
the sequence of luciferase catalyzed reactons which in a schematically
simplified and partly hypothetical form are shown in FIG. 1 will take
place. The reactions 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"., (A. 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 there is formed from luciferase (E), ATP and D-luciferin
(LH.sub.2), free pyrophosphate (PPi) and enzyme bound luciferyladenylate.
This reaction is not limiting for the rate of the complete reaction. The
enzyme-luciferyladenylate complex obtained is subect 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 Mc Elroy, W. D., (1974), Biochemistry, 13, 921-925). In reaction
number 2 luciferyladenylate is oxidized with oxygen under production of
AMP (adenosinel monophosphate) and excited oxyluciferin (P.sup.x), which
both remain enzyme bound, and carbon dioxide. Oxyluciferin is in reaction
3 transformed into its ground state while emitting a photon. The energy
for emitting the photon has been obtained from the oxidation of luciferin
and not from splitting of the pyrophosphate bound in ATP.
The enzyme-oxyluciferin-AMP-complex generated in reaction 3 is stable and
can be isolated by gel filtration if the reaction is performed in the
presence of pyrophosphatase (Gates, B. J., and DeLuca, M. (1975) Arch.,
Biochem, Biophys., 169, 616-621). In 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).
The stability of the enzyme-oxyluciferin-AMP-complex results in that the
mixture of luciferase, D-luciferin and ATP give rise to a light flash
rather than a constant light emission. The maximum light intensity will be
reached within one second and is thereafter declining to a steady-state
level, where the regeneration of free enzyme is obtained with essentially
the same speed as the light emission. The steady-state level is, however,
not completely constant mainly because of the fact that the free products
are inhibiting the reactions.
The determination of ATP has usually been made in such a way that the
sample with an unknown ATP-concentration has been mixed with the
bioluminiscent reagent which contains luciferase, D-luciferin and
magnesium ions. In order to obtain a maximum reliability of the analysis
the mixing should suitably take place with a given reaction rate and in a
measuring 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 used for the determination
has been registered, the analysis is repeated with a sample containing a
known ATP-concentration and with a blank without ATP. From these three
measurements the unknown ATP-concentration has been calculated. If the
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 and without addition of a known ATP concentration.
Already in 1952 it was shown that ATP dependent bioluminiscence 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 bioluminiscent reagent to an ATP
converting system in order to continuously follow the ATP concentration by
measuring the light intensity has, however, obtained very little practical
significance. This is due partly to the fact that the activity of the
luciferase during the reaction declined by means of product inhibition as
described above, partly because of the fact that the luciferase reagents
have been contaminated with ATP converting systems. The interest of the
method has, however, raised when it has become possible, by means of using
purified luciferase and with a synthetical preparation of the luciferin,
to obtain during a reaction time of several minutes a negligible decrease
of the light intensity as well as of the ATP concentration (Lundin, A. and
Thore A. (1975) Anal. Biochem. 66, 47-63. Suitable conditions for the
analysis have been investigated and the method has proven useable for ATP
concentrations up to 10.sup.-6 M (Lundin, A., Rickardsson, A. and Thore,
A., (1977), Anal. Biochem. 75, 611-620). 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 show the analytical use of a reagent with
the above cited properties for kinetic determinations of subtrates and
enzymes, for end-point determination of substrate, to monitor
photophosphorylation and to follow lytic reactions (Lundin, A.,
Rickardsson, A., and Thore, A. (1976), Anal. Biochem. 75, 611-620; Lundin
A., Rickardsson, A., and Thore, A. (1977), "Proceedings of the 2nd
Bi-Annual ATP Methodology Symposium," SAI Technology Company, San Diego;
Lundin, A., Thore, A. and Baltscheffsky, M. (1977), FEBS Lett. 79, 73-76;
Lundin, A. (1978), "Methods in Enzymology" (M. DeLuca, ed.) Vol. 57,
Academic Press, New York; Lundin, A. and Baltscheffsky, M. (1978) "Methods
in Enzymology"0 (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.
The difficulties in preparing in a reproducible way a reagent with the
above cited properties have, however, implied that the above cited
applications have not obtained any use outside the laboratory at which the
technique was developed.
Before the use of the present invention the stability of the light level
varied in different reagents from a decline of a few percent 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 properties, i.e. with a stable light level. The
effect of the D-luciferin analogs of the stability of the light level has
not previously been observed and it should in analytical applications be
closely at hand to avoid said analogs as they have been shown to inhibit
the light reaction competitively with the D-luciferin (Denburg, J. L.)
With a D-luciferin analog is meant in connection with this invention
substances which inhibit the previously described luciferase reaction and
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 at the addition of a D-luciferin analog, said
addition could mean that a smaller part of the total luciferase amount
will exist as an inactive enzyme-product-complex.
As the free enzyme concentration decreases by forming of an enzyme-product
complex, the enzyme-luciferin analog complex is probably dissociated under
formation of free enzyme as is shown in the reaction 5 in FIG. 1 where I
represents the D-luciferinanalog. By using the D-luciferin-analog, i.e.
the competitive inhibitor, the luciferase amount can be increased without
any corresponding increase of the reaction rate. This is true
irrespectively 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 are
reversible and are faster than reactions 2-4 (reaction 4 will be discussed
in detail below).
To the extent free AMP and free oxluciferin are formed in the reaction it
could also be of interest to study whether this would affect the product
inhibition. If e.g. 10.sup.-6 M ATP would lead to a generation of
10.sup.-6 M AMP and 10.sup.-6 M oxyluciferin the inhibition of AMP would,
as K.sub.i for AMP is 2.4.times.10.sup.-4 M (see Lee, R. T., Denburg, J.
L. and McElroy, W. D. (1970) Arch. Biochem. Biohpys, 141, 38-52) affect
the luciferase activity by less than 0.5%. On the other hand the forming
of oxyluciferin, the K.sub.i value on which is 2.3.times.10.sup.-7 M
(Goto, T., Kubota, I., Suzuki, N. and Kishi, Y. (1973) "Chemiluminiscence
and Bioluminiscence" (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 under certain conditions be
important to counteract the effect of the generation of free oxyluciferin.
The addition of D-luciferin analog makes the initial inhibition of the
luciferase so big 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 and apparent saturation can
be achieved at a low concentration of D-luciferin without affecting the
accuracy of the analysis in any other way than by a reducing of the
sensitivity. This is an additional advantage of the invention, since
D-luciferin is an expensive substance which only in exceptional cases can
be added in saturating concentrations.
The reduced luciferase activity obtained by the addition of an analog can
be compensated by an increase of the luciferase concentration. Thus, the
luciferin-luciferase ratio can be optimized with respect to e.g. cost for
reagent production without affecting the sensitivity of the analysis. The
importance 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 considerable
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 e.g production cost of the
reagent and also for increasing the stability of the light-level. A
suitable concentration of the D-luciferin analog is one which gives an
inhibition of the luciferase reaction, and thus the light intensity, by at
least 25% since at a lower degree of inhibition, the effect on the
stability of the light level and the required concentration of the
D-luciferin for 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 registration equipment unnecessary big and also makes it
necessary to increase the amount of the luciferase so much that the
analysis will be uneconomical. Furthermore, an analytical interference can
easily be obtained at high amounts of luciferase.
In accordance with a specifically preferred embodiment of the invention the
previously mentioned competitive inhibitor is added to the reagent
together with pyrophosphate. It is known per se that concentrations of
pyrophosphate which are inhibiting luciferase reaction, counteract the
decline of the light intensity (McElroy, 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 have
been used before, preferably in a highest concentration 10.sup.-4 M and
specifically not more than 10.sup.-5 M and a competitive inhibitor in the
concentration defined above a very stable light level can be obtained.
Through this combination the same stability of the light level can be
achieved as would require a considerably higher degree of inhibition of
the luciferase to achieve by use of either pyrophosphate of D-luciferin
analog. At a competitive inhibition with a D-luciferin analog of around
50% and a concentration of pyrophosphate of 10.sup.-6 M it has thus 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
pyrophosphate of 10.sup.-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
registration equipment can be used and above all it gives very good
prerequisite for continuous 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 the pyrophosphate reacts
with AMP in the enzyme-oxyluciferin-AMP complex according to reaction 4 of
FIG. 1. The formation of ATP from pyrophosphate and AMP requires energy.
This energy is probably obtained from the transformation of enzyme back to
its original conformation from the conformation which was obtained before
the oxidation reaction (reaction 2 of FIG. 1). The conformation change
would explain why the product inhibition is non-competitive (Lemasters, J.
J. and Hacknebrock, C. R. (1977) Biochemistry 16, 445-447) whereas
oxylucuiferin is a competitive inhibitor (see the above cited application
by Goto et al., 1973). The reaction with pyrophosphate would thus change a
strong non-competitive inhibition (see the above cited publication by Goto
et al., 1973) to a weaker competitive inhibition. The effect of the
competitive inhibition on the light level could according to the invention
be counteracted by means of adding a D-luciferin-analog (see above).
According to reaction 4 of FIG. 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 the pyrophosphate could in inhibiting
concentrations contribute by driving the reaction 1 of FIG. 1 backwards.
By adding pyrophosphate the total enzyme concentration could thus be
increased without a corresponding increase of the light intensity. A
smaller part of the total enzyme concentration will thereby be 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 one could to the
bioluminiscent reagent add co-enzyme A, i.e. either in combination with
the competitive inhibitor only or in combination with the competitive
inhibitor and pyrophosphate. Also, the addition of co-enzyme A has 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
e.g. by the publication of Airth, R. L., Rhodes, W. C. and McElroy, W. D.
(1958), Biochem. Biophys, Acta, 27, 519-, but the use of co-enzyme A for
improving the analysis conditions when determining ATP has not previously
been suggested.
The reaction mechanics for co-enzyme A is thus probably that the compound
reacts with oxyluciferin in the enzyme-oxyluciferin AMP-complex according
to reaction 4 in FIG. 1 (where co-enzyme A is denoted CoA). The reaction
between co-enzyme A and oxyluciferin changes the non-competitive product
inhibition of the enzyme and oxyluciferin -AMP-complex to a weak
competitive inhibition by AMP, which is negligible under normal analytical
conditions.
Thus, coenzyme 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 analogs
and pyrophosphate will however in many analytical applications not be of
importance since the sensitivity of luciferase method generally 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 to high proportions of
contaminants interfering with the analysis. In certain applications of the
present invention it could therefore be of special interest to use a
hgihly purified luciferase preparation. Different methods for purifying
luciferase are known per se and anyone of these methods could be used in
most cases. A preferred embodiment of the invention, when the demands of
purification are very high, woould 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 e.g. bovine serumalbumin, thiol compounds and/or EDTA
(Etylenediamin tetraacetic acid).
The use of pyrophosphate and coenzyme A may be limited by the existence in
certain biological samples of enzyme systems which degrade these
substances. In this cases the effect of such enzymes could be prevented by
adding an inhibitor which does not affect the luciferase reaction. The
pyrophosphatase activity could e.g be inhibited by mangan 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 bioluminiscence reagent with the desired properties the
choice of substances or the combination of substances within the group of
D-luciferin analogs, pyrophosphate and coenzyme 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 within a big group of substances it would, for the man
skilled in the art, not imply any difficulties 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 measurement of ATP
converting reactions with the improved bioluminiscence 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
spectrophotometrical analysis based on e.g. NAD.sup.+ /NADH-conversion.
Also when determining ATP in non-ATP-converting systems, i.e. in samples
with a constant ATP concentration, 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 position and the light measurement may
continue during any desired period of time. Thereby the sensitivity as
well as the reproducibility will be increased. Also when determining ATP
in cellular systems the reagent according to the invention has big
advantages since it makes it possible to measure the concentration of
extra cellular and intracellular ATP in the same sample. The concentration
of extra cellular 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 single components of the bioluminiscent reagent according to
the invention for simplicity's sake have 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.
The invention will now be further explained by means of the following
non-limiting example.
EXAMPLE
This example, which refers to FIG. 2, illustrates how D-luciferin analogs
(in this case L-luciferin) and pyrophosphate could be used together so
that a reagent with a stable light level is achieved already at a
reasonable degree of inhibition. The luciferase used in the example has
been purified by means of isoelectric focusing. In FIG. 2 the light
intensity is shown as a function of time after adding a final
concentration of 10.sup.-6 M ATP to the reaction mixture. In the reaction
mixture (final volume 1 ml) there is comprised in all cases luciferase,
D-luciferin (100 .mu.g/ml), magnesiumacetate (10mM), bovine serum albumin
(0.1%), EDTA (2mM), and 0.1 M tris (hydroxymethyl) aminometanbuffer
adjusted to pH 7.75 by using acetic acid.
FIG. 2a shows the light curve obtained without any further additives. A
reagent with a declining level according to FIG. 2a is not applicable for
continous measurement of ATP-converting systems. FIG. 2b shows the effect
of the addition of L-luciferin (10 .mu.g/ml) resulting in an inhibition of
about 70%. The decline of the curve is acceptable but the initial peak
makes the reagents not applicable at high ATP concentrations for
continuous measurements in ATP converting systems. When using higher
concentrations of the additive and thus a higher degree of inhibition one
will however obtain straight light curves and applicable reagents. In FIG.
2c there is shown the effect of pyrophosphate (10.sup.-6 M). The decline
of the light curve is still too big and there is a small initial peak. At
higher and more inhibiting concentrations of the additive the light curve
will be more straight. FIG. 2d shows the effect of L-luciferin (10
.mu.g/ml) and pyrophosphate (10.sup.-6 M). The decline as well as the
initial peak has in this case been almost completely eliminated. This
reagent is then well suitable for analytical purposes. Only the reagents
which contain L-luciferin (2b and 2d) apparently saturated with respect to
luciferin (D+L, see above) and only these reagents will consequently give
the maximum analytical accuracy.
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