United States Patent (19) 11 Patent Number: 5,891,659 Murakami Et Al

USOO589 1659A United States Patent (19) 11 Patent Number: 5,891,659 Murakami et al. (45) Date of Patent: Apr. 6, 1999

54) BIOLUMINESCENT ADENOSINE 4,960,696 10/1990 Imahori et al...... 435/42 PHOSPHATE ESTER ASSAY AND REAGENT OTHER PUBLICATIONS 75 Inventors: Seiji Murakami; Tatsuya Sakakibara; Database WPI, AN 88-318 079, Derwent Publications Ltd., Naoki Eisaki; Motoo Nakajima, all of London; & JP-A-63 223 799 (Eisai Co., Ltd.) 1988. Noda; Kazuhiro Imai, Tokyo, all of Soviet Patents Abstracts, B section, week 8805, 1985, Aug. Japan 30, Derwent Publications Ltd., London., & SU-A-1317027 (Moscow Lomonosov Univ). 73 Assignee: Kikkoman Corporation, Noda, Japan Primary Examiner Ralph Gitomer 21 Appl. No.: 805,613 Attorney, Agent, or Firm-Banner & Witcoff, Ltd. 57 ABSTRACT 22 Filed: Feb. 26, 1997 There is provided a bioluminescence reagent comprising at 30 Foreign Application Priority Data least pyruvate orthophosphate dikinase, phosphoenolpyru Mar. 4, 1996 JP Japan ...... 8-070911 Vic acid, pyrophosphoric acid, magnesium ion or another metallic ions, luciferin and luciferase, which reagent is Such

(51) Int. Cl." ...... C12O 1/66 that the amount of luminescence is maintained in a high 52 U.S. C...... 435/8; 435/15; 435/21 level and moreover Stably without decaying for a long time 58 Field of Search ...... 435/8, 4, 15, 17, in a bioluminescence reaction, and there is provided a 435/21, 25, 26, 92 method for quantitatively determining an adenosine phos phate ester or a Substance taking part in the ATP conversion 56) References Cited reaction in high Sensitivity and high accuracy using an U.S. PATENT DOCUMENTS inexpensive and Simple measuring apparatus. 4,246,340 1/1981 Lundin et al...... 435/8 7 Claims, 5 Drawing Sheets U.S. Patent Apr. 6, 1999 Sheet 1 of 5 5,891,659

FIG. 1 CHANGE WITH TIME LAPSE OF THE AMOUNT OF LUMNESCENCE CORRESPONDING TO EACH AMP CONCENTRATION 1OOOOOOO

1 OOOOOO 00000 0000 000000000000 00000000000

1 OOOOO

1 OOOO AAAAAAAAAAAAAAAAAAAAAAAAAA AA AA AAA

1 OOO XXX XXXXXX XXXX XXXXXXXXXXXXX XXXXXX

O 1 OO 2OO 3OO 400 500 600 TIME (SECOND)

AMP CONCENTRATION –0 - 4.0 x 10 M AMP CONCENTRATION -- 4.0 x 10' M AMP CONCENTRATION -A- 4.0 x 10' M AMP CONCENTRATION - x - 4.0 x 10' M AMP CONCENTRATION -k- 4.0 x 10' M U.S. Patent Apr. 6, 1999 Sheet 2 of 5 5,891,659

FG. 2 STANDARD CURVE OF AMP (10 MINUTES AFTER THE START OF REACTION)

1000000 g CB 1 OOOO O

O ? OOOO O (f) U Z OOO D -

H5 1 OO 2. D

sCC 10 1013 102 10' 100 O 10 AMP CONCENTRATION (M) U.S. Patent Apr. 6, 1999 Sheet 3 of 5 5,891,659

FIG 3

CHANGE WITH TIME LAPSE OF THE AMOUNT OF LUMNESCENCE CORRESPONDING TO EACH ATP CONCENTRATION 1 OOOOOOO 00000000000000000000000000000000000 000001" 10000 TAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 1000 *XXXxxxxxxxxxxxxxxxxxxxxxxxxxxxx

O OO 200 3OO 400 500 600 TIME (SECOND)

ATP CONCENTRATION -- 4.0 x 10 M ATP CONCENTRATION -- 4.0 x 1 O'M ATP CONCENTRATION -A- 4.0 x 1 O' M ATP CONCENTRATION - x- 4.0 x 10' M ATP CONCENTRATION - - 4.0 x 1 O'M U.S. Patent Apr. 6, 1999 Sheet 4 of 5 5,891,659

FIG. 4 STANDARD CURVE OF ATP (10 MINUTES AFTER THE START OF REACTION)

1000000 o D - CE OOOOO O Z O 1 OOOO (O U Z O 1 OOO - l O - OO 2 D

S.CC O 103 O'? 10 O 1O O ATP CONCENTRATION (M) U.S. Patent Apr. 6, 1999 Sheet 5 of 5 5,891,659

F.G. 5 CHANGE WITH TIME LAPSE OF RELATIVE AMOUNT OF LUMNESCENCE IN THE INVENTION AND COMPARATIVE EXAMPLE

120

100

O 100 200 300 400 500 600 700 TIME (SECOND)

THE INVENTION

m -a -um as s as a (PYRUVATE ORTHOPHOSPHATE DKINASE WASADDED) COMPARATIVE EXAMPLE (PYRURATE ORTHOPHOSPHATE DKINASE WAS NOT ADDED) 5,891,659 1 2 BOLUMNESCENT ADENOSINE Further, Since a thiol reagent has a SH group on the PHOSPHATE ESTER ASSAY AND REAGENT Structure, it, in general, is easily oxidized and has a problem BACKGROUND OF THE INVENTION on Storage as a Solution. Further, Since CoA on the market 1. Field of the Invention is one extracted from a yeast or the like, it is expensive, and This invention relates to a bioluminescence reagent that moreover a possibility that ATP, etc. are mixed is noted. The the amount of luminescence may be maintained in a high inclusion of ATP is not desired because it causes the increase level and moreover Stably without decaying for a long time of the background when ATP is an object to be assayed. in a bioluminescence method, a method for quantitatively determining an adenosine phosphate ester using the reagent, In a method which comprises adding pyrophosphoric acid and a method for quantitatively determining a Substance to the reaction System at the time when a bioluminescence taking part in an ATP conversion reaction System using the reagent. reaction progressed in Some extent and the amount of The ATP conversion reaction system in the invention luminescence Started to lower, and thereby forming intense means any reaction System which is composed, for example luminescence momentarily (Arch. Biochem. BiophyS. 46, of a combination of enzymes and Substrates, and wherein 15 399–416; 1955), the peak intensity of light is increased again ATP is formed or consumed at the time of reaction. in the middle of the luminescence reaction and thereby the For example, it includes, So far known ATP assaying extension of the luminescence time is attempted, but it is Systems each utilizing a bioluminescence reaction (a reac impossible to Stabilize luminescence over a long time. tion System wherein luciferase is made to act on ATP, luciferin, dissolved oxygen and magnesium ion to form In a method which comprises using, in a bioluminescence AMP, pyrophosphoric acid, oxyluciferin, carbon dioxide and method, a D-luciferin analog in the reaction System as a light), and an assaying System for an adenosine phosphate competitive inhibitor (Patent Kokai No. 55-13893), ester such as AMP is, ADP, ATP or cyclic AMP disclosed although the amount of luminescence can be maintained at in the invention, and in addition a reaction System wherein 25 a certain level and Stably for a long time, the intensity of another reaction System is combined with Such an assay luminescence is Suppressed or inhibited by at least 25%, System (reaction System) and in the reaction system after the particularly 50 to 90% by the addition of the inhibitor, and combination, ATP is formed or consumed at the time of thus the method has a drawback that the lowering of reaction. Sensitivity is compelled. 2. Description of the Related Prior Art Heretofore, there has been known an ATP-quantitatively In a method which comprises using a polyphosphoric acid determining method by a So-called bioluminescent method compound and a Sulfhydryl compound together in a biolu wherein a bioluminescence reagent comprising luciferin, luciferase and magnesium ions (or other metallic ions) is minescence method (Patent Kokai No. 8-47399), the peak reacted with a Sample containing adenosine triphosphate intensity of light formed in the luminescence reaction is (hereinafter referred to as ATP) and the amount of lumines 35 increased and thereby the extension of the luminescence cence formed is quantitatively determined. time is attempted, but it is impossible to maintain lumines However, although this method has an advantage that ATP cence Stably over a long time. can be determined quickly, it has a disadvantage that the Further, as to the above bioluminescence methods, there Stability of luminescence is poor, and more specifically 40 luminescence fades out in a very short time, and therefore it is no regeneration of ATP in any of them, and they each have has a problem that for Securing Sensitivity and accuracy, it a drawback that luminescence decays with time lapse as ATP gets necessary to control the reaction time Strictly and use a is consumed. luminometer, a measuring apparatus equipped with an auto injection function for capturing luminescence fading out in 45 Therefore, for maintaining the amount of luminescence a short time. Stably for a long time without decaying, it is compelled to Heretofore, Some techniques for Stabilizing luminescence examine the Sub Strate concentrations, enzyme over a long time in this bioluminescence method have been concentrations, pH, temperature, addition of a Suppressor or developed, but any of them has problems to be improved. inhibitor, etc., and even thereby Sufficiently Satisfactory Namely, in a method which comprises adding coenzyme 50 results cannot be expected. A (hereinafter abbreviated as CoA) to a reaction System (Patent Kohyo No. 6-500921), a luciferase-luciferin reaction On the other hand, a quantitative determination method of is carried out in the presence of CoA, under a condition of cyclic AMP using a bioluminescence method shown by the lowering the peak intensity of light formed in the reaction, following reaction formula is known. by a small amount (for example, by about 3 to 30% of the 55 peak intensity under the absence of this condition), and Reaction Formula thereby all the amount of light emitted in the reaction (i.e., Cyclic 3',5'-nucleotide amount measured by integrating the curve of intensity to hosphodiesterase time) is increased, and thereby all the light output can be Cyclic AMP (Reaction 1) AMP measured more simply and more accurately. 60 AMP - ATP Adenylate kinase G 2ADP However, in this method, it is compelled to lower the peak Mg2+ (Sensitivity) of light formed by the reaction, and the relations (Reaction 2) between the intensity of luminescence and time get to be curve relations, and the amount of luminescence gradually ADP + phosphoenol Pyruveinases Pyruvic acid pyruvic acid Mg: - ATP decayS. Therefore, it cannot be expected to maintain the 65 (Reaction 3) amount of luminescence at a high level stably for a long time. 5,891,659 3 4 -continued dikinase, phosphoenolpyruvic acid, pyrophosphoric acid, Reaction Formula magnesium ion or other metallic ions, luciferin and luciferase, the invention is also (3) a method for quantita ATP Luciferin + O2 tively determining an adenosine phosphate ester which comprises reacting the above bioluminescence reagent with Mg2+ Y Luciferase a Sample containing the adenosine phosphate ester, and measuring the amount of luminescence formed, the inven AMP + pyrophosphoric Oxyluciferin + CO2 + Light tion is also (4) a method for quantitatively determining ATP acid which compriseS reacting the above bioluminescence reagent with a Sample containing ATP, and measuring the (Reaction 4) amount of luminescence formed, the invention is also (5) a method for quantitatively determining AMP which com priseS reacting the above bioluminescence reagent with a This method is characterized by a method of quantita Sample containing AMP, and measuring the amount of tively determining cyclic AMP by Reaction 1 wherein cyclic luminescence formed, the invention is also (6) a method for AMP is hydrolyzed with cyclic-3',5'-nucleotide phosphodi 15 quantitatively determining ADP which comprises reacting a esterase to form AMP in the reaction system, Reaction 2 bioluminescence reagent comprising pyruvate orthophos wherein the AMP is reacted with adenylate kinase in the phate dikinase, phosphoenolpyruvic acid, pyrophosphoric presence of magnesium ion and a trace amount of ATP to acid, magnesium ion or other metallic ions, luciferin, convert it to ADP, Reaction 3 wherein the ADP is reacted luciferase and pyruvate kinase with a Sample containing ADP, and measuring the amount of luminescence formed, with pyruvate kinase in the presence of magnesium ion and the invention is also (7) a method for quantitatively deter phosphoenolpyruvic acid to convert it to ATP and pyruvic mining cyclic AMP which compriseS reacting a biolumines acid, Reaction 4 wherein the ATP is reacted with luciferase cence reagent comprising pyruvate orthophosphate dikinase, in the presence of luciferin, magnesium ion (or other metal phosphoenolpyruvic acid, pyrophosphoric acid, magnesium lic ions) and dissolved oxygen to form luminescence, and ion or other metallic ions, luciferin, luciferase and cyclic measuring the amount of luminescence formed in Reaction 25 3',5'-nucleotide phosphodiesterase with a Sample containing 4 (METHODS IN ENZYMOLOGY 38, 62–65; 1974). cyclic AMP, and measuring the amount of luminescence However, in this method, AMP formed from cyclic AMP formed, the invention is also (8) a method for quantitatively is converted to ADP by the reaction with adenylate kinase in determining a Substance taking part in an ATP conversion the presence of a trace amount of ATP, and the ADP is reaction System which comprises adding a bioluminescence converted to ATP with pyruvate kinase, and therefore, it is reagent comprising pyruvate orthophosphate dikinase, phos required to add a trace amount of ATP in advance to the phoenolpyruvic acid, pyrophosphoric acid, magnesium ion reaction System. or other metallic ions, luciferin and luciferase to the ATP This additions of ATP is not desirable because when conversion reaction System to cause reaction, and measuring the amount of luminescence formed, and invention is also cyclic AMP is the object to be assayed, the ATP causes the 35 (9) a method for quantitatively determining pyruvate ortho increase of the background. phosphate dikinase, phosphoenolpyruvic acid, pyrophos AS a result, a blank value corresponding to cyclic AMP of phoric acid, luciferin, luciferase, pyruvate kinase, acetate at least 1.3x10 M is formed, and thus the method has a kinase, creatine kinase or cyclic-3',5'-nucleotide phosphodi problem that cyclic AMP cannot be determined with high esterase utilizing the above quantitatively determining Sensitivity. 40 method. Thus the invention aims to provide a bioluminescence BRIEF DESCRIPTION OF THE DRAWINGS reagent that the amount of luminescence is maintained in a FIG. 1 shows the change with time lapse of the amount of high level and moreover Stably without decaying for a long luminescence corresponding to each AMP concentration. time in a bioluminescence reaction, and provide a method The axis of ordinate shows the amount of luminescence for quantitatively determining an adenosine phosphate ester 45 (RLU/s) and the axis of abscissa shows time (second). or a Substance taking part in an ATP conversion reaction in FIG. 2 shows a standard curve showing the relations high Sensitivity and high accuracy using an inexpensive and between the AMP concentration and the amount of lumi Simple measuring apparatus. nescence (at 10 minutes after the start of the reaction). The SUMMARY OF THE INVENTION axis of ordinate shows the amount of luminescence (RLU/s) 50 and the axis of abscissa shows the AMP concentration (M). The present inventors have intensely made Sequential FIG.3 shows the change with time lapse of the amount of researches to Solve these problems, and they have found that luminescence corresponding to each ATP concentration. The when a reagent comprising ATP regenerating enzyme, Sub axis of ordinate shows the amount of luminescence (RLU/s) Strates of ATP regenerating enzyme, magnesium ion, and the axis of abscissa shows time (second). luciferin and luciferase is reacted with a Sample containing 55 FIG. 4 shows a standard curve showing the relations an adenosine phosphate ester, the amount of luminescence is between the ATP concentration and the amount of lumines maintained in a high level and moreover Stable without cence (at 10 minutes after the start of the reaction). The axis decaying for a long time, and it gets possible to quantita of ordinate shows the amount of luminescence (RLU/s) and tively determine the adenosine phosphate ester in high the axis of abscissa shows the ATP concentration (M). Sensitivity and high accuracy using an inexpensive and 60 FIG. 5 shows the change with time lapse of the relative Simple measuring apparatus wherein Said ATP regenerating amount of luminescence in the case where the ATP concen enzyme catalyzes the formation of ATP from AMP. tration is 1.0x10' M. Thus the invention is (1) a bioluminescence reagent comprising ATP regenerating enzyme, Substrates of ATP DETAILED DESCRIPTION OF THE regenerating enzyme, magnesium ion or other metallic ions, 65 PREFERRED EMBODIMENTS luciferin and luciferase, the invention is also (2) a biolumi First, the bioluminescence reagent used in the invention is nescence reagent comprising pyruvate orthophosphate prepared using as constituents at least pyruvate orthophos 5,891,659 S 6 phate dikinase or phosphoenolpyruvate Synthase or other The fracture liquid was centrifuged at 7,000 rp.m. for 15 ATP regenerating enzyme, Substrates of ATP regenerating minutes, and the Supernatant part was collected whereby 620 enzyme, magnesium ion or other metallic ions, luciferin and ml of the liquid was obtained. luciferase. For example, a bioluminescence reagent used in Step 2 (the first QAE-Sephadex chromatography) the invention is prepared using as constituents at least Ammonium Sulfate was dissolved in the above 620 ml pyruvate orthophosphate dikinase, phosphoenolpyruvic Solution at a rate of 2 g/100 ml, and the Solution was put on acid, pyrophosphoric acid, magnesium ion or other metallic about 700 ml of QAE-Sephadex previously equilibrated ions, luciferin and luciferase. with the above Buffer A (pH 7.5) containing 0.15M ammo Among the above constituents of the bioluminescence nium Sulfate to adsorb the enzyme. The resin (gel) was reagent, pyruvate orthophosphate dikinase is a known washed with the above Buffer A (pH 7.5) containing 0.15M enzyme catalyzing Such a reaction that it acts on AMP, ammonium Sulfate to remove unabsorbed proteins, and then phosphoenolpyruvic acid and pyrophosphoric acid in the elution was carried out using the above Buffer A (pH 7.5) presence of magnesium ion to form ATP pyruvic acid and containing 0.6M ammonium Sulfate. phosphoric acid, and also catalyzing the reverse reaction Step 3 (the Second QAE-SephadeX chromatography) thereof. 15 The eluate was concentrated and dialyzed against Buffer The physical properties and production process thereof A (pH. 7.5) by a hollow fiber ultrafiltration apparatus (PAN are already known, and it is relatively easily available. 13-DX, made by Asahi Medical Co.), and then put on AS the enzyme, those derived from plants and from QAE-Sephadex column (diameter 6 cm x15 cm) equili microorganisms are known. brated in advance with the Buffer A (pH 7.5) to adsorb the AS those derived from plants, there can, for example, be enzyme. resin was washed with the Buffer A (pH 7.5) mentioned enzymes from the leaves of corn Biochemistry containing 0.15M ammonium sulfate to remove unabsorbed 12, 2862-2867 (1973) and the leaves of Sugarcane The proteins, and then the enzyme was eluted by a linear Biochemical Journal 114, 117-125 (1969)). concentration gradient using 1.5 L each of the Buffer A (pH AS those derived from microorganisms, there can, for 7.5) containing 0.15M ammonium sulfate and the Buffer A example, be mentioned enzymes produced by microorgan 25 (pH 7.5) containing 0.8M ammonium sulfate. iSms belonging to Propionibacterium Shermanii Step 4 (Butyl-Toyopearl chromatography) Biochemistry 10, 721-729 (1971), Acetobacter xylinum The active fractions were recovered, ammonium Sulfate Journal of Bacteriology (1970)), Bacteroides symbiosus was added to adjust the ammonium Sulfate concentration to Metoods in Enzymology 42, 199-212 (1975) and the 1M, and the mixture was put on Butyl-Toyopearl (made by genus Microbispora (for example, Microbispora thermoro Toso Co., Ltd.) column (diameter 4.5 cmx15 cm) equili sea IFO 14047), etc. brated with Buffer A (H 7.5) contained 1M ammonium A Specific example of production of the enzyme produced Sulfate to adsorb the enzyme. by a microorganism is shown below. The enzyme was eluted with a linear gradient of an 50 ml of a medium composed of 0.2% yeast extract, 0.2% ammonium sulfate concentration (1.0M-0M) in 1.2 L of the Casamino acid, 0.001% ferrous sulfate, 0.05% potassium 35 Buffer A. chloride, 0.1% dipotassium hydrogenphosphate, 0.05% Step 5 (gel filtration chromatography) magnesium sulfate and 0.3% lactic acid (pH 7.0) was put in The active fractions were concentrated to 2 ml using an a Sakaguchi flask (capacity: 500 ml), and sterilized at 121 ultrafiltration membrane apparatus made by Amicon Co. C. for 15 minutes. (cut-off: 10,000), and 100 ul of the concentrate was put on 40 TSKG 3000 SWX1 (diameter 0.76 cm x30 cm x2) equili Microbispora thermorosea IFO 14047 was inoculated in brated in advance with 20 mM HEPES buffer (pH 7.5) the medium, and Shaking cultured at 45 C. overnight to give containing 0.3M ammonium sulfate, whereby gel filtration a culture. was carried out. 50 ml of this culture was inoculated in 1 L of a medium The whole enzyme was subjected to gel filtration and the having the same composition as above in a 5-L Sakaguchi 45 active fraction were concentrated. The concentrate was flask, and cultured overnight to give a culture. Subjected again to the same column operations as above to 500 ml portions of this culture were inoculated in 20 L of carry out gel filtration. The whole enzyme was Subjected to a medium having the same composition as above in each of the gel filtration, and 5.4 ml of the active fraction eluted two 30-L jar fermenters, respectively, and aeration Stirring were collected. cultured at 45 C. for 24 hours under the conditions of an 50 The above fraction showed the single protein band on aeration rate of 20 L/min and a stirring rate of 300 rp.m. SDS-polyacrylamide gel electrophoresis and contained 6.65 After the completion of the culture, the cells were col mg protein with a specific activity of 9.92 U/mg. lected from 40 L of this culture using Microsa (made by ASSay method of pyruvate orthophosphate dikinase activ Asahi Chemical Industry Co., Ltd.). 20 mM HEPES buffer ity. (pH 7.5) containing 5 mM EDTA, 1 mM MgSO4 and 1 mM 55 (Method to determine ATP formed by a luminescence DTT (hereinafter referred to as Buffer A) was added to part method) (200 g) of the cells, and the cells were sufficiently suspended 180ul of 50 mM BIS-TRIS PROPANE buffer (pH 6.8) to make the total volume 700 ml. Purification of the enzyme containing 3 mM magnesium Sulfate, 25 mM ammonium 700 ml of the above cell suspension was subjected to the Sulfate, 2 mM 2-mercaptoethanol, 2 mM pyrophosphoric operations of each Stage shown below. 60 acid, 2 mM phosphoenolpyruvic acid and 0.1 mM AMP, Step 1 (preparation of a crude enzyme Solution) preincubated at 37 C., was pipetted into a microtube, and 20 8.75 g of lysozyme (made by Nagase Biochemical Indus All of an enzyme Solution having a appropriate activity was try Co.) was added to 700 ml of the above cell suspension, added to carry out reaction for 15 minutes, and the reaction the mixture was left alone at room temperature for 2 hours Solution was boiled for 3 minutes in boiling water to cease with gentle Stirring, 23.1 g of diammonium hydrogenphoS 65 the reaction. The reaction solution was diluted suitably, 50 phate was added, the mixture was stirred at room tempera All of the dilution was put in a test tube, 50 ul of “Lucifer LU” ture for further 2 hours, and then the cells were fractured. (made by KIKKOMAN CORPORATION) solution was 5,891,659 7 8 added dropwise, and then the amount of luminescence was dikinase, (h) dithiothreitol and (i) EDTA as stabilizers of measured. Separately, a graph was prepared in advance by luciferase, and (i) HEPES buffer (pH 7.0) as a stabilizer of examining the relations between the concentration of ATP the reaction System, and they are not essential components. and the amount of luminescence using ATP Standard Solu Description is made below on the mechanisms of the tions having known concentrations. The umol of ATP invention. formed per minute at 37 C. was calculated using this graph, The method for determination of AMP according to the and this value is assumed to be the activity unit in the invention can be shown by the following reaction formula. enzyme Solution used. The amount of enzyme which forms 1 tumol of ATP per minute at 37 C. is defined to be 1 unit (U). Pyruvic acid + Phosphoenolpyruvic Preferred concentration ranges of the constituents of the phosphoric acid acid bioluminescence reagent of the invention are shown below. Pyruvate (a) pyruvate orthophosphate dikinase; a concentration of 2+ orthophosphate 0.001 U/ml (final concentration) or more, particularly Mg dikinase 0.002 to 100 U/ml (final concentration). 15 ------(b) phosphoenolpyruvic acid; a concentration of 0.1 mM ATP AMP + Pyrophosphoric (final concentration) or more, particularly 0.5 to 8.0 ------as------mM (final concentration). (c) pyrophosphoric acid; a concentration of 1.0 uM (final concentration) or more, particularly 5.0 to 100 uM (final concentration). (d) magnesium ion; a concentration of 1.0 mM (final Luciferin + O2 Oxyluciferin + CO2 + Light concentration) or more, particularly 5.0 to 100 mM S (final concentration). 25 (e) luciferin; a concentration of 5.0 uM (final In the above reaction, as shown in the broken line frame, concentration) or more, particularly 50.0 to 10,000 uM a reaction (a) to make pyruvate orthophosphate dikinase act (final concentration). on AMP, pyrophosphoric acid, phosphoenolpyruvic acid and (f) luciferase; a concentration of 0.1 mg/ml (final magnesium ion and thereby form ATP, pyruvic acid and concentration) or more, particularly 0.5 to 20 mg/ml phosphoric acid is carried out, and then, as shown in the (final concentration). alternate long and short dash line frame, a reaction (b) to (g) ammonium Sulfate; a concentration of 0.1 mM (final make luciferase act on ATP, luciferin, dissolved oxygen and concentration) or more, particularly 0.5 to 100 mM magnesium ion and thereby form AMP, oxyluciferin, carbon (final concentration). dioxide and light is carried out. Thus, the determination of AMP is carried out by com (h) dithiothreitol; a concentration of 0.1 mM (final 35 bining Reaction (a) and Reaction (b) to form an ATP concentration) or more, particularly 0.5 to 10 mM (final conversion reaction System and measuring the amount of concentration). luminescence formed. (i) EDTA; a concentration of 0.1 mM (final concentration) Description is made below on the main points of this or more, particularly 0.5 to 10 mM (final reaction. concentration). 40 First, AMP is converted to ATP by Reaction (a), and then (i) HEPES buffer (pH 7.0); a concentration of 10 mM by Reaction (b) ATP is consumed to form luminescence and (final concentration) or more, particularly 20 to 200 AMP, and this AMP is subjected to Reaction (a) to regen mM (final concentration). erate ATP (k) cyclic-3',5'-nucleotide phosphodiesterase; a concen This ATP is subjected again to Reaction (b) to consume tration of 0.01 U/ml (final concentration) or more, 45 ATP and cause luminescence. particularly 0.02 to 10 U/ml (final concentration). Thereafter, these two reactions occur Simultaneously, con (l) pyruvate kinase; a concentration of 0.01 U/ml (final tinuously and repeatedly. concentration) or more, particularly 0.02 to 10 U/ml It is confirmed that luminescence formed by this series of (final concentration). the ATP conversion reaction system is stable over 10 min AS luciferase used herein, any luciferase used in ATP 50 utes or more in a high level without decaying, and Still Stable assay by luciferin-luciferase Systems may be used. after the time lapse of 5 hours almost without decaying. For example, there can be mentioned luciferases derived Next, description is made on the method for determina from Luciola cruciata, Luciola lateralis, American firefly, tion of ATP according to the invention. etc., these luciferaseS produced by genetic recombination The method for determination of ATP of the invention can methods, etc. 55 be represented by the same reaction formula as in the above AS buffers, there can further be used Tris-Succinic acid method for determination of AMP. buffer, BIS-Tris PROPANE buffer, MES buffer, etc. In this reaction, first ATP is consumed by Reaction (b) to AS preferred concentration, the same concentration range cause luminescence and form AMP, this AMP formed is as in the above HEPES buffer can be mentioned, and as converted to ATP by Reaction (a), and this ATP regenerated preferred pH range, 6.5 to 8.0 can be mentioned. 60 is then Subjected again to Reaction (b) to consume ATP and Various compounds may be added to the bioluminescence cause luminescence. reagent of the invention to effect the enzymatic reaction and Thereafter, these two reactions occur Simultaneously, con luminescence reaction Smoothly. tinuously and repeatedly. AS Such compounds, there can, for example, be men Luminescence formed in the ATP conversion reaction tioned Stabilizers, Surfactants, activators, etc. 65 System of the invention is stable over at least minutes or Namely, among the above constituents, (g) ammonium more in a high level without decaying, and Still Stable after Sulfate is used as an activator of pyruvate orthophosphate the time lapse of 5 hours almost without decaying. 5,891,659 9 Next, description is made on the method for determina -continued tion of cyclic AMP according to the invention. This determination method can be represented by the Pyruvic acid.PhosphoenolpyruvicS” following reaction formula. phosphoric acid acid Pyruvate Mg2+ orthophosphate Cyclic 3',5'-nucleotide dikinase Cyclic AMP phosphodiesterase Ge. *AMP ------r * ATP AMP + Pyrophosphoric acid es ------Pyruvic acid TPhosphoenolpyruvics phosphoric acid acid 15 Luciferin + O2 Oxyluciferin + CO2 + Light

Pyruvate ...... scr ...i.41.Mg2. N. orthophosphatedikinase :- i i This determination method is characterized in that another ATP *AMP + Pyrophosphoric acid : reaction (d) to react ADP with pyruvate kinase in the ------presence of magnesium ion and phosphoenolpyruvic acid to form ATP and pyruvic acid, shown in the alternative long Mg2+ : and three Short dashes line frame, is combined with Reaction (a) (as shown above) and Reaction (b) (as shown above). : : 25 In this connection, acetate kinase or creatine kinase may Luciferin + O2 Oxyluciferin + CO2+Light : be used in place of pyruvate kinase used. In the above reaction, first, as shown in the alternative S long and three short dashes line frame, Reaction (d) to react ADP with pyruvate kinase in the presence of magnesium ion This determination method is characterized in that an ATP and phosphoenolpyruvic acid and thereby form ATP and conversion reaction System is constituted by combining pyruvic acid is carried out, thereby ADP is converted to ATP, another reaction (c) to hydrolyze cyclic AMP with cyclic then by Reaction (b) this ATP is consumed to cause lumi 3',5'-nucleotide phosphodiesterase and thereby form AMP, nescence and form AMP, and then by Reaction (a) this AMP shown in the alternative long and two short dashes line is converted again to ATP. This ATP is subjected again to 35 Reaction (b), to ATP is consumed, luminescence occurs and frame, with Reaction (a) (as shown above) and Reaction (b) ATP is converted to AMP. (as shown above). Thereafter, these two reactions occur Simultaneously, con In the above reaction, first, as shown in the alternative tinuously and repeatedly. long and two short dashes line frame, Reaction (c) to Luminescence formed in this series of ATP conversion hydrolyze cyclic AMP with cyclic-3',5'-nucleotide phos 40 reaction System is Stable over at least 10 minutes or more in phodiesterase to form AMP is carried out and thereby cyclic a high level without decaying, and Still Stable after the time AMP is converted to AMP, then by Reaction (a) AMP is lapse of 5 hours almost without decaying. converted to ATP, and then by Reaction (b) ATP is consumed Since luminescence is formed exactly depending on to cause luminescence and form AMP. This AMP is con (directly proportional to) the concentration of any of a Series 45 of the enzymes and Substrates taking part in the above ATP verted to ATP by Reaction (a). This ATP regenerated is conversion reaction Systems, it is possible to determine a Subjected again to Reaction (b) to consume ATP and cause Substance taking part in the ATP conversion reactions Such luminescence. as any of the enzymes and Substrates in high Sensitivity and Thereafter, these two reactions occur Simultaneously, con high accuracy and moreover in a short time by measuring the tinuously and repeatedly. 50 amount of luminescence. Luminescence formed in the ATP conversion reaction Thus, according to the invention, it is possible to deter System is stable over at least 10 minutes or more in a high mine many Substances taking part in ATP conversion reac level without decaying, and Still Stable after the time lapse tion Systems, for example, adenosine phosphate esterS Such of 5 hours almost without decaying. as AMP, ADP, ATP and cyclic AMP, pyruvate orthophos Next, description is made on the method for determina 55 phate dikinase, phosphoenolpyruvic acid, pyrophosphoric tion of ADP according to the invention. acid, luciferin, luciferase, pyruvate kinase, acetate kinase, This determination method can be represented by the creatine kinase, cyclic-3',5'-nucleotide phosphodiesterase, following reaction formula. etc. in high Sensitivity and high accuracy and moreover in a Short time. 60 ADP+ phosphoenol Pyruveinases Pyruvic acid EXAMPLE 1. Pyruvic acid Mg2+ + ATP (Reaction 3) (Preparation of a bioluminescence reagent for AMP determination) (S 65 The Substances mentioned in the following (a) to () were dissolved in ultra-pure water So that the concentration thereof could be the respective final concentrations, whereby 5,891,659 11 12 a bioluminescence reagent for AMP determination was -continued prepared. (b) phosphoenolpyruvic acid 4.2 mM (final concentration) (made by Boehringer (a) pyruvate orthophosphate 0.4 U/ml Mannheim GmbH) dikinase (final concentration) (c) pyrophosphoric acid 42 mM (final concentration) (the preparation process is (d) magnesium ion 15 mM (final concentration) as described above) (e) luciferin 1.5 mM (final concentration) (b) phosphoenolpyruvic acid 4.2 mM (final concentration) (made by Sigma Co.) (made by Boehringer (f) luciferase 1.4 mg/ml (final concentration) Mannheim GmbH) (made by (c) pyrophosphoric acid 42 uM (final concentration) KIKKOMAN (d) magnesium ion 15 mM (final concentration) CORPORATION) (e) luciferin 1.5 mM (final concentration) (g) ammonium sulfate 7.5 mM (final concentration) (made by Sigma Co.) (h) dithiothreitol 1.0 mM (final concentration) (f) luciferase 1.4 mg/ml (final concentration) (i) EDTA 1.0 mM (final concentration) (made by (i) HEPES buffer (pH 7.0) 50 mM (final concentration) KIKKOMAN 15 CORPORATION) (g) ammonium sulfate 7.5 mM (final concentration) In the above, (g) ammonium Sulfate is used to strengthen (h) dithiothreitol 1.0 mM (final concentration) the activation of pyruvate orthophosphate dikinase, (h) (i) EDTA 1.0 mM (final concentration) dithiothreitol and (i) EDTA to stabilize luciferase and () (i) HEPES buffer (pH 7.0) 50 mM (final concentration) HEPES buffer (pH 7.0) to stabilize the reaction system. In the above, (g) ammonium Sulfate is used to strengthen EXAMPLE 4 the activation of pyruvate orthophosphate dikinase, (h) (Method for determination of ATP) dithiothreitol and (i) EDTA to stabilize luciferase and () 100ul of the bioluminescence reagent prepared in HEPES buffer (pH 7.0) to stabilize the reaction system. Example 3 and 100 ul of each of samples containing ATP in 25 Several concentrations (standard Solutions) were reacted, EXAMPLE 2 and the amount of luminescence formed was measured by Lumat LB 9501, luminometer made by Berthold Co. to carry (Method for determination of AMP) out the determination of ATP. 100 ul of the bioluminescence reagent prepared in The measurement of the amount of luminescence was Example 1 and 100 ul of each of samples containing AMP carried out by a method to measure the amount of lumines in Several concentrations (standard Solutions) were reacted, cence per one Second at intervals of 20 Seconds over 10 and the amount of luminescence formed was measured by minutes after the Start of the reaction. Lumat LB9501, luminometer made by Berthold Co. to carry The change with time lapse of the amount of lumines out the determination of AMP. cence corresponding to each ATP concentration was The measurement of the amount of luminescence was 35 examined, and the results were shown in FIG. 3. carried out by a method to measure the amount of lumines Further, the relations between the ATP concentration and cence per one Second at intervals of 20 Seconds over 10 the amount of luminescence (value at 10 minutes after the minutes after the Start of the reaction. Start of the reaction) were examined, and the results were The change with time lapse of the amount of lumines shown in FIG. 4 as an ATP calibration curve. cence corresponding to each AMP concentration was 40 It is understood from the results of FIG. 3 that according examined, and the results were shown in FIG. 1. to the invention, in the method for determination of ATP Further, the relations between the AMP concentration and utilizing the bioluminescence reaction, the amount of lumi the amount of luminescence (value at 10 minutes after the neScence is stable in a high level and moreover without Start of the reaction) were examined, and the results were decaying for a long time. shown in FIG. 2 as an AMP calibration curve. 45 Further, it is understood from the results of FIG. 4 that It is understood from the results of FIG. 1 that according according to the invention, 10 to 10M of ATP can be to the invention, in the method for determination of AMP determined in high Sensitivity and high accuracy. utilizing the bioluminescence reaction, the amount of lumi neScence is stable in a high level and moreover without EXAMPLE 5 decaying for a long time. 50 (Preparation of a bioluminescence reagent for ADP Further, it is understood from the results of FIG. 2 that determination) according to the invention, 10 to 10M of AMP can be The Substances mentioned in the following (a) to (k) were determined in extremely high Sensitivity and high accuracy. dissolved in ultra-pure water So that the concentrations thereof could be the respective final concentrations, whereby EXAMPLE 3 55 a bioluminescence reagent for ADP determination was pre (Preparation of a bioluminescence reagent for ATP pared. determination) The Substances mentioned in the following (a) to (j) were (a) pyruvate orthophosphate 0.4 U/ml dissolved in ultra-pure water So that the concentrations dikinase (final concentration) thereof could be the respective final concentrations, whereby 60 (the preparation process is as described above) a bioluminescence reagent for ATP determination was pre (b) phosphoenolpyruvic acid 4.2 mM (final concentration) pared. (made by Boehringer Mannheim GmbH) (c) pyrophosphoric acid 42 uM (final concentration) (a) pyruvate orthophosphate 0.4 U/ml 65 (d) magnesium ion 15 mM (final concentration) dikinase (final concentration) (e) luciferin 1.5 mM (final concentration) 5,891,659 13 14 -continued Seconds later becoming /100, 200 Seconds later becoming (made by Sigma Co.) /1,000). Therefore, it will be understood that for measuring (f) luciferase 1.4 mg/ml (final concentration) the rapid flash at the initial Stage of the reaction, particularly (made by immediately after the Start of the reaction, an injection KIKKOMAN method for quickly mixing a Sample containing ATP with the CORPORATION) (g) ammonium sulfate 7.5 mM (final concentration) bioluminescence reagent, work requiring special trouble or (h) dithiothreitol 1.0 mM (final concentration) a delicate measuring apparatuS Such as a luminometer gets (i) EDTA 1.0 mM (final concentration) neceSSary. (i) HEPES buffer (pH 7.0) 50 mM (final concentration) On the other hand, it will be understood that in the method (k) pyruvate kinase 0.4 U/ml (final concentration) (made by Boehringer for determination of ATP of the invention using pyruvate Mannheim GmbH) orthophosphate dikinase (shown by the broken line), the amount of luminescence is maintained in a high level and Stably for a long time (10 minutes) without decaying. EXAMPLE 6 Therefore, it will be understood that according to the (Preparation of a bioluminescence reagent for cyclic AMP 15 invention, Since a condition very excellent for the continu determination) ouS analysis of an ATP conversion reaction is given, and the The Substances mentioned in the following (a) to (k) were amount of luminescence measured by integrating the locus dissolved in ultra-pure water So that the concentrations of the intensity of luminescence to time is remarkably thereof could be the respective final concentrations, whereby increased compared with the comparative example, deter a bioluminescence reagent for cyclic AMP determination mination in high Sensitivity and high accuracy gets possible was prepared. using an inexpensive and Simple measuring apparatus. Further, it will be understood from the results of FIG. 1 to FIG. 5 obtained in the above Example 2, 4 and 7 that since (a) pyruvate orthophosphate 0.4 U/ml luminescence is formed extremely exactly depending on dikinase (final concentration) 25 (the preparation process is (directly proportional to) the concentration of any of a Series as described above) of the enzymes and Substrates taking part in the ATP (b) phosphoenolpyruvic acid 4.2 mM (final concentration) conversion reaction Systems, it is possible to determine a (made by Boehringer Mannheim GmbH) Substance taking part in the ATP conversion reaction Such as (c) pyrophosphoric acid 42 uM (final concentration) any of the enzymes and Substrates in high Sensitivity and (d) magnesium ion 15 mM (final concentration) high accuracy and moreover in a short time by measuring the (e) luciferin 1.5 mM (final concentration) amount of luminescence. Namely, it will be understood that (made by Sigma Co.) (f) luciferase 1.4 mg/ml (final concentration) if the bioluminescence reagent of the invention is used, it is (made by possible to determine any of many Substances taking part in KIKKOMAN the ATP conversion reaction Systems, for example, adenos CORPORATION) 35 ine phosphate esters such as AMP, ADP, ATP and and cyclic (g) ammonium sulfate 7.5 mM (final concentration) AMP, pyruvate orthophosphate dikinase, phosphoenolpyru (h) dithiothreitol 1.0 mM (final concentration) (i) EDTA 1.0 mM (final concentration) Vic acid, pyrophosphoric acid, luciferin, luciferase, pyruvate (i) HEPES buffer (pH 7.0) 50 mM (final concentration) kinase, acetate kinase, creatine kinase, cyclic-3',5'- (k) cyclic-3',5'-nucleotide 0.4 U/ml (final concentration) nucleotide phosphodiesterase, etc. in high Sensitivity and phosphodiesterase (made by 40 high accuracy and moreover in a short time. Sigma Co.) Further, it will also be understood that the biolumines cence reagent of the invention can be utilized preferably in the assay of adenosine phosphate esterS Such as ATP, and EXAMPLE 7 Substances and enzymes taking part in ATP conversion (Method for determination of ATP) 45 100 ul of the bioluminescence reagent for ATP determi reaction Systems in clinical chemistry, clinical microbiology, nation prepared in Example 3 and 100 ul of a Sample biochemical research, biological research and bioenergetics. containing ATP in a concentration of 1.0x10"M (final What is claimed is: concentration) (a standard Solution) were reacted, and the 1. A bioluminescence reagent comprising: amount of luminescence formed was measured by Lumat 50 (a) ATP regenerating enzyme which catalyzes the forma LB 9501, luminometer made by Berthold Co., and thereby tion of ATP from AMP, the change of luminescence with time lapse was examined. (b) Substrates of ATP regenerating enzyme, The measurement of the amount of luminescence was carried out by a method to measure the amount of lumines (c) magnesium ion or another divalent metallic cation, cence per one Second at intervals of one Second over 10 55 (d) luciferin and minutes after the Start of the reaction. (e) luciferase. For comparison, the same operations as in the above 2. A bioluminescence reagent comprising: method for determination of ATP were carried out to deter (a) pyruvate orthophosphate dikinase, mine ATP except that (a) pyruvate orthophosphate dikinase (b) phosphoenolpyruvic acid, was not used. 60 The results are shown in FIG. 5. (c) pyrophosphoric acid, As apparent from the results of FIG. 5, in the case of the (d) magnesium ion or another divalent metallic cations, method for determination of ATP where pyruvate orthophos (e) luciferin and phate dikinase was not used (continuous line; the method of (f) luciferase. the comparative example), the amount of luminescence 65 3. A method for quantitatively determining an adenosine reaches the peak extremely rapidly, namely a flash of light phosphate ester which comprises reacting a biolumines is emitted, and then luminescence decays rapidly (100 cence reagent comprising: 5,891,659 15 16 (a) pyruvate orthophosphate dikinase, (f) luciferase (b) phosphoenolpyruvic acid, with a Sample containing AMP, and measuring the amount (c) pyrophosphoric acid, of luminescence formed to determine AMP quantitatively. (d) magnesium ion or another divalent metallic cation, 6. The method according to claim 3 which comprises (e) luciferin and reacting a bioluminescence reagent comprising: (f) luciferase (a) pyruvate orthophosphate dikinase, with a Sample containing the adenosine phosphate ester, (b) phosphoenolpyruvic acid, measuring the amount of luminescence formed and then (c) pyrophosphoric acid, quantitatively determining an amount of the adenosine phos phate ester with a Standard curve obtained by using an (d) magnesium ion or other divalent metallic cation, adenosine phosphate ester Standard Solution having a pre (e) luciferin, determined concentration. (f) luciferase and 4. The method according to claim 3 which comprises (g) pyruvate kinase reacting a bioluminescence reagent comprising: 15 with a Sample containing ADP, and measuring the amount of (a) pyruvate orthophosphate dikinase, luminescence formed to determine ADP quantitatively. (b) phosphoenolpyruvic acid, 7. The method according to claim 3 which comprises (c) pyrophosphoric acid, reacting a bioluminescence reagent comprising: (d) magnesium ion or another divalent metallic cation, (a) pyruvate orthophosphate dikinase, (e) luciferin and (b) phosphoenolpyruvic acid, (f) luciferase (c) pyrophosphoric acid, with a Sample containing ATP, and measuring the amount of (d) magnesium ion or another divalent metallic cation, luminescence formed to determine ATP quantitatively. (e) luciferin, 5. The method according to claim 3 which comprises 25 (f) luciferase and reacting a bioluminescence reagent comprising: (g) cyclic-3',5'-nucleotide phosphodiesterase (a) pyruvate orthophosphate dikinase, with a Sample containing cyclic AMP, and measuring the (b) phosphoenolpyruvic acid, amount of luminescence formed to determine cyclic AMP (c) pyrophosphoric acid, quantitatively. (d) magnesium ion or another divalent metallic cation, (e) luciferin and