ANALYTICAL
BIOCHEMISTRY
Continuous
Defense
61 I-620 (1976)
Monitoring Purified
ARNE LUNDIN, National
75,
Research
of ATP-Converting Firefly Luciferase
Reactions
by
ANNE RICKARDSSON, AND ANDERSTHORE Institute,
Department
4, S-172 04 Sundbyberg
4, Sweden
Received April 5, 1976; accepted June 8, 1976 The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 1O-6 M the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenient for routine applications.
The wide analytical usefulness of the firefly luciferin-luciferase system was first demonstrated in 1952 by Strehler and Totter (1). Using a partially purified preparation of luciferase they demonstrated that the firefly assay of ATP may be used for the assay of metabolites and enzymes participating in ATP-converting reactions. Due to its high sensitivity and specificity the firefly system has frequently been used for analytical purposes as described in several reviews (2-4). One obvious way of assaying ATP-converting reactions with firefly luciferase is to take out aliquots at various times to perform the ATP assay separately. This procedure has been used, for example, for the assay of adenine nucleotides (5-7) and phosphodiesterase (8). The firefly system may also be added directly to the ATP-converting system allowing continuous monitoring of the ATP level by measuring the bioluminescence. A few reports on such directly coupled assays have been published (9- 12). A serious problem in this type of assay is the presence of ATP-converting enzymes in crude preparations of firefly luciferase (6,13). Purification of the firefly reagent by gel filtration on Sephadex G-100 removes most of these enzymes (14) resulting in increased specificity (6). The luciferase preparation obtained results in an almost constant light intensity proportional to the ATP concentration (6). This gel-filtered luciferase preparation is particularly suitable for continuous monitoring of ATP-converting reactions. The present work was conducted to find 611 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
612
LUNDIN,
RICKARDSSON,
6
7 PH
AND THORE
8
/
9
FIG. 1. The influence of pH on intensity and decay rate of the bioluminescence. Peak height (O), light intensity after 2 min (A), and decay rate of the light after 2 min (0) were measured using 10 ~1 of luciferase, 1.4 x 10-5~luciferin, 1OWMATP, 10rn~ MgSO,, 2 mMEDTA, and 20 mM Tris-H,SO, buffer in a total volume of 1 ml.
optimal conditions for the continuous illustrate its analytical usefulness. MATERIALS
monitoring
technique
and to
AND METHODS
Crude firefly lantern extract (FLE-50), crystalline pyruvate kinase and adenylate kinase from rabbit muscle, crude apyrase, bovine serum albumin, ATP, ADP, AMP, cyclic AMP, phosphoenolpyruvate, and phosphocreatine were obtained from Sigma Chemical Co. Creatine phosphokinase was obtained from Dade Reagents Inc. (Miami) and bovine heart phosphodiesterase from Boehringer/Mannheim. Luciferin was kindly synthesized by Drs. Monica Kowalska and Borje Gstman from our institute, essentially according to the method of White ef al. (15). Luciferase was purified by gel filtration essentially according to Nielsen and Rasmussen (14). Crude firefly lantern extract, FLE-50, corresponding to 200 mg of dried lanterns, was dissolved in 10 ml of a solution containing 0.1% bovine serum albumin, 10 mM MgS04, and 1 mM EDTA. The pH was adjusted to 7.4 with KOH. Particulate matter was removed by centrifugation, 27,000g for 15 min. The supernatant was applied to a Sephadex G-100 column (2.6 x 100 cm) and eluted with a buffer consisting of 50 mM glycine, 10 mM Na,HAsO,, and 1 mM EDTA, pH 7.4. The gel filtration was performed at 4°C with a flow of 20 ml/hr. Previous results (14)
CONTINUOUS
MONITORING
613
OF ATP
c
I
16'
If3 CONCENTRATION (relatwe
10-Z OF LUCIFERASE unks)
11
FIG. 2. The influence of luciferase concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in Fig. 1, but with pH 7.75. Dilutions of luciferase are relative to the original preparation obtained as described in Materials and Methods.
on the relative positions of peaks for nucleoside diphosphokinase, luciferase, and adenylate kinase were confirmed. Peak fractions containing more than 50% of the maximal luciferase activity were pooled. The preparation obtained in this way contained 80% of the total activity eluted from the column, corresponding to 40% of the activity applied. All assays with luciferase were performed at room temperature, measuring the bioluminescence with equipment described previously (6).
c
I
10-6 CONCENTRATION
F----T
10-5 d OF LUCIFERIN [Ml
10
FIG. 3. The influence of luciferin concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in FIG. 1, but with lo-’ M ATP and pH 7.75.
614
LUNDIN,
1
( 10-B
RICKARDSSON,
AND THORE
I 10-l
1o-6 1o-5 1o-4 CONCENTRATION OF ATP(M)
1o-3
1o-2
FIG. 4. The inlluence of ATP concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in Fig. 1, but with pH 7.75.
Light emission was registered on a Servograph REC Sl/REA 112 recorder (Radiometer, Copenhagen). Enzymes were dissolved or diluted in 20 mM Tris-H,SO,, pH 7.75, containing 0.1% bovine serum albumin. Metabolites were dissolved and diluted in the same buffer containing 2 mM EDTA instead of albumin (7). The latter buffer was also used in all assays with luciferase. Unless otherwise stated bioluminescence assays were per-
TIME [min)
FIG. 5. Endpoint analysis of a mixture of adenine nucleotides by continuous monitoring of the ATP level by purified firefly luciferase. Luciferase (10 CJ) was added to a solution containing, in 1 ml, 2.0 x lo+ M AMP, 2.5 x 10-r M ADP, 2.4 x l&’ M AMP, 2.0 x 10-r M cyclic AMP, 0.1 mM phosphoenolpyruvate, 10 mM MgSO,, 12.5 mM K,SO,, 1.4 x 10e5 M luciferin, 2 mM EDTA, and 20 mM Tris-H,SO, buffer, pH 7.75. After recording the ATP level, pyruvate kinase (approximately 5 units), adenylate kinase (approximately 250 units), and phosphodiesterase (approximately 0.06 units) were added to convert ADP, AMP, and cyclic AMP, respectively, to ATP.
CONTINUOUS
0
MONITORING
1
2
615
OF ATP
3
TIME [mm)
FIG. 6. Continuous monitoring of apyrase activity by purified firefly luciferase. The addition of 10 ~1 of luciferase to 1 ml of a solution containing 2 X 10-O M ATP, 5 mM CaCI,, 5 mM MgSO,, and 1.4 x 1O-5 M luciferin resulted in a constant light intensity. When apyrase (approximately 5 x 10m3units) was added, light intensity decreased at a constant rate.
formed in a total volume of 1 ml using 10 (~1 of the purified luciferase preparation and 1.4 x 10~~ M luciferin. The addition of 5 or 10 mM MgS04 resulted in favorable reaction conditions with only slight inhibition of any of the enzyme systems involved. The ATP level corresponding to a certain amount of light was determined by adding a known amount of ATP to the sample and measuring the increase of the light intensity. ATP solutions for these calibrations were standardized by the 340-nm spectrophotometric method using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. For this purpose the ATP determination kit 366-UV from Sigma was used according to the manufacturer (16). RESULTS
The first series of experiments was performed to find optimal assay conditions for the continuous monitoring technique. After injection of ATP, the peak light intensity, the intensity after 2 min, and the rate of decay of the light after 2 min were determined under various conditions.
616
LUNDIN,
RICKARDSSON,
CPK
CONCENTRATION
AND THORE
(m I U /ml)
FIG. 7. Kinetic determination of creatine phosphokinase activity by the continuous monitoring technique. The initial rate of ATP formation was determined using purified luciferase, 1.4 x 1O-5 M luciferin, 10m5M ADP, 7 mM phosphocreatine, 5 mM MgSO,, and 0.1 M triethanolamine buffer, pH 7.0, and various amounts of creatine phosphokinase (CPK).
The first experiment (Fig. 1) confirmed within limits of error a pH optimum of 7.75, as obtained by others (17). This pH also resulted in a slow decay of the light and was therefore used in subsequent experiments. The rapid decay of the light at pH values less than 6 or higher than 8 was probably mainly due to inactivation of luciferase. This was shown by measuring the luciferase activity by the peak-height method after incubation at extreme pH values for various times. The second experiment (Fig. 2) showed that the luciferase concentration should be kept within certain limits to avoid a rapid decay of the light. At high concentrations of luciferase, the decay of the light is probably mainly due to product inhibition, since aliquots from the luminescent mixture diluted at various times following initiation of the reaction had the same luciferase activity as measured by the peak-height method. A low concentrations of luciferase the decay is most likely due to inactivation of luciferase, since the luciferase activity decreased with the same kinetics in buffer without added substrates. In all subsequent experiments, the luciferase preparation obtained as described in Materials and Methods was diluted 100 times to obtain a slow decay and the highest possible activity. The third experiment (Fig. 3) showed that 7 x 10 +-2 x 10e4 M luciferin resulted in maximal light intensity. The decay rate increased with the luciferin concentration. In subsequent experiments the luciferin concentration was 1.4 x 1OW M. Under conditions arrived at in the previous experiments, ATP concentrations less than lO-(j M resulted in a linear relationship between
CONTINUOUS
MONITORING
617
OF ATP
t lo-’ ADP
8. Kinetic assay of ADP formation was determined phosphoenolpyruvate, 12.5 mM buffer, pH 7.75, pyruvate kinase of ADP. FIG.
ATP
10-G CONCENTRATION
10-S (M)
by the continuous monitoring technique. The initial rate of using purified luciferase, 1.4 x 10e5 M luciferin, 1 mM K,SO,, 5 mM MgSO,, 2 mM EDTA. 20 mM Tris-H,SO, (approximately 3 x lo-* units), and various concentrations
ATP concentration and bioluminescence and a slow decay of the light (Fig. 4). At ATP concentrations higher than approximately 10e6 M, the linear relationship ceases and the decay rate increases, making the continuous monitoring technique unsuitable in most cases. In subsequent experiments, the ATP concentration never exceeded 10e6 M. The experiment shown in Fig. 5 illustrates how the continuous monitoring technique may be used for endpoint analysis of metabolites. The ATP level in a solution containing ATP, ADP, AMP, and cyclicAMP was monitored during stepwise conversion of the various nucleotides to ATP. The increase of the light after addition of pyruvate kinase, adenylate kinase, and phosphodiesterase is proportional to the amount of ATP formed from ADP, AMP, and cyclicAMP, respectively. Results on nucleotide levels obtained with the continuous monitoring technique were identical to those obtained with the sampling technique previously described (7). The time needed for completion of the adenylate kinase reaction increased when nucleotide concentrations were lowered. Since the decay of the bioluminescence was not completely eliminated even with the purified luciferase, concentrations of AMP and cyclicAMP less than lo-’ M will result in inconveniently long incubation times under the present assay conditions. However, the sensitivity limit for ATP and ADP is identical to that reported previously for ATP (6). The partially purified luciferase may also be used for kinetic
618
LUNDIN,
RICKARDSSON,
AND
THORE
determinations of enzymes and metabolites. In the experiment depicted in Fig. 6, the addition of apyrase to a mixture with constant bioluminescence resulted in a linear decrease of the light, reflecting the decomposition of ATP to ADP. Thus, the technique may be used for measurements of initial rates of enzymic reactions resulting in formation or decomposition of ATP. The use of the continuous monitoring technique for kinetic assays of enzymes is exemplified in Fig. 7. In this figure initial rates of ATP formation from ADP and phosphocreatine have been plotted against concentration of creatine phosphokinase, demonstrating a linear relationship. Although the assay was not optimized with respect to, for example, substrate concentrations, lop4 enzymic units were easily assayed. Metabolite levels may also be assayed kinetically as shown in Fig. 8. Pyruvate kinase was added to solutions containing phosphoenolpyruvate, cofactors, and various concentrations of ADP, and the initial rate of the rise of bioluminescence was measured. As shown in the figure, the rate of ATP production corresponding to the rise of bioluminescence was proportional to the ADP concentration. DISCUSSION
Mechanisms and kinetics of the firefly luciferase-catalyzed reactions have recently been described by DeLuca and McElroy (18). The reactions start with the formation of an enzyme-luciferyl adenylate complex from Mg-ATP, luciferin, and luciferase. Before the addition of oxygen, there are conformational changes within the complex resulting in a lag of 25 msec before any light is emitted and a rise to the maximum light emission requiring approximately 0.3 sec. The oxidative decarboxylation of luciferin results in the light-emitting product. After maximum light emission the light decreases due to product inhibition of the enzyme (18,19). From these findings it may be concluded that the continuous monitoring of ATP by firefly luciferase will have a time resolution of about 0.3 sec. It may also be concluded that analytical conditions should be arranged so as to avoid product inhibition. Since the rate of product formation and the rate of photon emission are identical (18), product inhibition will develop rapidly when light emission, i.e., reaction rate, is high. This might explain the correlation between decay rate and light intensity obtained at high concentrations of luciferase (Fig. 2) and ATP (Fig. 4). The increased decay rate at high levels of luciferin (Fig. 3) does not seem to be due to product inhibition, since light intensity is not increased. A rapid decay of the light may also be due to unspecific inactivation of luciferase, for example, by dilution (Fig. 2) or by extreme pH values (Fig. 1). Under conditions not resulting in product inhibition or other types of inactivation, the light intensity is constant and proportional to the ATP concentration (6). Furthermore, firefly luciferase is specific for ATP (20),
CONTINUOUS
MONITORING
OF ATP
619
and ATP-converting enzymes may be removed by gel filtration (14). Thus, purified firefly luciferase may be used for nondestructive, continuous monitoring of ATP levels in complex biological systems. The present work is an attempt to illustrate how the continuous monitoring technique may be used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. As compared to sampling followed by separate luciferase assay, the continuous monitoring technique using purified luciferase involves fewer analytical steps. This results in less consumption of reagents, less manual work, and fewer sources of error. Furthermore, purified luciferase has a higher specificity and a lower background luminescence than the commercial luciferase preparation (6). The number of known ATP-converting enzymatic reactions means that the continuous monitoring technique might be developed to an analytical tool as versatile as the 340-nm spectrophotometric family of assays for nicotinamide adenine dinucleotide-linked reactions. The main advantage of the luciferase system would be the extreme sensitivity, which also means that analytical interference may be diminished by dilution. Furthermore, the equipment necessary for measuring bioluminescence is inexpensive and simple in operation and construction. Turbidity in the sample would be expected to be of little importance since light emission from distant parts of the sample is measured with less efficiency. At present we are working on further improvements of the luciferase reagent and the purification of luciferase. Some of the more promising applications of the continuous monitoring technique in areas such as clinical biochemistry are also being explored. ACKNOWLEDGMENTS This work was supported by the Swedish Board for Technical Development and by a gram to one of us (A.L) from Forsvarsmedicinska Forskningsdelegationen, Stockholm. We gratefully acknowledge the skillful technical assistance of Miss Ulrika Karnell.
REFERENCES 1. Strehler, B. L., and Totter, J. R. (1952) Arch. Biochem. Biophys. 2. Strehler, B. L. (1%8) in Methods of Biochemical Analysis (Glick, 99-181, Interscience, New York. 3. Stanley, P. E. (1974) in Liquid Scintillation Counting (Crook, M. eds.), Vol. 3, pp. 253-272, Heyden, London. 4. Schram, E. (1974) in Liquid Scintillation Counting (Stanley, P. E., eds.). pp. 383-402, Academic Press, New York. 5. Pradet, A. (1%7) Physiol. Veg. 5. 209-221, 6. Lundin, A., and Thore, A. (1975) Anal. Biochem. 66, 47-63. 7. Lundin. A., and Thore, A. (1975) Appl. Mcrobiol. 30, 713-721. 8. Johnson, R. A., Hardman. J. G., Broadus, A. E., and Sutherland, Biochem. 35, 91-97.
40, 28-41. D., ed.), Vol. 16. pp.
A.. and Johnson, P.. and Scoggins, B. A.,
E. W. (1970) Anal.
620
LUNDIN,
RICKARDSSON,
AND THORE
9. Weiss, B., Lehne, R., and Strada, S. (1972) Anal. Biochem. 45, 222-235. 10. Witteveen, S. A. G. J., Sobel, B. E., and DeLuca, M. (1974) Proc. Nat. Acad. Sci. USA 71, 1384- 1387. 11. Balharry, G. J. E., and Nicholas, D. J. D. (1971) Anal. Biochem. 40, 1-17. 12. Lemasters, J. J., and Hackenbrock, C. R. (1973) Biochem. Biophys. Res. Commun. 55, 1262- 1270. 13. Rasmussen, H., and Nielsen, R. (1%8) Acfa Chem. Stand. 22, 1745-1756. 14. Nielsen, R., and Rasmussen, H. (1968) Acta Chem. Stand. 22, 1757- 1762. 15. White, E. H., McCapra, F., and Field, G. F. (1%3)J. Amer. Chem. Sot. 85, 337-343. 16. Sigma Technical Bulletin 366UV. 17. McElroy, W. D., and Strehler, B. L. (1949) Arch. Biochem. 22, 420-433. 18. DeLuca. M.. and McElroy, W. D. (1974) Biochemisrvy 13, 921-925. 19. Gates, B. J., and DeLuca, M. (1975) Arch. Biochem. Biophys. 169, 616-621. 20. McElroy, W. D., and Green, A. (1956) Arch. Biochem. Biophys. 64, 257-271.
BIOCHEMISTRY
Continuous
Defense
61 I-620 (1976)
Monitoring Purified
ARNE LUNDIN, National
75,
Research
of ATP-Converting Firefly Luciferase
Reactions
by
ANNE RICKARDSSON, AND ANDERSTHORE Institute,
Department
4, S-172 04 Sundbyberg
4, Sweden
Received April 5, 1976; accepted June 8, 1976 The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 1O-6 M the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenient for routine applications.
The wide analytical usefulness of the firefly luciferin-luciferase system was first demonstrated in 1952 by Strehler and Totter (1). Using a partially purified preparation of luciferase they demonstrated that the firefly assay of ATP may be used for the assay of metabolites and enzymes participating in ATP-converting reactions. Due to its high sensitivity and specificity the firefly system has frequently been used for analytical purposes as described in several reviews (2-4). One obvious way of assaying ATP-converting reactions with firefly luciferase is to take out aliquots at various times to perform the ATP assay separately. This procedure has been used, for example, for the assay of adenine nucleotides (5-7) and phosphodiesterase (8). The firefly system may also be added directly to the ATP-converting system allowing continuous monitoring of the ATP level by measuring the bioluminescence. A few reports on such directly coupled assays have been published (9- 12). A serious problem in this type of assay is the presence of ATP-converting enzymes in crude preparations of firefly luciferase (6,13). Purification of the firefly reagent by gel filtration on Sephadex G-100 removes most of these enzymes (14) resulting in increased specificity (6). The luciferase preparation obtained results in an almost constant light intensity proportional to the ATP concentration (6). This gel-filtered luciferase preparation is particularly suitable for continuous monitoring of ATP-converting reactions. The present work was conducted to find 611 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
612
LUNDIN,
RICKARDSSON,
6
7 PH
AND THORE
8
/
9
FIG. 1. The influence of pH on intensity and decay rate of the bioluminescence. Peak height (O), light intensity after 2 min (A), and decay rate of the light after 2 min (0) were measured using 10 ~1 of luciferase, 1.4 x 10-5~luciferin, 1OWMATP, 10rn~ MgSO,, 2 mMEDTA, and 20 mM Tris-H,SO, buffer in a total volume of 1 ml.
optimal conditions for the continuous illustrate its analytical usefulness. MATERIALS
monitoring
technique
and to
AND METHODS
Crude firefly lantern extract (FLE-50), crystalline pyruvate kinase and adenylate kinase from rabbit muscle, crude apyrase, bovine serum albumin, ATP, ADP, AMP, cyclic AMP, phosphoenolpyruvate, and phosphocreatine were obtained from Sigma Chemical Co. Creatine phosphokinase was obtained from Dade Reagents Inc. (Miami) and bovine heart phosphodiesterase from Boehringer/Mannheim. Luciferin was kindly synthesized by Drs. Monica Kowalska and Borje Gstman from our institute, essentially according to the method of White ef al. (15). Luciferase was purified by gel filtration essentially according to Nielsen and Rasmussen (14). Crude firefly lantern extract, FLE-50, corresponding to 200 mg of dried lanterns, was dissolved in 10 ml of a solution containing 0.1% bovine serum albumin, 10 mM MgS04, and 1 mM EDTA. The pH was adjusted to 7.4 with KOH. Particulate matter was removed by centrifugation, 27,000g for 15 min. The supernatant was applied to a Sephadex G-100 column (2.6 x 100 cm) and eluted with a buffer consisting of 50 mM glycine, 10 mM Na,HAsO,, and 1 mM EDTA, pH 7.4. The gel filtration was performed at 4°C with a flow of 20 ml/hr. Previous results (14)
CONTINUOUS
MONITORING
613
OF ATP
c
I
16'
If3 CONCENTRATION (relatwe
10-Z OF LUCIFERASE unks)
11
FIG. 2. The influence of luciferase concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in Fig. 1, but with pH 7.75. Dilutions of luciferase are relative to the original preparation obtained as described in Materials and Methods.
on the relative positions of peaks for nucleoside diphosphokinase, luciferase, and adenylate kinase were confirmed. Peak fractions containing more than 50% of the maximal luciferase activity were pooled. The preparation obtained in this way contained 80% of the total activity eluted from the column, corresponding to 40% of the activity applied. All assays with luciferase were performed at room temperature, measuring the bioluminescence with equipment described previously (6).
c
I
10-6 CONCENTRATION
F----T
10-5 d OF LUCIFERIN [Ml
10
FIG. 3. The influence of luciferin concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in FIG. 1, but with lo-’ M ATP and pH 7.75.
614
LUNDIN,
1
( 10-B
RICKARDSSON,
AND THORE
I 10-l
1o-6 1o-5 1o-4 CONCENTRATION OF ATP(M)
1o-3
1o-2
FIG. 4. The inlluence of ATP concentration on intensity and decay rate of the bioluminescence. Symbols and experimental conditions as in Fig. 1, but with pH 7.75.
Light emission was registered on a Servograph REC Sl/REA 112 recorder (Radiometer, Copenhagen). Enzymes were dissolved or diluted in 20 mM Tris-H,SO,, pH 7.75, containing 0.1% bovine serum albumin. Metabolites were dissolved and diluted in the same buffer containing 2 mM EDTA instead of albumin (7). The latter buffer was also used in all assays with luciferase. Unless otherwise stated bioluminescence assays were per-
TIME [min)
FIG. 5. Endpoint analysis of a mixture of adenine nucleotides by continuous monitoring of the ATP level by purified firefly luciferase. Luciferase (10 CJ) was added to a solution containing, in 1 ml, 2.0 x lo+ M AMP, 2.5 x 10-r M ADP, 2.4 x l&’ M AMP, 2.0 x 10-r M cyclic AMP, 0.1 mM phosphoenolpyruvate, 10 mM MgSO,, 12.5 mM K,SO,, 1.4 x 10e5 M luciferin, 2 mM EDTA, and 20 mM Tris-H,SO, buffer, pH 7.75. After recording the ATP level, pyruvate kinase (approximately 5 units), adenylate kinase (approximately 250 units), and phosphodiesterase (approximately 0.06 units) were added to convert ADP, AMP, and cyclic AMP, respectively, to ATP.
CONTINUOUS
0
MONITORING
1
2
615
OF ATP
3
TIME [mm)
FIG. 6. Continuous monitoring of apyrase activity by purified firefly luciferase. The addition of 10 ~1 of luciferase to 1 ml of a solution containing 2 X 10-O M ATP, 5 mM CaCI,, 5 mM MgSO,, and 1.4 x 1O-5 M luciferin resulted in a constant light intensity. When apyrase (approximately 5 x 10m3units) was added, light intensity decreased at a constant rate.
formed in a total volume of 1 ml using 10 (~1 of the purified luciferase preparation and 1.4 x 10~~ M luciferin. The addition of 5 or 10 mM MgS04 resulted in favorable reaction conditions with only slight inhibition of any of the enzyme systems involved. The ATP level corresponding to a certain amount of light was determined by adding a known amount of ATP to the sample and measuring the increase of the light intensity. ATP solutions for these calibrations were standardized by the 340-nm spectrophotometric method using glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. For this purpose the ATP determination kit 366-UV from Sigma was used according to the manufacturer (16). RESULTS
The first series of experiments was performed to find optimal assay conditions for the continuous monitoring technique. After injection of ATP, the peak light intensity, the intensity after 2 min, and the rate of decay of the light after 2 min were determined under various conditions.
616
LUNDIN,
RICKARDSSON,
CPK
CONCENTRATION
AND THORE
(m I U /ml)
FIG. 7. Kinetic determination of creatine phosphokinase activity by the continuous monitoring technique. The initial rate of ATP formation was determined using purified luciferase, 1.4 x 1O-5 M luciferin, 10m5M ADP, 7 mM phosphocreatine, 5 mM MgSO,, and 0.1 M triethanolamine buffer, pH 7.0, and various amounts of creatine phosphokinase (CPK).
The first experiment (Fig. 1) confirmed within limits of error a pH optimum of 7.75, as obtained by others (17). This pH also resulted in a slow decay of the light and was therefore used in subsequent experiments. The rapid decay of the light at pH values less than 6 or higher than 8 was probably mainly due to inactivation of luciferase. This was shown by measuring the luciferase activity by the peak-height method after incubation at extreme pH values for various times. The second experiment (Fig. 2) showed that the luciferase concentration should be kept within certain limits to avoid a rapid decay of the light. At high concentrations of luciferase, the decay of the light is probably mainly due to product inhibition, since aliquots from the luminescent mixture diluted at various times following initiation of the reaction had the same luciferase activity as measured by the peak-height method. A low concentrations of luciferase the decay is most likely due to inactivation of luciferase, since the luciferase activity decreased with the same kinetics in buffer without added substrates. In all subsequent experiments, the luciferase preparation obtained as described in Materials and Methods was diluted 100 times to obtain a slow decay and the highest possible activity. The third experiment (Fig. 3) showed that 7 x 10 +-2 x 10e4 M luciferin resulted in maximal light intensity. The decay rate increased with the luciferin concentration. In subsequent experiments the luciferin concentration was 1.4 x 1OW M. Under conditions arrived at in the previous experiments, ATP concentrations less than lO-(j M resulted in a linear relationship between
CONTINUOUS
MONITORING
617
OF ATP
t lo-’ ADP
8. Kinetic assay of ADP formation was determined phosphoenolpyruvate, 12.5 mM buffer, pH 7.75, pyruvate kinase of ADP. FIG.
ATP
10-G CONCENTRATION
10-S (M)
by the continuous monitoring technique. The initial rate of using purified luciferase, 1.4 x 10e5 M luciferin, 1 mM K,SO,, 5 mM MgSO,, 2 mM EDTA. 20 mM Tris-H,SO, (approximately 3 x lo-* units), and various concentrations
ATP concentration and bioluminescence and a slow decay of the light (Fig. 4). At ATP concentrations higher than approximately 10e6 M, the linear relationship ceases and the decay rate increases, making the continuous monitoring technique unsuitable in most cases. In subsequent experiments, the ATP concentration never exceeded 10e6 M. The experiment shown in Fig. 5 illustrates how the continuous monitoring technique may be used for endpoint analysis of metabolites. The ATP level in a solution containing ATP, ADP, AMP, and cyclicAMP was monitored during stepwise conversion of the various nucleotides to ATP. The increase of the light after addition of pyruvate kinase, adenylate kinase, and phosphodiesterase is proportional to the amount of ATP formed from ADP, AMP, and cyclicAMP, respectively. Results on nucleotide levels obtained with the continuous monitoring technique were identical to those obtained with the sampling technique previously described (7). The time needed for completion of the adenylate kinase reaction increased when nucleotide concentrations were lowered. Since the decay of the bioluminescence was not completely eliminated even with the purified luciferase, concentrations of AMP and cyclicAMP less than lo-’ M will result in inconveniently long incubation times under the present assay conditions. However, the sensitivity limit for ATP and ADP is identical to that reported previously for ATP (6). The partially purified luciferase may also be used for kinetic
618
LUNDIN,
RICKARDSSON,
AND
THORE
determinations of enzymes and metabolites. In the experiment depicted in Fig. 6, the addition of apyrase to a mixture with constant bioluminescence resulted in a linear decrease of the light, reflecting the decomposition of ATP to ADP. Thus, the technique may be used for measurements of initial rates of enzymic reactions resulting in formation or decomposition of ATP. The use of the continuous monitoring technique for kinetic assays of enzymes is exemplified in Fig. 7. In this figure initial rates of ATP formation from ADP and phosphocreatine have been plotted against concentration of creatine phosphokinase, demonstrating a linear relationship. Although the assay was not optimized with respect to, for example, substrate concentrations, lop4 enzymic units were easily assayed. Metabolite levels may also be assayed kinetically as shown in Fig. 8. Pyruvate kinase was added to solutions containing phosphoenolpyruvate, cofactors, and various concentrations of ADP, and the initial rate of the rise of bioluminescence was measured. As shown in the figure, the rate of ATP production corresponding to the rise of bioluminescence was proportional to the ADP concentration. DISCUSSION
Mechanisms and kinetics of the firefly luciferase-catalyzed reactions have recently been described by DeLuca and McElroy (18). The reactions start with the formation of an enzyme-luciferyl adenylate complex from Mg-ATP, luciferin, and luciferase. Before the addition of oxygen, there are conformational changes within the complex resulting in a lag of 25 msec before any light is emitted and a rise to the maximum light emission requiring approximately 0.3 sec. The oxidative decarboxylation of luciferin results in the light-emitting product. After maximum light emission the light decreases due to product inhibition of the enzyme (18,19). From these findings it may be concluded that the continuous monitoring of ATP by firefly luciferase will have a time resolution of about 0.3 sec. It may also be concluded that analytical conditions should be arranged so as to avoid product inhibition. Since the rate of product formation and the rate of photon emission are identical (18), product inhibition will develop rapidly when light emission, i.e., reaction rate, is high. This might explain the correlation between decay rate and light intensity obtained at high concentrations of luciferase (Fig. 2) and ATP (Fig. 4). The increased decay rate at high levels of luciferin (Fig. 3) does not seem to be due to product inhibition, since light intensity is not increased. A rapid decay of the light may also be due to unspecific inactivation of luciferase, for example, by dilution (Fig. 2) or by extreme pH values (Fig. 1). Under conditions not resulting in product inhibition or other types of inactivation, the light intensity is constant and proportional to the ATP concentration (6). Furthermore, firefly luciferase is specific for ATP (20),
CONTINUOUS
MONITORING
OF ATP
619
and ATP-converting enzymes may be removed by gel filtration (14). Thus, purified firefly luciferase may be used for nondestructive, continuous monitoring of ATP levels in complex biological systems. The present work is an attempt to illustrate how the continuous monitoring technique may be used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. As compared to sampling followed by separate luciferase assay, the continuous monitoring technique using purified luciferase involves fewer analytical steps. This results in less consumption of reagents, less manual work, and fewer sources of error. Furthermore, purified luciferase has a higher specificity and a lower background luminescence than the commercial luciferase preparation (6). The number of known ATP-converting enzymatic reactions means that the continuous monitoring technique might be developed to an analytical tool as versatile as the 340-nm spectrophotometric family of assays for nicotinamide adenine dinucleotide-linked reactions. The main advantage of the luciferase system would be the extreme sensitivity, which also means that analytical interference may be diminished by dilution. Furthermore, the equipment necessary for measuring bioluminescence is inexpensive and simple in operation and construction. Turbidity in the sample would be expected to be of little importance since light emission from distant parts of the sample is measured with less efficiency. At present we are working on further improvements of the luciferase reagent and the purification of luciferase. Some of the more promising applications of the continuous monitoring technique in areas such as clinical biochemistry are also being explored. ACKNOWLEDGMENTS This work was supported by the Swedish Board for Technical Development and by a gram to one of us (A.L) from Forsvarsmedicinska Forskningsdelegationen, Stockholm. We gratefully acknowledge the skillful technical assistance of Miss Ulrika Karnell.
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