High-sensitivity activity measuring method of enzyme having nadph as coenzyme

Abstract

To provide a method for measuring the activity of an enzyme having NADPH as a coenzyme with high sensitivity by utilizing an NADP enzyme cycling method, without requiring any special facility or equipment, and by using an absorbance method generally used in a laboratory or a clinical inspection room.SOLUTION: A method for measuring the enzymatic activity of a target enzyme having NADPH in a sample as a coenzyme includes: (1) bringing a substrate of the target enzyme and NADPH in contact with a target enzyme in the sample and performing enzymatic reaction of the target enzyme having NADPH as a coenzyme; (2) decomposing unreacted NADPH in reaction liquid after enzymatic reaction by using perchloric acid; (3) removing perchloric acid from solution after unreacted NADPH decomposition by using a weak base containing potassium; and (4) quantitatively determining NADP by performing NADP enzyme cycling reaction by using solution from which perchloric acid is removed.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a highly sensitive method for measuring the activity of an enzyme that uses NADPH as a coenzyme. [Background technology]

[0002] To detect trace amounts of biological components, it is necessary to amplify the detection signal. When the target is a nucleic acid, high sensitivity can be achieved by amplifying the target sequence itself using the polymerase chain reaction (PCR). This makes it possible to detect the activity of a target molecule. Furthermore, in immunoassays where the target is considered an antigen, the detection signal from the target molecule can be amplified several thousand-fold by labeling the antibody with peroxidase or alkaline phosphatase. However, no general-purpose method for amplifying the detection signal has been reported for measuring the activity of enzymes contained in biological samples, which is frequently used in biochemical research and clinical testing. Therefore, measuring the activity of trace amounts of enzymes or when the activity of the target enzyme is extremely low can be extremely difficult.

[0003] Enzyme cycling is one of the few methods for amplifying detection signals targeting small molecule compounds. A typical target of enzymatic cycling is nicotinamide adenine dinucleotide phosphate (NADP). NADP enzymatic cycling is a method for amplifying the oxidized form (NADP + ) is added NADP is produced by combining a reaction with an enzyme and a reaction with a reduced form (NADPH) as a coenzyme. + and NADPH The electrons generated during this process are used in the pigment-producing reaction. + As long as the NADPH cycling reaction continues, pigment accumulates. Therefore, this enzyme cycling method is expected to significantly increase the sensitivity. + If this method can be applied to measuring the activity of enzymes that use NADP as a coenzyme (NADP enzyme), it could become a method that can measure even small amounts of enzyme activity with high sensitivity. However, there is a major problem when applying this enzyme cycling method to measuring the activity of NADP enzymes. Generally, when measuring the activity of enzymes that use NADPH as a coenzyme, the reaction starts Excess substrate and NADPH are added to the reaction mixture. As the reaction proceeds, NADP is released along with the product. + is produced, but unreacted NADPH remains in the reaction mixture, and the NADP + In enzyme cycling reactions, the sum of NADPH and NADP does not change. + Since both of these are substrates, even if this enzyme reaction mixture is subjected to an enzyme cycling reaction, the enzyme activity will not be reflected in the results of the enzyme cycling method. Therefore, measuring enzyme activity using the NADP enzyme cycling method is not easy, and a general-purpose method to solve this problem has not yet been developed.

[0004] The present inventors have also been developing methods for measuring enzyme activity, and have so far developed a method for measuring the enzyme activity of dihydropyrimidine dehydrogenase (DPD) (particularly DPD is a compound that is similar to 5-fluorouracil, an anti-cancer drug widely used in cancer chemotherapy. DPD is an enzyme that converts 5-FU into 5,6-dihydro-5-fluorouracil (5-FUH2). DPD is responsible for the metabolism of 5-FU in the liver, but in cases of DPD deficiency, the metabolism of 5-FU after administration is poor. Due to metabolic insufficiency, the blood concentration of 5-FU becomes abnormally high, inducing fatal side effects such as bone marrow suppression. Therefore, to avoid such side effects, DPD activity is required before administering 5-FU. It is required to measure the following. Here, DPD involves two reactions: a forward reaction that converts 5-FU to 5-FUH2 using NADPH as a coenzyme, and a reverse reaction that converts 5-FU to 5-FUH2 using NADP + Supplement There is a reverse reaction in which 5-FUH2 is converted into 5-FU by the enzyme. The method for measuring the activity is NADP +The 5-FU produced by the reverse reaction of DPD with 5-FU as a coenzyme is separated by high performance liquid chromatography (HPLC), and then DPD activity is measured by detecting absorbance. However, for widespread use in research and clinical laboratories, a simpler method for measuring enzyme activity is required. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2021-153494 [Non-patent literature]

[0006] [Non-Patent Document 1] Analytical Chemistry, 2006;55(1):45-49 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention utilizes the NADP enzyme cycling method, which does not require special facilities or equipment, and can measure the activity of enzymes that use NADPH as a coenzyme using an absorbance method commonly used in laboratories and clinical laboratories. The present invention aims to provide a method for measuring the above with high sensitivity. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that NADPH can be used as a coenzyme When measuring the activity of the enzyme, after the reaction of the coenzyme NADPH, the unreacted NADPH is removed with perchloric acid, and then the excess perchloric acid is removed with a weak base containing potassium. +We have found that only ATP can be used in enzyme cycling reactions. This has led to the discovery of a method for measuring enzyme activity with high sensitivity even in situations where activity measurement is difficult, such as when the amount of enzyme is small or the enzyme activity is low, and has completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme in a sample, comprising: (1) contacting a target enzyme in the sample with a substrate of the target enzyme and NADPH to carry out an enzymatic reaction of the target enzyme using NADPH as a coenzyme; (2) Decomposing unreacted NADPH in the reaction solution after the enzymatic reaction using perchloric acid; (3) Using a weak base containing potassium, perchlorate is removed from the solution after decomposition of unreacted NADPH. To do; (4) Quantifying NADP by performing an NADP enzyme cycling reaction using a solution from which perchlorate has been removed. A method comprising: [2] The target enzyme is dihydropyrimidine dehydrogenase (DPD) or glutamate The method according to [1], wherein the enzyme is a dehydrogenase (GLDH). [3] When the target enzyme is DPD, the substrate of the target enzyme is 5-fluorouracil (5-FU) or or uracil; or, when the target enzyme is GLDH, the substrate of the target enzyme is α-ketoglutarate. [4] The method according to any one of [1] to [3] above, wherein in (2), the final concentration of perchloric acid added to the reaction solution after the enzyme reaction is 0.1 M to 1.2 M. [5] In the above (2), the decomposition of unreacted NADPH is carried out by reacting with perchloric acid for 5 minutes to 2 hours. The method according to any one of [1] to [4], which is carried out by [6] The method according to any one of [1] to [5], wherein in (3) above, the potassium-containing weak base is potassium bicarbonate or potassium carbonate. [7] In the above (3), potassium is added to the solution after decomposition of the unreacted NADPH. The method according to any one of [1] to [6], wherein the final concentration of the weak base is 0.2 M to 2.4 M. [8] In the above (4), the NADP enzyme cycling reaction is glucose-6-phosphate dehydrogenase. The method according to any one of [1] to [7], which is carried out using glucosease (G6PDH). [9] The method according to any one of [1] to [8], wherein the sample is a blood sample or a peripheral blood mononuclear cell (PBMC) lysate.

[10] Using any of the methods described in [1] to [9], NADPH in the sample is used as a coenzyme. and varying the abundance of the target enzyme in the sample, including measuring the enzymatic activity of the target enzyme. A method for testing for diseases that cause abnormalities in the enzymatic activity of a target enzyme.

[11] For measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme, and / or in a sample A kit for testing for a disease that changes the abundance of a target enzyme in a subject or a disease that causes abnormalities in the enzymatic activity of the target enzyme, comprising: NADPH, the substrate of the target enzyme, perchlorate, a weak base containing potassium, NADP enzyme cycling A kit including reagents for:

[12] When the target enzyme is DPD, the substrate of the target enzyme is 5-fluorouracil (5-FU). or uracil; or, when the target enzyme is GLDH, the substrate of the target enzyme is α-ketoglutarate.

[13] The kit according to

[11] or

[12] , wherein the potassium-containing weak base is potassium bicarbonate or potassium carbonate.

[14] The kit according to any one of

[11] to

[13] , wherein the NADP enzyme cycling reagent comprises glucose-6-phosphate (G6P); glucose-6-phosphate dehydrogenase (G6PDH); 1-methoxyphenazine methosulfate (1-Methoxy PMS), 5-methylphenazinium methylsulfate (PMS), or diaphorase; and a diazonium salt. [Effects of the Invention]

[0010] According to the present invention, the activity of an enzyme that uses NADPH as a coenzyme can be measured with high sensitivity. It is expected that this technology will be used as a new research tool to discover previously undiscovered biological phenomena and as a new method for diagnosing diseases. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the procedure for measuring enzyme activity when dihydropyrimidine dehydrogenase (DPD) is used as the enzyme that requires NADPH as a coenzyme. [Figure 2] FIG. 2 is a schematic diagram showing the enzymatic cycling reaction carried out in the examples. [Figure 3] Figure 3 is a graph showing the results of enzyme activity measurements using dihydropyrimidine dehydrogenase (DPD) as the enzyme requiring NADPH as a coenzyme. The horizontal axis represents wavelength (nm), and the vertical axis represents absorbance. Each graph shows the time (minutes) of the enzyme cycling reaction. [Figure 4] FIG. 4 is a schematic diagram showing the enzyme reaction and the enzyme cycling reaction when glutamate dehydrogenase (GLDH) is used as the enzyme requiring NADPH as a coenzyme. [Figure 5]5A and 5B are graphs showing the results of enzyme activity measurements when glutamate dehydrogenase (GLDH) was used as the enzyme requiring NADPH as a coenzyme. Graph A shows the results of measuring absorbance at 340 nm over time during the GLDH reaction. Graph B shows the results of measuring absorbance at 450 nm over time during the enzyme cycling reaction. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Method for measuring enzyme activity> One embodiment of the present invention is a method for measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme in a sample. 1. A method comprising: (1) contacting a target enzyme in the sample with a substrate of the target enzyme and NADPH to carry out an enzymatic reaction of the target enzyme using NADPH as a coenzyme; (2) Decomposing unreacted NADPH in the reaction solution after the enzymatic reaction using perchloric acid; (3) Using a weak base containing potassium, perchlorate is removed from the solution after decomposition of unreacted NADPH. To do; (4) Quantifying NADP by performing an NADP enzyme cycling reaction using a solution from which perchlorate has been removed. By using this method, enzyme activity can be easily measured without using HPLC.

[0013] (enzymatic reaction of target enzyme) The target enzyme is not particularly limited as long as it is an enzyme that uses NADPH as a coenzyme. Dropyrimidine dehydrogenase (DPD), glutamate dehydrogenase (GLDH), etc. Examples include:

[0014] When the target enzyme is DPD, the substrate of the target enzyme is 5-fluorouracil (5-FU) or uracil. That is, the target enzyme DPD is reacted with the substrate of the target enzyme 5-FU and the coenzyme As a result of the enzymatic reaction, 5,6-dihydro-5-fluorouracil (5-FUH2) and NADP + The target enzyme DPD was contacted with the substrate uracil and coenzyme NADPH to carry out an enzymatic reaction, resulting in the production of 5,6-dihydrouracil and and NADP + is generated. When the target enzyme is GLDH, the substrate of the target enzyme is α-ketoglutarate. That is, the target enzyme GLDH is contacted with the substrate of the target enzyme, α-ketoglutarate, and the coenzyme NADPH to carry out an enzymatic reaction, resulting in the production of glutamate and NADP. + is generated.

[0015] The sample is not particularly limited as long as it contains the enzyme to be measured, and may be, for example, a biological sample such as a blood sample or a peripheral blood mononuclear cell (PBMC) lysate, or any aqueous medium that contains the enzyme to be measured and maintains the enzyme activity. When the sample is a blood sample, it may be, for example, a whole blood sample, a plasma sample, or a serum sample. Furthermore, the PBMC lysate can be obtained by subjecting PBMCs collected by a method known to those skilled in the art to disruption by a method known to those skilled in the art. Examples of disruption methods include those using an ultrasonic disrupter, a bead-type disrupter, or a French press. The total protein amount in a sample is not particularly limited, but may be, for example, per 500 μL of sample, with a lower limit of 10 μg or more, 100 μg or more, 150 μg or more, 180 μg or more, or 200 μg or more, and an upper limit of 1000 μg or less, 500 μg or less, 250 μg or less, 220 μg or less, or 200 μg or less. Furthermore, any of these upper and lower limits may be combined to express a range, such as 10 μg to 1000 μg, 100 μg to 500 μg, 150 μg to 250 μg, or 180 μg to 220 μg. Alternatively, the amount may be specifically specified as 200 μg. The lower limit per 1 L of sample may be 20 mg or more, 200 mg or more, 300 mg or more, 360 mg or more, or 400 mg or more, and the upper limit may be 2 g or less, 1 g or less, 500 mg or less, 440 mg or less, or 400 mg or less, and the range may be 20 mg to 2 g, 200 mg to 1 g, 300 mg to 500 mg, or 360 mg to 440 mg. It may also be specifically specified as 400 mg.

[0016] The amount of the substrate for the target enzyme is not particularly limited, as long as it is an appropriate amount for the target enzyme to undergo an enzymatic reaction. The final concentration of the substrate added to the sample may be, for example, 0.5 μM or more, 5.0 μM or more, 7.5 μM or more, 9.0 μM or more, or 10.0 μM or more at the lower limit, and 50.0 μM or less, 25.0 μM or less, 12.5 μM or less, 11.0 μM or less, or 10.0 μM or less at the upper limit. Furthermore, any of these upper and lower limits may be combined to express a range, such as 0.5 μM to 50.0 μM, 5.0 μM to 25.0 μM, 7.5 μM to 12.5 μM, or 9.0 μM to 11.0 μM. Alternatively, the final concentration may be specifically specified as 10 μM.

[0017] NADPH is specifically limited in the amount required to allow the target enzyme to carry out the enzymatic reaction. The final concentration of NADPH added to a sample may have a lower limit of, for example, 10 μM or more, 100 μM or more, 150 μM or more, 180 μM or more, or 200 μM or more, and an upper limit of 2000 μM or less, 1000 μM or less, 500 μM or less, 250 μM or less, 220 μM or less, or 200 μM or less. Furthermore, any of these upper and lower limits may be combined to express a range, such as 10 μM to 2000 μM, 10 μM to 1000 μM, 100 μM to 500 μM, 150 μM to 250 μM, 180 μM to 220 μM, or 200 μM to 250 μM. Alternatively, the range may be specifically specified as 200 μM.

[0018] The reaction time of the target enzyme is not particularly limited as long as it is a time appropriate for the target enzyme to carry out the enzymatic reaction. For example, the lower limit may be 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, The time may be 30 minutes or more, 1 hour or more, 2 hours or more, or 3 hours or more, and the upper limit may be 12 hours or less, 6 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less. Alternatively, any of these upper and lower limits may be combined to represent a range, such as 1 minute to 12 hours, 5 minutes to 6 hours, 10 minutes to 3 hours, 20 minutes to 2 hours, 30 minutes to 1 hour, 1 minute to 1 hour, or 5 minutes to 1 hour. Alternatively, the range may be specifically set to 30 minutes.

[0019] The reaction temperature of the target enzyme is not particularly limited as long as it is a temperature appropriate for the target enzyme to undergo an enzymatic reaction. For example, the lower limit may be 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 36°C or higher, or 37°C or higher, and the upper limit may be 50°C or lower, 45°C or lower, 42°C or lower, 41°C or lower, or 40°C or lower. Furthermore, any of these upper and lower limits may be combined to express a range, such as 15°C to 50°C, 20°C to 45°C, 35°C to 42°C, 36°C to 41°C, or 37°C to 40°C.

[0020] (Decomposition of unreacted NADPH) The present invention relates to NADP produced by an enzymatic reaction catalyzed by a target enzyme. + NADP enzyme cycling However, in the NADP enzyme cycling reaction, NADP + Since both NADPH and NADPH act as coenzymes, if unreacted NADPH remains in the measurement solution, it is impossible to distinguish between the NADPH produced by the enzymatic cycling reaction and the unreacted NADPH, making it impossible to accurately measure the enzyme activity. Therefore, it is necessary to remove unreacted NADPH from the reaction solution after the enzymatic reaction before the NADP enzymatic cycling reaction.

[0021] In the present invention, perchloric acid is used to remove unreacted NADPH by decomposition. NADPH is weak in acid, and NADP + Since it is known that NADPH is sensitive to bases, the use of perchloric acid decomposes unreacted NADPH, eliminating the effect of NADPH in the subsequent enzyme cycling reaction. Furthermore, by using perchloric acid, not only is unreacted NADPH decomposed, but the enzyme reaction is accelerated. This has the advantage that proteins (for example, cell-derived proteins) present in the reaction solution after the reaction can be removed.

[0022] The amount of perchloric acid to be added is not particularly limited, but an excess amount is used to decompose unreacted NADPH. It is preferable to add perchloric acid to the reaction solution after the enzymatic reaction. The final concentration of perchloric acid added to the reaction solution after the enzymatic reaction may have a lower limit of 0.1 M or more, 0.2 M or more, 0.3 M or more, 0.4 M or more, or 0.5 M or more, and an upper limit of 1.2 M or less, 1.0 M or less, 0.8 M or less, 0.6 M or less, 0.5 M or less, or 0.4 M or less. Furthermore, any of these upper and lower limits may be combined to express a range, such as 0.1 M to 1.2 M or 0.3 M to 0.6 M. Alternatively, the range may be specifically specified as 0.4 M.

[0023] The reaction time with perchloric acid for decomposing unreacted NADPH is not particularly limited as long as the unreacted NADPH after the enzyme reaction is decomposed. For example, the lower limit can be set to 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or the like. The time may be 1 minute or more, 45 minutes or more, or 1 hour or more, with the upper limit being 2 hours or less, 1.5 hours or less, Alternatively, the range may be set to 1 hour or less. For example, it may be expressed as 5 minutes to 2 hours, or 30 minutes to 2 hours. Alternatively, the time period may be specifically specified as 1 hour, and unreacted NADPH may be treated with perchloric acid for about 1 hour. This allows for almost 100% decomposition.

[0024] The reaction temperature with perchloric acid for decomposing unreacted NADPH is not particularly limited. For example, room temperature This can be considered.

[0025] Furthermore, proteins (for example, cell-derived proteins) present in the reaction solution after the enzyme reaction may be removed by centrifugation.

[0026] (Removal of perchloric acid) The perchloric acid used in the decomposition of the unreacted NADPH is a strong acid, and the solution after the reaction contains Because it contains a large amount of excess perchlorate, it cannot be used directly in the enzyme cycling reaction. Therefore, it is necessary to remove the perchlorate. However, the NADP produced by the enzyme reaction + is weak to bases, so when a strong base is used, NADP + is likely to decompose.

[0027] The present invention uses a weak base containing potassium to remove excess perchloric acid. The weak base containing potassium is not particularly limited as long as it can remove perchloric acid. Examples include potassium bicarbonate and potassium carbonate. Being a weak base, it can gently neutralize perchloric acid and convert it to potassium perchlorate, which is sparingly soluble in water. Potassium perchlorate has very low water solubility and immediately forms a precipitate when potassium bicarbonate is added to an aqueous perchloric acid solution, which has the advantage of being easily removed.

[0028] The amount of the weak base containing potassium to be added is not particularly limited, but it is preferable to add an amount sufficient to remove perchloric acid. The final concentration of the potassium-containing weak base may have a lower limit of, for example, 0.2 M or more, 0.4 M or more, 0.6 M or more, 0.8 M or more, or 1.0 M or more, and an upper limit of 2.4 M or less, 2.0 M or less, 1.6 M or less, 1.2 M or less, 1.0 M or less, or 0.8 M or less. A range may be expressed by combining any of these upper and lower limits, such as 0.2 M to 2.4 M or 0.6 M to 1.2 M. The range may also be specifically set to 0.8 M. The molar concentration ratio of perchloric acid to potassium-containing weak base may be, for example, about 1:2.

[0029] The reaction time and reaction temperature for removing perchloric acid are not particularly limited as long as they can remove perchloric acid.

[0030] Furthermore, potassium perchlorate may be removed by additional centrifugation, which allows for more efficient removal of potassium perchlorate.

[0031] (Quantification of NADP by NADP enzyme cycling reaction) The supernatant of the solution after removing potassium perchlorate was used in the enzyme cycling reaction. + Only NADP enzyme can be quantified with high sensitivity. The NADP enzyme cycling reaction is not particularly limited and can be carried out by a method known to those skilled in the art. A commercially available kit may also be used. Specifically, for example, the following reactions are carried out successively: (i) NADP + Electron transfer to NADPH through an enzymatic reaction using as a coenzyme; (ii) transferring electrons from NADPH to an electron mediator; and (iii) Transferring electrons from the electron mediator to the chromogen to produce a pigment.

[0032] The enzymatic reaction (i) above is not particularly limited as long as it is used in an NADP enzyme cycling reaction. For example, Enzyme reaction of hydrogenase (G6PDH), glycerol dehydrogenase using glycerol as a substrate Examples of the enzyme reaction include an enzyme reaction of malic acid, and an enzyme reaction of malate dehydrogenase using malic acid as a substrate.

[0033] The electron mediator (ii) above is not particularly limited as long as it is used in the NADP enzyme cycling reaction, and examples thereof include 1-methoxyphenazine methosulfate (1-Methoxy PMS), 5-methylphenazinium methylsulfate (PMS), and diaphorase.

[0034] The chromogen (iii) above is not particularly limited as long as it is used in the NADP enzyme cycling reaction. For example, it may be a diazonium salt or a tetrazolium salt. For example, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2-(4-iodophenyl)-3-(4-nitril)-2H-tetrazolium, monosodium salt (WST-8), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2-(4-iodophenyl)-3-(4-nitril)-2H-tetrazolium, monosodium salt (WST-8), (2,4-diphenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) , 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (NTB), or 2-benzothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (WST-4).

[0035] The reaction time for the enzymatic cycling reaction is not particularly limited as long as it is an appropriate time for carrying out the enzymatic cycling reaction. For example, the lower limit may be 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more, or 1.5 hours or more, and the upper limit may be 2 hours or less, 1.5 hours or less, 1 hour or less, 45 minutes or less, or 30 minutes or less. Furthermore, any of these upper and lower limits may be combined to express a range, such as 1 minute to 2 hours, 15 minutes to 2 hours, or 45 minutes to 1.5 hours. It may also be specifically specified as 1 hour.

[0036] By detecting the dye accumulated by the enzyme cycling reaction, the enzymatic activity of the target enzyme contained in the sample can be determined. The dye is not particularly limited as long as it can be used in the enzymatic cycling reaction. For example, when a diazonium salt is used as a chromogen in the enzymatic cycling reaction, a formazan dye can be detected. The detection of the dye can be carried out by a method known to those skilled in the art, and is not particularly limited, but can be carried out by measuring absorbance, for example.

[0037] <Disease testing methods> Another aspect of the present invention is to measure NADPH in a sample as a coenzyme by using the above-mentioned method for measuring enzyme activity. The method for testing for a disease that changes the amount of a target enzyme present in a sample or a disease that causes an abnormality in the enzymatic activity of a target enzyme comprises measuring the enzymatic activity of the target enzyme.

[0038] The disease is not particularly limited as long as it changes the amount of the target enzyme in a biological sample or causes abnormalities in the enzymatic activity of the target enzyme. For example, in the case of DPD deficiency, Since DPD is not present in any biological samples, the detection of DPD deficiency is difficult when the target enzyme is DPD. Furthermore, for example, since the amount of the target enzyme increases in a biological sample such as a blood sample in the case of liver damage, liver damage can be examined when the target enzyme is GLDH.

[0039] <Kit> Another aspect of the present invention is a method for measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme, and A kit for testing for a disease that changes the amount of a target enzyme present in a sample and / or a disease that causes an abnormality in the enzymatic activity of the target enzyme, comprising: NADPH, the substrate of the target enzyme, perchlorate, a weak base containing potassium, NADP enzyme cycling The kit may be used as a clinical test kit or a research kit.

[0040] When the target enzyme is DPD, its substrate is 5-fluorouracil (5-FU) or uracil. When the target enzyme is GLDH, it may contain α-ketoglutarate as its substrate.

[0041] Perchloric acid decomposes the unreacted NADPH after the enzyme reaction and also decomposes the NADPH that is involved in the enzyme reaction. It is a reagent for removing proteins.

[0042] The potassium-containing weak base is a reagent that precipitates excess perchloric acid as an insoluble substance, potassium perchlorate, and neutralizes the reaction solution. Examples of the weak base include, but are not limited to, potassium bicarbonate and potassium carbonate.

[0043] The reagent for NADP enzyme cycling is not particularly limited, but for example, glucose-6-phosphate (G6P); glucose-6-phosphate dehydrogenase (G6PDH); 1-methoxyphenazinemeth sulfate (1-Methoxy PMS), 5-methylphenazinium methyl sulfate (PMS), or diaphorase; and diazonium salts.

[0044] The kit may further include reagents such as buffers, diluents, and / or standards, and may include equipment such as test plates. [Example]

[0045] The present invention will be described in more detail below with reference to examples, but it goes without saying that the scope of the present invention is not limited to the examples.

[0046] Example 1: Measurement of dihydropyrimidine dehydrogenase (DPD) activity Using dihydropyrimidine dehydrogenase (DPD) as an enzyme that requires NADPH as a coenzyme, the enzyme activity was measured according to the following procedure (Fig. 1). 1. Peripheral blood mononuclear cells (PBMC) lysate (total protein amount 200 μg) derived from a healthy individual was diluted with 100 μM HCl. The mixture was mixed with Rasil (50 μL; Fujifilm Wako Pure Chemical Industries, Ltd., 212-00062) and 2 mM NADPH (50 μL; Oriental Yeast Co., Ltd., 44330000) in phosphate buffer to a total volume of 500 μL, and then incubated at 37°C for 30 minutes. The phosphate buffer was prepared using sodium dihydrogen phosphate dihydrate (Kanto Chemical Co., Ltd., 37239-00) and disodium hydrogen phosphate 12-hydrate (Kanto Chemical Co., Ltd., 37240-00). 2. To the reaction solution in 1 above, an aqueous solution of perchloric acid (Kanto Chemical, 32060-00) was added to a final concentration of 0.4 M, and the mixture was incubated for 1 hour. 3. To the reaction mixture in step 2 above, an aqueous solution of potassium bicarbonate (Kanto Chemical, 32307-00) was added to a final concentration of 0.8M. 4. After centrifugation, the supernatant was diluted 50-fold and subjected to an enzymatic cycling reaction (NADP / NADPH Assay Kit-WST: Dojindo Laboratories, N510) (Figure 2). 5. Measure the absorbance at 450 nm to determine the NADP present in the sample. + The amount was calculated and converted to DPD activity. Ta.

[0047] The results of the enzyme activity measurement are shown in Figure 3. The graph in which uracil was added as a substrate showed higher absorbance than the graph in which uracil was not added (control). In other words, the DPD activity in the sample was measured by the above method. It was shown that the measured enzyme activity increased depending on the ring time.

[0048] Furthermore, the results of repeated experiments on the same specimens are shown in Table 1. The values ​​were similar to the DPD activity values ​​reported previously for Japanese people (Non-Patent Document 1). Furthermore, the day-to-day reproducibility of the DPD activity measurement value according to the present invention was 13.4%, which was shown to be higher than the day-to-day reproducibility (24.5%) in Non-Patent Document 1, allowing measurement with higher reproducibility. [Table 1]

[0049] Example 2: Measurement of glutamate dehydrogenase (GLDH) activity Glutamate dehydrogenase (GLDH) was used as the enzyme that requires NADPH as a coenzyme. NADPH-dependent GLDH reduces α-ketoglutarate to glutamate in the presence of NADPH. A typical activity measurement involves tracking the decrease in absorbance at 340 nm of the NADPH consumed during the reduction. Therefore, a reaction solution was prepared by excessively diluting GLDH and adding an excess of NADPH. Using the change in absorbance at 340 nm and the enzyme reaction solution after 30 minutes, enzyme activity was measured according to the following procedure (Figure 4). 1. Commercially available GLDH (2.5 U / mL; Oriental Yeast, 46486003) was diluted to prepare 0.5 U / mL GLDH (1 mL). This was mixed with 100 mM α-ketoglutaric acid (10 μL; Fujifilm Wako Pure Chemical Industries, 115-00082), 10 mM NADPH (10 μL), and 500 mM NH4Cl (100 μL; Kanto Chemical, 01287-30) in Tris buffer (Nacalai Tesque, 35406-75) to a total volume of 1 mL. 2. The absorbance at 340 nm was measured over a 30 minute period. 3. Add a perchloric acid solution to the reaction mixture in step 2 above to a final concentration of 0.4 M and incubate for 1 hour. I was mobilized. 4. To the reaction mixture from step 3 above, an aqueous solution of potassium bicarbonate was added to a final concentration of 0.8 M, and the mixture was centrifuged. 5. The supernatant was diluted 50-fold and subjected to an enzyme cycling reaction, and the absorbance at 460 nm was measured for 1 hour.

[0050] When very dilute GLDH was used, the absorbance of the NADPH used was large and the change in NADPH was small, making it difficult to track the activity by the change in absorbance at 340 nm (Figure 1). 5A). On the other hand, the activity measurement method of the present invention can be used to separate unreacted NADPH after the enzyme reaction. Then, the NADP produced by the enzyme reaction + Only the signal caused by the enzyme cyclin It was found that amplification by the enzyme-linked enzyme method enabled even small changes in activity to be detected as large signal changes (Fig. 5B). + The measured concentration was 74.3 nM, which was almost equal to the theoretical value (75.0 nM) calculated from the activity value provided. This indicates that the present invention can be used as a general method for measuring the activity of enzymes that use NADPH as a coenzyme.

Claims

1. A method for measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme in a sample, comprising: (1) contacting a target enzyme in the sample with a substrate of the target enzyme and NADPH to carry out an enzymatic reaction of the target enzyme using NADPH as a coenzyme; (2) Decomposing unreacted NADPH in the reaction solution after the enzymatic reaction using perchloric acid; (3) Using a weak base containing potassium, perchloric acid is removed from the solution after decomposition of unreacted NADPH. To do; (4) Quantifying NADP by performing an NADP enzyme cycling reaction using a solution from which perchloric acid has been removed. A method comprising:

2. The target enzyme is dihydropyrimidine dehydrogenase (DPD) or glutamate dehydrogenase (GD). The method of claim 1, wherein the enzyme is hydroxylase (GLDH).

3. When the target enzyme is DPD, the substrate of the target enzyme is 5-fluorouracil (5-FU) or is uracil; or, when the target enzyme is GLDH, the substrate of the target enzyme is α-ketoglutarate.

4. The method according to claim 1, wherein in (2), the final concentration of perchloric acid added to the reaction solution after the enzymatic reaction is 0.1 M to 1.2 M.

5. In the above (2), the decomposition of unreacted NADPH is carried out by reacting with perchloric acid for 5 minutes to 2 hours. The method of claim 1 , wherein the method is carried out by

6. The method according to claim 1, wherein in (3), the potassium-containing weak base is potassium bicarbonate or potassium carbonate.

7. In the above (3), a weak solution containing potassium is added to the solution after decomposition of the unreacted NADPH.

2. The method of claim 1, wherein the final concentration of the base is 0.2 M to 2.4 M.

8. In the above (4), the NADP enzyme cycling reaction is glucose-6-phosphate dehydrogenase. The method of claim 1, wherein the method is carried out using a genase (G6PDH).

9. The method of claim 1, wherein the sample is a blood sample or a peripheral blood mononuclear cell (PBMC) lysate.

10. A method for detecting a target that uses NADPH as a coenzyme in a sample, using the method according to any one of claims 1 to 9. A method for testing for a disease that changes the amount of a target enzyme present in a sample or a disease that causes an abnormality in the enzymatic activity of a target enzyme, comprising measuring the enzymatic activity of an enzyme.

11. For measuring the enzymatic activity of a target enzyme that uses NADPH as a coenzyme, and / or in a sample A kit for testing for a disease that changes the abundance of a target enzyme or a disease that causes abnormalities in the enzymatic activity of the target enzyme, comprising: NADPH, the substrate of the target enzyme, perchlorate, a weak base containing potassium, and the NADP enzyme cyclin A kit including a reagent for performing the assay.

12. When the target enzyme is DPD, the substrate of the target enzyme is 5-fluorouracil (5-FU) or is uracil; or, when the target enzyme is GLDH, the substrate of the target enzyme is α-ketoglutarate.

13. The kit according to claim 11, wherein the potassium-containing weak base is potassium bicarbonate or potassium carbonate.

14. The NADP enzyme cycling reagents include glucose-6-phosphate (G6P); Glycine 6-phosphate dehydrogenase (G6PDH); 1-methoxyphenazine methosulfate (1-Methoxy PMS), 5-methylphenazinium methylsulfate (PMS), or diaphorase; and dimethicone The kit of claim 11 comprising an azonium salt.

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