JP7333547B2 - Amino acid quantification method and amino acid quantification kit - Google Patents

Description

The present invention relates to an amino acid quantification method, an amino acid quantification kit, and the like.

L-amino acids (L-AA) play an important role as constituents of proteins in vivo, and there are 20 types of them. Many studies have been made on the functionality of L-amino acids, and L-amino acids are used in various industries such as pharmaceuticals, processed foods, and health foods. For example, free L-amino acids in food are related to taste, aroma after heating, preservability, bioregulatory function after ingestion, etc., and are attracting attention as an important factor in the fields of food science and nutritional science. . In recent years, it has been found that the L-amino acid concentration in the blood changes depending on the disease, and by measuring the amino acid concentration in the blood, it is a biomarker that enables cancer diagnosis such as lung cancer, stomach cancer, and colon cancer. It is also used as Therefore, amino acid quantification technology has become an indispensable technology in various fields such as product development, quality control, and diagnosis using amino acids.

As an amino acid quantification technique for L-amino acids, a method is known in which amino acids are separated by liquid chromatography and detected by color reaction with ninhydrin or a fluorescent derivatizing agent, orthophthalaldehyde (Non-Patent Document 1). However, this method has the problem that it takes about 2 hours to analyze one specimen, and thus it is not suitable for measuring a large number of specimens.

As another amino acid quantification technique, a method for measuring amino acids using an enzyme acting on L-amino acids is known (Patent Document 1). However, this method has problems in that it has low selectivity for amino acids as target substrates, reacts with amino acids other than target amino acids, and there is no enzyme corresponding to 20 types of amino acids.

Aminoacyl-tRNA synthetase (AARS) is an enzyme involved in protein synthesis in vivo, and produces aminoacyl-tRNA according to reaction schemes 1 and 2 below. There are 20 AARS specific for 20 L-amino acids (L-AA). Therefore, it is considered to be an enzyme with extremely high selectivity for target amino acids and transfer RNA (tRNA).

Among the AARS, glutamyl-tRNA synthetase that acts on glutamic acid, glutaminyl-tRNA synthetase that acts on glutamine, and arginyl-tRNA synthetase that acts on arginine are three types of AARS. It has been reported that the AARS reaction does not occur unless is pre-associated and activated (Non-Patent Document 2).

In the reaction, pyrophosphate (PPi) is produced, and adenosine triphosphate (ATP), L-amino acid (L-AA), and tRNA act on AARS one molecule each to produce aminoacyl adenylic acid (aminoacyl AMP )-AARS-tRNA complex. In the complex, aminoacyl AMP is usually strongly bound to AARS, so the above AARS reaction does not proceed unless tRNA or a nucleophilic agent (amines) is added to decompose the complex. (Patent Document 2, Non-Patent Documents 3-4).

Such reactions involving AARS (AARS reactions) have been confirmed by methods using radiolabels, as in Non-Patent Documents 5 and 6, and L-amino acid quantification techniques using this method. has been developed so far. For example, in Non-Patent Document 7, based on the AARS reaction of Reaction Schemes 1 and 2, a radiolabeled amino acid to be measured is used, ATP, tRNA and AARS are allowed to act, and the produced aminoacyl-tRNA is adsorbed to beads, A method for measuring an amino acid to be measured by measuring the radiation dose is described. However, in this method, a radiolabeled amino acid to be measured is indispensable, and facilities and equipment capable of handling the radiolabeled amino acid to be measured are required. In addition, complicated operations such as adsorption and separation of beads are required. Furthermore, since it is based on the AARS reactions of Reaction Schemes 1 and 2, AARS reacts with one molecule of amino acid and ATP to produce one molecule of aminoacyl-tRNA, so that pyrophosphate is produced in an amount equal to or less than that of tRNA in the sample. not. Therefore, in this method, it is necessary to detect a minute amount of the reaction product, aminoacyl-tRNA, and it is said that a radiolabeled amino acid to be measured is essential (Patent Document 2, paragraph 0012).

Further, for example, Patent Document 3 describes an amino acid analysis method based on the reaction shown in Reaction Formula 6 below.

In this method, L-amino acids are analyzed using pyrophosphate produced when AARS binds to ATP and L-amino acids as an index (Patent Document 3, Non-Patent Documents 8, 9, 10). However, as can be seen from reaction formula 6, this method is a method of reacting without adding tRNA, and is a method in which tRNA and AARS are pre-associated with AARS, which must be activated, so that no reaction occurs. .

Furthermore, Patent Documents 2 and 4 describe methods for quantifying L-amino acids based on the AARS reactions of Reaction Schemes 1 and 2 without adding tRNA. That is, as described above, aminoacyl AMP normally binds strongly to AARS to form an aminoacyl AMP-AARS complex. Therefore, in this method, by adding an amine (nucleophilic agent) such as hydroxylamine as an aminoacyl AMP-AARS complex decomposing reagent, AARS is again made to be in a state capable of reacting, and as a result, a small amount of AARS can It is characterized by quantifying amino acids. However, the complex-dissolving reagent described in Patent Documents 2 and 4 reacts with the L-amino acid of the complex to produce a compound (for example, as shown in paragraph 0037 of Patent Document 2, the When hydroxylamine is used as a decomplexing reagent, "amino acid hydroxamic acid" is produced), so L-amino acids cannot be reused, and as a result, the same amount of L-amino acids in the sample Only pyrophosphate, which is a product, can be obtained (Paragraph No. 0023 of Patent Document 2).

In addition, this method is a method of reacting without adding tRNA, and reaction does not occur with AARS, which must be pre-associated with tRNA and AARS and activated. Incidentally, in Patent Document 2, tRNA and AARS must associate in advance and activate AARS, so it is sufficient to add tRNA. It is described that it is sufficient to add a low molar concentration equivalent to AARS (Paragraph No. 0012 of Patent Document 2). In addition, when tRNA is used in the method based on the AARS reaction of Reaction Schemes 1 and 2, the tRNA that constitutes the aminoacyl-tRNA cannot be reused, and as a result, the amount of tRNA in the sample limits the reaction. As a result, only pyrophosphate, which is a product equivalent to tRNA, can be obtained. From the above, since the method based on the AARS reactions of Reaction Schemes 1 and 2 does not reuse tRNA or L-amino acids, it is possible to obtain pyrophosphate in an amount equivalent to tRNA or L-amino acids. Since the pyrophosphate generated in the AARS reaction is detected by multistep enzymatic reactions, it is complicated and there is concern that each enzyme is susceptible to contaminants in the blood and other external factors.

In fact, in Patent Document 2, when an aminoacyl AMP-AARS-tRNA complex formed from AARS pre-associated with tRNA is reacted with an amine (nucleophilic agent) such as hydroxylamine, the complex AA and at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complex, and tRNA are released from the body, and these are again in a state capable of reacting, and as a result, L-amino acids can be quantified. This fact is not described or suggested at all, nor is any embodiment showing such a fact disclosed.

On the other hand, as another AARS reaction, for example, reactions represented by reaction formulas 7 and 8 below are known. In this reaction, one molecule each of ATP and L-amino acid acts on AARS to form an aminoacyl AMP-AARS complex. Then, by allowing ATP, GTP, etc. to act on the complex, diadenosine polyphosphate (ApnA) such as diadenosine tetraphosphate (Ap4A) and adenosine anosine tetraphosphate (Ap4G), etc., which are expected as pharmaceuticals and drug raw materials is synthesized and manufactured (Patent Document 5, Patent Document 6, Non-Patent Document 11, Non-Patent Document 12).

In Reaction Scheme 7 of the reaction, the reverse reaction is promoted in the presence of pyrophosphate to produce amino acids, AARS and ATP from the aminoacyl AMP-AARS complex. Therefore, in order to proceed with the reaction, it is necessary to prevent the reverse reaction from occurring. Inorganic pyrophosphatase is used to degrade Furthermore, the techniques described in these publications relate only to synthesis and production of ApnA, Ap4G, etc., and are not intended to solve technical problems related to quantification of amino acids. In fact, these known documents do not mention the quantification of amino acids utilizing the AARS reactions of the above reaction schemes 7 and 8, and there is no mention of the reuse of the amino acids and AARS generated in reaction scheme 8. not touched.

Further, a method for quantifying L- and D-amino acids based on the AARS reactions of Reaction Schemes 7 and 8 is described in Patent Document 7. In this method, the AARS and amino acids are released from the once-formed aminoacyl AMP-AARS conjugate, and these are again used to form the aminoacyl AMP-AARS conjugate, thereby finally obtaining pyrophosphate, which is the object of measurement. It is characterized in that the reaction product such as is produced up to the number of moles greater than the number of moles of amino acids contained in the sample. However, the invention makes no mention of AARS, in which tRNA and AARS must be pre-associated and activated. Furthermore, in Patent Document 7, AA and at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complex and tRNA are released from the aminoacyl AMP-AARS-tRNA complex, and these are regenerated. No mention or suggestion of availability.

JP 2013-146264 A Patent No. 5305208 JP 2011-50357 A WO2013118894A1 JP-A-2000-69990 Patent No. 3135649 WO2017170469A1

"Chemistry and Biology", (Japan), 2015, Vol.53, p. 192-197 Structure, 14, 1791-1799, 2006 "Vought Biochemistry (Part 2)", (Japan), 3rd edition, 1024-1029 J. Biol. Chem., 241, 839-845, 1966 J. Biol. Chem., 240, 432-438, 1965 Eur. J. Biochem., 70, 137-145, 1976 Analytical Biochem., 363, 246-254, 2007 Analytical Biochem., 443, 22-26, 2013 Appl. Biochem. Biotechnol., 174, 2527-2536, 2014 J. Chem. Chem. Eng. 6,397-400,2012 "Tokyo Medical University Journal", (Japan), 1993, Vol.51, p. 469-480 Agric. Biol. Chem., 53, 615-623, 1989

The present invention solves various problems in the prior art related to the above amino acid quantification method, and provides a method and a kit for amino acid quantification that selectively, easily, and highly sensitively quantifies an amino acid to be measured using AARS. intended to provide

As a result of various studies, the inventors have found a method for quantifying the amount of amino acids (L-form and D-form) in a sample using AARS, particularly glutamic acid, glutamine and arginine, as shown in FIG. As described above, aminoacyl-tRNA synthetase (AARS) and transfer RNA (tRNA) corresponding to amino acids (AA) in a sample are reacted to form a complex (AARS-tRNA complex) composed of AARS and tRNA. The AARS-tRNA complex is reacted with amino acids and ATP to form an aminoacyl AMP-AARS-tRNA complex, and then the once-formed aminoacyl AMP-AARS-tRNA complex (AARS and aminoacyl tRNA) and an amino acid regeneration reagent (nucleotide or alkaline compound) are reacted to obtain at least one selected from the group consisting of AA, AARS, AARS-tRNA complex and tRNA from the complex. By liberating the compounds and using them again to form an aminoacyl AMP-AARS-tRNA complex, the reaction product such as pyrophosphate to be measured is finally converted into tRNA and / Or, they found that they can be produced up to a larger number of moles than amino acids, and completed the present invention.

The present invention relates to the following aspects [1] to [7].
[1] Step (I) including the following steps:
(Step I-1) Aminoacyl-tRNA synthetase (AARS) corresponding to an amino acid (AA) in a sample and transfer RNA (tRNA) are reacted to form a complex composed of AARS and tRNA (AARS-tRNA complex) A step comprising a reaction (Reaction 1) to cause;
(Step I-2) reacting the AA, the AARS-tRNA complex corresponding to the AA, and adenosine triphosphate (ATP) in the presence of a divalent cation to form aminoacyl adenylic acid (aminoacyl AMP); a step comprising a reaction (reaction 2) to form a complex consisting of an AARS-tRNA complex (aminoacyl AMP-AARS-tRNA complex);
(Step I-3) The aminoacyl AMP-AARS-tRNA complex formed in Reaction 2 and/or Reaction 4 (including AARS and aminoacyl-tRNA that can be produced from the complex) is allowed to act with an amino acid regeneration reagent to and a reaction (reaction 3) for releasing at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complexes and tRNA;
(Step I-4) Reusing the AA released in Reaction 3 and at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complex and tRNA in Reaction 1 and/or Reaction 2 forming an aminoacyl AMP-AARS-tRNA complex reaction (reaction 4); and
(Step I-5) repeating steps I-3 and I-4, measuring the amount of the reaction product produced in step (I), and determining the amount of amino acid based on the measured amount of the reaction product; step (II) comprising determining
A method for quantifying amino acids in a sample, comprising:
[2] The method for quantifying amino acids according to aspect [1], wherein the amino acid regeneration reagent used in step (I) is a nucleotide and an alkaline compound.
[3] The method for quantifying amino acids according to aspect [1], wherein the amount of tRNA used in step (I) is 0.25 or more times the number of moles of AARS.
[4] The method for quantifying amino acids according to aspects [1] to [3], wherein the amount of the reaction product produced in step (I) is measured by measuring absorbance change by an absorbance method.
[5] The method for quantifying amino acids according to any one of aspects [1] to [4], wherein at least one of pyrophosphate and hydrogen ions is measured as the reaction product generated in step (I).
[6] Any one of aspects [1] to [5], wherein the number of moles of the reaction product produced in step (I) is greater than the number of moles of tRNA and/or amino acid contained in the reaction solution. The method for quantifying amino acids according to item 1.
[7] An amino acid quantification kit for performing the amino acid quantification method according to aspects [1] to [6], comprising ATP, an amino acid regeneration reagent, and AARS and tRNA corresponding to the amino acid. Quantitative kit.

In the method for quantifying amino acids according to the present invention, AARS and tRNA corresponding to amino acids in a sample are reacted to form a complex (AARS-tRNA complex) composed of AARS and tRNA, and divalent cations are produced. In the presence, the AARS-tRNA complex, the amino acid and ATP react to form an aminoacyl AMP-AARS-tRNA complex. Next, the once-formed aminoacyl AMP-AARS-tRNA complex (including AARS and aminoacyl-tRNA that can be produced from the complex) is reacted with an amino acid regeneration reagent (nucleotide or alkaline compound) to produce AA, AARS, By liberating at least one or more compounds selected from the group consisting of AARS-tRNA complexes and tRNAs and repeatedly using them to form aminoacyl AMP-AARS-tRNA complexes, pyrophosphate, etc. to be measured Reaction products can be produced up to more moles than the tRNA and/or amino acids contained in the reaction. As a result, radiolabeled amino acids to be measured as in the prior art are no longer necessary. In addition, even if these reaction products are measured by a simpler means than the conventional technology, the amino acid quantification range of the amino acid quantification method of high-sensitivity analysis by fluorescence method using multi-step enzymatic reaction of the conventional technology can be quantified in a range equivalent to , and such high-sensitivity analysis becomes unnecessary. Therefore, according to the present invention, it is possible to provide a method and kit for quantifying glutamic acid, glutamine, and arginine specifically, simply, and with high sensitivity.

FIG. 2 shows the reaction steps considered to be involved in the method of the invention. FIG. 4 shows the amount of pyrophosphate produced by various AARS reactions using tRNA. FIG. 3 shows the amount of pyrophosphate produced by various AARS reactions using tRNA mixtures. FIG. 3 is a diagram showing the amount of pyrophosphate produced at various concentrations of tRNA in ArgRS. FIG. 4 is a diagram showing the amount of pyrophosphate produced at various concentrations of tRNA in GluRS. FIG. 4 is a diagram showing the amount of pyrophosphate produced by various concentrations of tRNA in GlnRS. FIG. 10 is a diagram showing pyrophosphate production at various concentrations of tRNA mixtures in ArgRS. FIG. 10 shows the amount of pyrophosphate produced at various concentrations of tRNA mixtures in GlnRS. FIG. 4 is a diagram showing the amount of pyrophosphate produced in the AARS reaction of the present invention and known documents (Non-Patent Document 6, Patent Document 2). FIG. 4 is a diagram showing pyrophosphate production amounts caused by various ATP concentrations. FIG. 4 is a diagram showing pyrophosphate production amounts caused by various divalent ion concentrations. It is a figure which shows the pyrophosphate production amount by each reaction temperature. It is a figure which shows the pyrophosphate production amount by each pH. FIG. 4 shows an amino acid calibration curve in pyrophosphate measurement by the molybdenum blue method.

In reaction 1 of the method of the present invention, an AARS corresponding to an amino acid (AA) in a sample and a transfer RNA (tRNA) corresponding to the AARS are reacted to form an AARS-tRNA complex consisting of the AARS and tRNA. The AARS used in the method of the present invention are glutamyl-tRNA synthetase (GluRS) that specifically acts on glutamic acid, glutaminyl-tRNA synthetase (GlnRS) that specifically acts on glutamine, and arginyl-tRNA synthetase that specifically acts on arginine. An AARS that specifically acts on amino acids such as (ArgRS) is used. In addition, the AARS used in the present invention is derived from animals such as bovine, rat, and mouse, from plants such as Lupin Seed and Phaseolus aurus, and from microorganisms such as Escherchia, Thermus, Thermotoga, and Saccharomyces. Any AARS may be used as long as it is AARS, and microorganism-derived AARS is particularly preferable from the viewpoint of handling and productivity. Alternatively, it may be a recombinant AARS or a synthetic AARS. Soluble enzymes are preferred, but surfactants may be combined with insoluble enzymes, and enzymes obtained by solubilizing insoluble enzymes by fusion with solubilizing proteins or deletion of membrane-binding portions may be used. Known amino acid sequences of AARS are available, and recombinant AARS have sequences with greater than 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity. , proteins with AARS activity may be used.

The AARS used in the present invention can be any method or means known to those skilled in the art, for example, adding water to an object containing AARS, crushing it with a crusher, ultrasonic crusher, etc., and then centrifuging the crushed product. , an extract from which solid matter has been removed by filtration or the like, and AARS obtained by purifying and isolating the extract by column chromatography or the like can be used. That is, the main technical feature of the present invention is that, in an amino acid quantification method using AARS, AA from the formed aminoacyl AMP-AARS-tRNA complex, and from AARS, AARS-tRNA complex and tRNA By liberating at least one or more compounds selected from the group consisting of and repeatedly using them to form an aminoacyl AMP-AARS-tRNA complex, the reaction product such as pyrophosphate to be measured is converted into the reaction solution. The method of preparing AARS is not limited in any way, as it is to produce more moles than the tRNA and/or amino acids contained therein.

The AARS concentration in the reaction solution used for the reaction can be appropriately determined by a person skilled in the art according to the type of sample, the estimated amino acid concentration in the sample, the ATP concentration, and various reaction conditions such as reaction time and temperature. be done. In order to suppress the reverse reaction in reaction 2 of step (I) as much as possible, when the amino acid concentration in the sample is expected to be low, it is preferable to increase the AARS concentration. If the concentration is expected to be high, the AARS concentration may be low. Further, by increasing the AARS concentration, the reaction can be completed in a short time. Conversely, if a long reaction time is acceptable, the AARS concentration may be low. For example, the concentration of AARS derived from microorganisms such as Escherchia, Thermus, and Thermotoga is 0.1 μM or higher, more preferably 0.25 μM or higher, even more preferably 1.0 μM or higher, particularly preferably 3.0 μM or higher, and most preferably 3.0 μM or higher. Preferably, it can be 5.0 μM or more. In any case, the method of the present invention has the advantage that the AARS is used repeatedly, so there is no need to add an excess amount of AARS relative to the expected amount of amino acids in the sample. Therefore, the upper limit of the AARS concentration can be appropriately set by those skilled in the art in consideration of economic efficiency and the like.

Also, in Reaction 1 of the method of the present invention, tRNA is used together with AARS. The tRNA used in the present invention is a tRNA corresponding to AARS that specifically acts on each amino acid. Examples thereof include tRNA corresponding to GluRS that specifically acts on glutamic acid, tRNA corresponding to GlnRS that specifically acts on glutamine, and tRNA corresponding to ArgRS that specifically acts on arginine. In addition, the tRNA used in the present invention is derived from organisms such as animal-derived bovine, rat, mouse and the like, plant-derived such as Lupin Seed and Phaseolus aurus, and microorganism-derived such as Escherchia, Thermus, Thermotoga and Saccharomyces. Any tRNA may be used as long as it is tRNA, and microorganism-derived tRNA is particularly preferable from the viewpoint of handling and productivity. Alternatively, a synthesized tRNA may be used. A known nucleotide sequence of tRNA can be used, and the synthesized tRNA has a sequence identity of 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, and the AARS Any tRNA that binds to and causes an AARS reaction may be used.

As the tRNA used in the present invention, any method or means known to those skilled in the art can be used, for example, a subject containing tRNA is mixed with a mixture of an extraction buffer and phenol, pulverized with an ultrasonic homogenizer or the like, and then pulverized. tRNA obtained by centrifugation, filtration, or the like of the disrupted product, and further purified and isolated tRNA by column chromatography or the like can be used. That is, the main technical feature of the present invention is that, in an amino acid quantification method using AARS, AA from the formed aminoacyl AMP-AARS-tRNA complex, and from AARS, AARS-tRNA complex and tRNA By liberating at least one or more compounds selected from the group consisting of and repeatedly using them to form an aminoacyl AMP-AARS-tRNA complex, the reaction product such as pyrophosphate to be measured is converted into the reaction solution. The method of preparing tRNA is not limited at all, as it is to produce more moles than the tRNA and/or amino acids contained therein. Furthermore, a mixture in which multiple types of tRNAs corresponding to each amino acid are mixed in an arbitrary ratio, as shown in the following examples as a "tRNA mixture", is used as the "tRNA" in the method of the present invention. It is also possible to

The concentration of tRNA in the reaction mixture used in the reaction will vary depending on the type of sample, expected amino acid concentration, AARS concentration, nucleotide concentration, and various reaction conditions such as reaction time and temperature. determined by the contractor as appropriate. For example, the tRNA concentration can be 0.01 μM or higher, more preferably 0.1 μM or higher, even more preferably 0.5 μM or higher, particularly preferably 1.0 μM or higher, and most preferably 2.0 μM or higher. In the case of a tRNA mixture, the concentration of the total tRNA can be 1 μM or higher, more preferably 5 μM or higher, still more preferably 10 μM or higher, particularly preferably 50 μM or higher, and most preferably 100 μM or higher. The concentration of tRNA was calculated based on the average molecular weight of 25,000. The upper limit of the tRNA concentration can be appropriately set by those skilled in the art in consideration of economic efficiency and the like. In addition, although it depends on the reaction conditions, in order to produce reaction products such as pyrophosphate to be measured in more moles than amino acids contained in the sample, the number of moles of tRNA should be 0.5 times the number of moles of AARS. It is desirable to be 25 times or more, more preferably 0.5 times or more, still more preferably 1 time or more. Furthermore, although it depends on the reaction conditions, in order to produce reaction products such as pyrophosphate to be measured up to a larger number of moles than tRNA contained in the sample, the number of moles of tRNA should be adjusted to 0.5 moles of AARS. It is desirably 1 time or more, more preferably 0.25 times or more, still more preferably 0.5 times or more.

In Reaction 2 of the method of the present invention, an amino acid (AA) in a sample, an AARS-tRNA complex corresponding to the AA, and adenosine triphosphate (ATP) are reacted in the presence of a divalent cation to obtain an aminoacyl AMP-AARS-tRNA complexes are formed.
Samples used in the present invention are not particularly limited, and examples thereof include blood, fresh foods, processed foods and beverages. The amino acid concentration in each sample is different for each amino acid. For example, blood glutamic acid is 11-44 nmol/mL and glutamine is 488-733 nmol/mL. The content of free amino acids in fresh food, for example, in garlic is 136 mg/100 g for arginine. In tomatoes, glutamine is 94 mg/100 g. In dried shiitake, glutamic acid is 386 mg/100 g. The content of free amino acids in processed foods and beverages, for example, in soy sauce, is 782 mg/100 g of glutamic acid. Sencha contains 314 mg/100 g of arginine and 258 mg/100 g of glutamic acid. Depending on the expected amino acid concentration in each of these samples, appropriate dilutions can be prepared and used as the samples of the present invention.

ATP used in the present invention may be sodium salt, lithium salt, or the like. The concentration of ATP in the reaction solution used in the reaction depends on various reaction conditions such as the type of sample, expected amino acid concentration, AARS concentration, nucleotide concentration, and reaction time and temperature in the sample. The manufacturer can appropriately determine the amount, but it is preferable to add the amino acid in an excess amount relative to the expected amino acid concentration in the sample. For example, when the sample is blood, the ATP concentration should be 0.01 mM or higher, more preferably 0.1 mM or higher, still more preferably 1.0 mM or higher, particularly preferably 5.0 mM or higher, and most preferably 10.0 mM or higher. can be done. The upper limit of the ATP concentration can be appropriately set by those skilled in the art in consideration of economic efficiency and the like.

Divalent ions used in the present invention can be magnesium, manganese, cobalt, calcium and the like. Since each AARS has different requirements for divalent ions, divalent ions suitable for the AARS to be used may be appropriately used, but it is more preferable to use magnesium and manganese, which are required in common by AARS. Furthermore, polyamines such as spermine, spermidine, and putrescine, which act similarly to divalent ions, can also be used. The concentration of divalent ions in the reaction solution used in the reaction can be appropriately determined, but it is preferable to add the divalent ions in an amount equal to or higher than the ATP concentration. For example, the ATP: divalent cation ratio in AARS from microorganisms such as Escherchia, Thermus, Thermotoga is at least 1:1, more preferably at least 1:3, even more preferably at least 1:5, particularly preferably can be at least 1:7, most preferably at least 1:10.

Subsequently, in Reaction 3 of the method of the present invention, the aminoacyl AMP-AARS-tRNA complex formed in Reaction 2 and/or Reaction 4, and the aminoacyl tRNA that can be produced from the complex depending on the reaction conditions, etc. A regeneration reagent is allowed to act to decompose the complex to release AA and at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complex and tRNA from the complex. Examples of amino acid regeneration reagents for the reaction include nucleotides such as ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP) and guanosine triphosphate (GTP), sodium hydroxide, potassium hydroxide, carbonate Alkaline compounds that generate hydroxide ions (aqueous alkaline solutions) such as sodium and buffers can be used. Further, the alkaline compound should be able to make the reaction field (reaction system) pH 7 or higher. For example, in the case of AARS of the genus Escherchia, Thermus and Thermotoga, the pH is preferably 7.0 or higher, more preferably 7.5 or higher, and still more preferably 8.0 or higher. Incidentally, the reaction pH can be adjusted using any buffer known to those skilled in the art. The concentration of the amino acid regeneration reagent in the reaction solution used in the reaction depends on various reaction conditions such as the type of sample, the expected amino acid concentration in the sample, the AARS concentration, the ATP concentration, the tRNA concentration, and the reaction time and temperature. Depending on the situation, the total amount of ATP used in Reaction 2 and the amino acid regeneration reagent used in the reaction should preferably be added in excess with respect to the concentration of amino acids in the sample. For example, when the sample is blood, the nucleotide concentration is 0.01 mM or higher, more preferably 0.1 mM or higher, still more preferably 1.0 mM or higher, particularly preferably 5.0 mM or higher, and most preferably 10.0 mM or higher. be able to. Therefore, in the method of the present invention, there is no need to add an aminoacyl AMP-AARS complex degrading reagent such as amines as described in Patent Document 2 in order to restore the AARS to a state capable of reacting. Since the released amino acid does not react with the reagent, the released amino acid can be reused to form the aminoacyl AMP-AARS-tRNA complex.

In Reaction 4 of the method of the present invention, the AA liberated in Reaction 3 and at least one compound selected from the group consisting of AARS, AARS-tRNA complex and tRNA are used again in Reaction 1 and/or Reaction 2. to form an aminoacyl AMP-AARS-tRNA complex reaction. Furthermore, in step (I) of the method of the present invention, the pyrophosphate generated in Reaction 2 or 4 and AMP in the reaction system are reacted with phosphoenolpyruvate and pyruvate dikinase by any method known to those skilled in the art. ATP produced by the formation of an aminoacyl AMP-AARS-tRNA complex, AA from the complex, and release of at least one or more compounds selected from the group consisting of AARS, AARS-tRNA complexes and tRNA and/or ADP produced by allowing Ap4A pyrophosphohydrolase to act on Ap4A produced from nucleotides in Reaction 3 can be reused as nucleotides in Reaction 3.

As a result, under the reaction conditions such as the composition of nucleotides such as ATP and the AARS corresponding to the amino acids, the reaction in step (I) results in the production of respective reaction products such as pyrophosphate and/or hydrogen ions. For each, more moles of molecules can be produced than the moles of tRNA or amino acid to be measured contained in the reaction solution. As a result, the method of the present invention enables quantification from extremely low amino acid concentrations of about 1 μM, which are lower than those of the prior art. Therefore, this point can be said to be a remarkable effect of the present invention as compared with the prior art.

However, even if the amount of reaction products such as pyrophosphate produced under the reaction conditions is less than the number of moles of tRNA and amino acids in the reaction solution, it is possible to quantify amino acids based on the reaction products. For example, reaction products such as pyrophosphate need not be produced in amounts greater than or equal to the number of moles of the amino acid. Therefore, the amounts of pyrophosphate and hydrogen ions produced in step (I) of the present invention are as long as amino acids can be quantified by an appropriate method for measuring the reaction product in step (II). It is not particularly limited. Therefore, the number of repetitions of reaction 3 (step I-3) and reaction 4 (step I-4) in (step I-5) of the method of the present invention is not particularly limited.

The reaction temperature in step (I) of the process of the present invention may be any temperature at which each reaction occurs. For example, AARS of the genus Escherchia is 10 to 60°C, preferably 40 to 50°C, and AARS of the genus Thermus and Thermotoga is 10 to 95°C, preferably 40 to 70°C. In addition, the reaction time may be any time during which the amino acids in the sample react with the AARS, preferably about 1 to 90 minutes, more preferably about 5 to 70 minutes, still more preferably about 10 to 60 minutes. is desirable.

Reaction components such as reagents and enzymes used in each step included in step (I) of the method of the present invention can be added to the reaction system by any means, procedures, etc. known to those skilled in the art, as long as the addition method causes the AARS reaction. can be added to For example, each component may be added to the reaction system at once before starting the reaction, or the AARS or amino acid may be added last and allowed to react.

In step (II) of the method of the present invention, the amount of each reaction product such as pyrophosphate and hydrogen ion produced in step (I) is measured, and the amount of amino acid is determined based on the measured amount of the reaction product. do. Step (II) is performed after stopping the reaction between the amino acid in the sample and AARS in step (I) by any method or means known to those skilled in the art, or after step ( It can be suitably carried out at any stage during the reaction in I).

The amount of pyrophosphate produced in step (I) of the present invention can be measured by any method or means known to those skilled in the art, for example, by measuring the absorbance of a blue complex produced by reacting molybdic acid and pyrophosphate. The molybdenum blue method, a method combining hypoxanthine-guanine phosphoribosyltransferase, xanthine oxidase or xanthine dehydrogenase can be used. Further, the pyrophosphate produced in the step (I) of the present invention is treated with inorganic pyrophosphatase or the like to convert it into two molecules of phosphoric acid, and the resulting phosphoric acid is measured, whereby a more sensitive measurement can be performed. For example, an enzymatic method that can measure pyrophosphate, such as a method that combines luminol with inorganic pyrophosphatase, pyruvate oxidase, and peroxidase and measures the luminescence of the product can be used. Furthermore, a measurement method of causing an oxidation-reduction reaction by an enzyme reaction or the like and measuring a potential change by a multi-electrode potential measuring instrument that detects a current value derived from the oxidation-reduction reaction can be used. In addition, for the measurement of the hydrogen ions produced in the reaction, it is possible to use, for example, a measurement method of measuring a potential change using a glass electrode or an ion-sensitive field effect transistor that detects hydrogen ions. Adenosine monophosphate (AMP) produced in the reaction can be measured using an AMP sensor that uses luminescence that detects adenosine monophosphate. Pyrophosphate, hydrogen ions, AMP, etc. generated in step (I) of the present invention can be separated from the reaction solution and measured. The method for separating pyrophosphate, hydrogen ions, AMP, etc. from the reaction solution is not particularly limited as long as it is a method that does not affect the measurement. separation and the like. The main technical features of the present invention are, in the amino acid quantification method using AARS, AA from the formed aminoacyl AMP-AARS-tRNA complex, and from the group consisting of AARS, AARS-tRNA complex and tRNA By liberating at least one or more selected compounds and repeatedly using these compounds to form the complex, the reaction product such as pyrophosphate to be measured is included in the reaction solution. The method of measuring the amount of the reaction product is not limited at all, as it is to produce more moles than tRNA and/or amino acids.

The present invention provides an amino acid quantification kit for carrying out the above-described method of the present invention, which contains the aforementioned components necessary for quantifying amino acids in a sample. The kit may appropriately contain other optional components known to those skilled in the art, such as stabilizers or buffers, to enhance the stability of reagent components such as enzymes. Although there are no particular limitations as long as it is a component that does not affect the measurement, for example, bovine serum albumin (BSA), ovalbumin, sugars, sugar alcohols, carboxyl group-containing compounds, antioxidants, surfactants, or enzymes and activity can be exemplified by amino acids without Further, as an example of the kit, the aforementioned kit for measuring pyrophosphate and hydrogen ions can be mentioned.
EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited by the following examples.

(Preparation of AARS derived from E. coli)
A plasmid (pET28b) having an AARS sequence of a mutant strain (NBRC3992) derived from E. coli K12 strain was introduced into E. coli BL21(DE3) pLys strain and used as an expression strain. Each expression strain was cultured at 37°C in LB medium containing kanamycin and chloramphenicol as selection markers, and after OD600 reached about 0.6, IPTG was added to a final concentration of 1 mM. Induction culture was performed overnight at 25°C. After the culture was completed, the cells were collected, and the obtained cells were sonicated to prepare a cell-free extract. After further centrifugation, expression of the target enzyme was confirmed by electrophoresis using a portion of the resulting supernatant. Next, the remaining supernatant was treated with a His-tag column (trade name: TALON superflow, manufactured by GE Healthcare) to remove contaminant proteins, resulting in ArgRS (SEQ ID NO: 1), GluRS (SEQ ID NO: 2), and GlnRS (SEQ ID NO: 3). Obtained.

(Preparation of tRNA derived from E. coli)
tRNA was transcribed in vitro from an oligonucleotide encoding the tRNA gene sequence from E. coli strain K12. After confirming transcription of the target tRNA by electrophoresis, unreacted NTPs, salts and the like were removed using a gel filtration column. Next, by removing nucleic acid fragments other than the target tRNA using an ion exchanger column, tRNAs (SEQ ID NOS: 4 to 6) corresponding to amino acids of arginine, glutamic acid and glutamine, respectively, were prepared.

(Preparation of E. coli-derived tRNA mixture)
A tRNA mixture was prepared by dissolving a mixture of dozens of E. coli-derived tRNAs (trade name: tRNA, from E. coli MRE600, 100 mg, manufactured by Roche) in nuclease-free water.

(Amount of pyrophosphate produced by various AARS reactions using tRNA: Step (I) of the method of the present invention)
150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 10 mM ATP, 100 mM MgCl ₂ , 50 μM L-glutamic acid, 2.0 μM GluRS and 18 μM tRNA was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was stopped, the precipitate was removed by centrifugation to prepare Working Product 1 (the product of the present invention).

150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 12.5 mM MnCl ₂ , 50 μM L-arginine, 2.5 μM ArgRS, and 10 μM tRNA was prepared and treated at 50° C. for 60 minutes. . After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After the reaction was terminated, the precipitate was removed by centrifugation to prepare a working product 2 (product of the present invention).

150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 3 mM ATP, 30 mM MgCl ₂ , 50 μM L-glutamine, 2.0 μM GlnRS and 8.2 μM tRNA was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Working Product 3 (product of the present invention).

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
150 μL of the reaction solution of Examples 1 to 3 prepared in Examples 4, 5, and 6, 15 μL of 1 M mercaptoethanol, and 60 μL of a coloring solution (2.5% ammonium molybdate/5N sulfuric acid) were mixed and allowed to stand at room temperature for 20 minutes. After that, absorbance at 580 nm was measured. The amount of pyrophosphate in the reaction solution was determined from the value obtained by subtracting the absorbance value of the sample to which water was added instead of the substrate amino acid as a blank from the absorbance value of each sample. As a result, as shown in FIG. 2, in GluRS, ArgRS and GlnRS, pyrroline produced only when the tRNA molecule and/or each substrate amino acid in the reaction solution was used (not reused) in the enzymatic reaction only once. More pyrophosphate was produced than the theoretical amount of acid.

(Amount of pyrophosphate produced by various AARS reactions using tRNA mixtures: step (I) of the method of the present invention)
150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Working Product 4 (the product of the present invention).

150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 125 mM MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mixture was prepared and treated at 50° C. for 60 minutes. . After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Working Product 5 (the product of the present invention).

150 μL of a reaction solution containing a mixture of 200 mM HEPES-KOH (pH 8.0), 3 mM ATP, 30 mM MgCl ₂ , 20 μM L-glutamine, 2.0 μM GlnRS and 320 μM tRNA was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Working Product 6 (product of the present invention).

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphate of Examples 4-6 prepared in Examples 8, 9 and 10 was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 3, in GluRS, ArgRS and GlnRS, the theoretical value of the amount of pyrophosphate produced when each substrate amino acid in the reaction solution was used (not reused) in the enzymatic reaction only once More pyrophosphate was produced.

(Amount of Pyrophosphate Produced at Various tRNA Concentrations: Step (I) of the Method of the Present Invention)
0 μM tRNA, _0.25 μM tRNA, 0.63 μM tRNA, 0.63 μM 150 μL of each reaction solution containing tRNA, 1.25 μM tRNA, 1.88 μM tRNA, 2.5 μM tRNA, or 5.0 μM tRNA was prepared and treated at 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 7, 8, 9, 10, 11, 12, and 13 were prepared.

0 μM tRNA, 6 μM tRNA, 12 μM tRNA, or 18 μM tRNA was added to a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 10 mM ATP, 100 mM MgCl ₂ , 50 μM L-glutamic acid, and 2.0 μM GluRS, respectively. 150 μL of each reaction solution was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 14, 15, 16 and 17 were prepared.

0 μM tRNA, 4 μM tRNA, 8 μM tRNA, or 16 μM tRNA was added to a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 3 mM ATP, 30 mM MgCl ₂ , 50 μM L-glutamine, and 2.0 μM GlnRS, respectively. 150 μL of each reaction solution was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 18, 19, 20, and 21 were prepared.

A reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 125 mM MgCl ₂ , 20 μM L-arginine, and 2.5 μM ArgRS was added with 0 μM tRNA mixture, 100 μM tRNA mixture, and 200 μM tRNA mixture, respectively. 150 μL of each reaction solution to which the product or 400 μM tRNA mixture was added was prepared and treated at 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 22, 23, 24, and 25 were prepared.

0 μM tRNA mixture, 160 μM tRNA mixture, 240 _μM tRNA mixture, Alternatively, 150 μL of each reaction solution to which 320 μM tRNA mixture was added was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 26, 27, 28, and 29 were prepared.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphate of Examples 7-29 prepared in Examples 12, 13, 14, 15 and 16 was determined by the molybdenum blue method described in Example 7. As a result, as shown in FIGS. 4, 5, 6, 7 and 8, pyrophosphate increased with increasing tRNA concentration in ArgRS, GluRS and GlnRS. In the absence of tRNA, no increase in pyrophosphate was observed in any of ArgRS, GluRS and GlnRS, indicating that tRNA is essential for these AARS reactions. In addition, more pyrophosphate is produced than the theoretical value of the amount of pyrophosphate produced simply when all of the tRNA and/or each substrate amino acid in the reaction solution is used once (not reused) in the enzyme reaction. It had been.

[Comparative Example 1]
(Comparison of the amount of pyrophosphate produced in the AARS reaction described in the present invention and known literature)
100 mM HEPES-KOH (pH 7.4), 2 mM ATP, 10 mM 150 μL of reaction solution containing MgCl ₂ , 20 μM L-arginine, 15.4 μM ArgRS and 2 μM tRNA was prepared and treated at 37° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare a comparative product 1.

[Comparative Example 2]
150 μL of a reaction solution containing 20 mM HEPES-KOH (pH 8.0), 0.5 mM ATP, 5 mM MgCl ₂ , 20 μM L-arginine, 1.0 μM ArgRS, and 400 mM hydroxylamine was prepared according to the reaction conditions described in Patent Document 2. , 50° C., 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare a comparative product 2.

150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 12.5 mM MnCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 10 μM tRNA was prepared and treated at 50° C. for 60 minutes. . After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare Working Product 30 (product of the present invention).

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphates of comparative products 1, 2 and 30 prepared in Comparative Examples 1 and 2 and Example 18 were measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 9, in the method of the present invention, the amount of pyrophosphate estimated to be produced when all of the added tRNA and amino acids were used (not reused) in a one-time reaction. More pyrophosphate was produced than the theoretical value. On the other hand, the amount of pyrophosphate produced by Comparative Product 1 was close to the theoretical value of tRNA and less than the theoretical value of amino acids. Moreover, in the comparative product 2, pyrophosphate was not produced at all.

(Amount of Pyrophosphate Produced at Various ATP Concentrations: Step (I) of the Method of the Present Invention)
Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 1.25 mM ATP, 1.25 mM MnCl ₂ , 50 μM L-arginine, 2.5 μM ArgRS, and 10.0 μM tRNA, 200 mM HEPES-KOH (pH 8.0) , 6.25 mM ATP, 6.25 mM MnCl ₂ , 50 μM L-arginine, 2.5 μM ArgRS, and 10.0 μM tRNA, or 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 12 150 μL of reaction solution containing 5 mM MnCl ₂ , 50 μM L-arginine, 2.5 μM ArgRS and 10.0 μM tRNA was prepared and treated at 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation to prepare working products (products of the present invention) 31, 32, and 33.

Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 1.25 mM ATP, 12.5 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mix, 200 mM HEPES-KOH (pH 8.0) , 2.5 mM ATP, 25 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture, 200 mM HEPES-KOH (pH 8.0), 5 mM ATP, 50 mM MgCl ₂ , 20 μM L- Reaction solutions containing glutamate, 5.0 μM GluRS, and 600 μM tRNA mix, or 200 mM HEPES-KOH (pH 8.0), 10 mM ATP, 100 mM MgCl ₂ , 20 μM L-glutamate, 5.0 μM GluRS, and 600 μM tRNA mix. 150 μL of reaction solution containing the product was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 34, 35, 36, and 37 were prepared.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
Pyrophosphate of Examples 31 to 37 prepared in Examples 20 and 21 was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 10, pyrophosphate increased with an increase in ATP concentration in ArgRS and GluRS. In addition, more pyrophosphate was produced than the theoretical amount of pyrophosphate produced when tRNA and/or all of the substrate amino acids were used (not reused) in the enzymatic reaction only once.

(Pyrophosphate production amount by various divalent ion concentrations: step (I) of the method of the present invention)
Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 18.75 mM MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mix, 200 mM HEPES-KOH (pH 8.0) , 12.5 mM ATP, 31.25 mM MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mixture, 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 62.5 mM Reaction solutions containing MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mix or 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 93.75 mM MgCl ₂ , 20 μM L-arginine , 2.5 μM ArgRS, and 400 μM tRNA mixture (150 μL) was prepared and treated at 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 38, 39, 40, and 41 were prepared.

Reaction solution containing 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 37.5 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mix, 200 mM HEPES-KOH (pH 8.0), 25 mM Reaction solution containing ATP, 125 mM MgCl ₂ , 20 μM L-glutamate, 5.0 μM GluRS, and 600 μM tRNA mixture, 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 187.5 mM MgCl ₂ , 20 μM L-glutamate, Reaction solutions containing 5.0 μM GluRS and 600 μM tRNA mixture, or 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture. 150 μL of the reaction solution containing was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and working products (products of the present invention) 42, 43, 44, and 45 were prepared.

(Measurement of pyrophosphate by molybdenum blue method: step (II) of the method of the present invention)
The pyrophosphate of Examples 38-45 prepared in Examples 23 and 24 was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 11, for both ArgRS and GluRS, at ATP: divalent ion ratios of 1:1.5 to 1:10, all of each substrate amino acid was used in a single enzymatic reaction (repeated). more pyrophosphate was produced than the theoretical amount of pyrophosphate produced in the case of non-utilization.

(Pyrophosphate production amount at each reaction temperature: step (I) of the method of the present invention)
150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 12.5 mM MnCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 2.5 μM tRNA was prepared, and 20 or 30, or 40 or 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. As a result, as shown in FIG. 12, good pyrophosphate production was observed over a wide range of 20 to 50.degree. In addition, more pyrophosphate was produced than the theoretical amount of pyrophosphate produced when all of the tRNA and/or each substrate amino acid were used (not reused) in the enzymatic reaction only once.

150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture was prepared and incubated at 30, 40, or 50°C. Treated for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. As a result, as shown in FIG. 12, good pyrophosphate production was observed over a wide range of 30 to 50.degree. In addition, more pyrophosphate was produced than the theoretical amount of pyrophosphate produced when all of the substrate amino acids were used in the enzymatic reaction only once (not reused).

(Pyrophosphate production amount at each pH: step (I) of the method of the present invention)
Reaction solution containing 200 mM MES-KOH (pH 6.0), 12.5 mM ATP, 125 mM MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mix, 200 mM HEPES-KOH (pH 7.0), 12 Reaction solution containing 5 mM ATP, 125 mM MgCl ₂ , 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mixture, 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 125 mM MgCl ₂ , 20 μM L- Reaction solutions containing Arginine, 2.5 μM ArgRS, and 400 μM tRNA mix or 200 mM CHES-KOH (pH 9.0), 12.5 mM ATP, 125 mM MgCl ₂ , 20 μM L-Arginine, 2.5 μM ArgRS, and 400 μM 150 μL of reaction solution containing the tRNA mixture was prepared and treated at 50° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. As a result, as shown in FIG. 13, at pH 7 to 8, more pyrophosphate than the theoretical value of the amount of pyrophosphate produced when all of the substrate amino acids were used (not reused) in the enzymatic reaction only once. was produced.

Reaction solution containing 200 mM MES-KOH (pH 6.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mix, 200 mM HEPES-KOH (pH 7.0), 25 mM ATP, Reaction solution containing 250 mM MgCl ₂ , 20 μM L-glutamate, 5.0 μM GluRS, and 600 μM tRNA mixture, 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamate, 5.0 μM GluRS , and a reaction solution containing 600 μM tRNA mixture, or a reaction solution containing 200 mM CHES-KOH (pH 9.0), 25 mM ATP, 250 mM MgCl ₂ , 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture. 150 μL was prepared and treated at 40° C. for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. As a result, as shown in FIG. 13, at pH 7 to 9, pyrophosphate more than the theoretical value of the amount of pyrophosphate produced when all of the substrate amino acids were used (not reused) in the enzymatic reaction only once. was produced.

(Standard curve of arginine in pyrophosphate measurement by molybdenum blue method using tRNA)
Prepare 150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 12.5 mM MnCl ₂ , 0, 10, 20, or 30 μM L-arginine, 2.5 μM ArgRS, and 10 μM tRNA, The mixture was reacted at 50°C for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 14, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.96) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 30 μM, indicating that L-arginine can be quantified.

(Standard curve of glutamine in pyrophosphate measurement by molybdenum blue method using tRNA)
150 μL of a reaction solution containing 200 mM HEPES-KOH (pH 8.0), 3 mM ATP, 30 mM MgCl ₂ , 0, 5, 10, 20, or 30 μM L-glutamine, 2 μM GlnRS, and 16 μM tRNA was prepared, heated at 40° C., 60° C. reacted for a minute. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 14, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.99) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 30 μM, indicating that L-glutamine can be quantified.

(Arginine calibration curve in pyrophosphate measurement by molybdenum blue method using tRNA mixture)
Prepare 150 μL reaction solution containing 200 mM HEPES-KOH (pH 8.0), 12.5 mM ATP, 125 mM MgCl ₂ , 0, 5, 10, or 20 μM L-arginine, 2.5 μM ArgRS, and 400 μM tRNA mixture; The mixture was reacted at 50°C for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 14, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.99) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 20 μM, indicating that L-arginine can be quantified.

(Standard curve of glutamic acid in pyrophosphate measurement by molybdenum blue method using tRNA mixture)
Prepare 150 μL of reaction solution containing 200 mM HEPES-KOH (pH 8.0), 25 mM ATP, 250 mM MgCl ₂ , 0, 5, 10, or 20 μM L-glutamic acid, 5.0 μM GluRS, and 600 μM tRNA mixture, at 40° C. , reacted for 60 minutes. After the reaction, 30 μL of trichloroacetic acid was added to a final concentration of 4% to terminate the reaction. After stopping the reaction, the precipitate was removed by centrifugation, and pyrophosphate in the supernatant was measured by the molybdenum blue method described in Example 7. As a result, as shown in FIG. 14, more pyrophosphate was produced than the theoretical amount of pyrophosphate that would be produced if all the added amino acids were used in the reaction. Moreover, a correlation (R=0.99) was observed between the amino acid concentration and the amount of pyrophosphate in the amino acid concentration range of 0 to 20 μM, indicating that L-glutamic acid can be quantified.

From the above results, it was shown that the AARS reaction in the method of the present invention can amplify the pyrophosphate produced by repeatedly using AARS, tRNA and amino acids in the reaction. As shown in Examples 4 to 11, all of ArgRS, GluRS and GlnRS were able to produce more pyrophosphate in the reaction than the number of moles of tRNA and/or amino acids in the reaction solution. As shown in Examples 12 to 17, it was found that the amount of pyrophosphate produced varies depending on the tRNA concentration. In addition, when no tRNA was added, no increase in pyrophosphate was observed in any of ArgRS, GluRS and GlnRS, indicating that tRNA is essential for these AARS reactions. As shown in Comparative Examples 1 and 2, in the conventional AARS reaction, the amount of pyrophosphate produced was less than the number of tRNA and/or amino acid molecules in the reaction solution. , it was found that more pyrophosphate was produced than the number of tRNA and/or amino acid molecules in the reaction solution. As shown in Examples 20 to 25, it was found that the amount of pyrophosphate produced varies depending on the ATP concentration and magnesium concentration. In addition, as shown in Examples 26-29, it was found that the amount of pyrophosphate produced varies depending on the enzyme reaction temperature and pH, and pyrophosphate is produced favorably at 30-50°C and pH 7-9. Furthermore, as shown in Examples 30 to 33, it was found that pyrophosphate increased linearly depending on the amino acid concentration, that is, there was a correlation between the amino acid concentration and the amount of pyrophosphate. It was confirmed that it was possible to quantify various amino acids from the produced pyrophosphate using the molybdenum blue method, which is a simple method.

In the conventional amino acid quantification method using AARS, which requires association of tRNA, it is necessary to detect trace amounts of reaction products, and radiolabeled target amino acids and highly sensitive analysis are essential. In contrast, according to the method for quantifying amino acids according to the present invention, it is possible to produce reaction products such as pyrophosphate, which are the objects to be measured, to a larger number of moles than tRNA and/or amino acids contained in the reaction solution. can. As a result, radiolabeled amino acids to be measured as in the prior art are unnecessary. In addition, even if these reaction products are measured by a simpler means than the conventional technology, the amino acid quantification range of the amino acid quantification method of high-sensitivity analysis by fluorescence method using multi-step enzymatic reaction of the conventional technology can be quantified in a range equivalent to , and such high-sensitivity analysis is unnecessary. Therefore, according to the present invention, it has become possible to provide a method for quantifying amino acids specifically, easily, and with high sensitivity using AARS, and a kit for amino acid quantification.

Claims (6)

Step (I) comprising the following steps:
(Step I-1) Aminoacyl-tRNA synthetase (AARS) corresponding to an amino acid (AA) in a sample and transfer RNA (tRNA) are reacted to form a complex consisting of AARS and tRNA (AARS-tRNA complex). A step comprising a reaction (Reaction 1) to cause;
(Step I-2) reacting the AA, the AARS-tRNA complex corresponding to the AA, and adenosine triphosphate (ATP) in the presence of a divalent cation to form aminoacyl adenylic acid (aminoacyl AMP); a step comprising a reaction (reaction 2) to form a complex consisting of an AARS-tRNA complex (aminoacyl AMP-AARS-tRNA complex);
(Step I-3) The aminoacyl AMP-AARS-tRNA complex formed in Reaction 2 and/or Reaction 4 (including AARS and aminoacyl-tRNA that can be produced from the complex) is allowed to act with an amino acid renaturing reagent to obtain AA. , and a step comprising a reaction (reaction 3) for releasing at least one compound selected from the group consisting of AARS, AARS-tRNA complexes and tRNA;
(Step I-4) Reusing the AA released in Reaction 3 and at least one compound selected from the group consisting of AARS, AARS-tRNA complex and tRNA in Reaction 1 and/or Reaction 2 forming an aminoacyl AMP-AARS-tRNA complex reaction (reaction 4); and
(Step I-5) repeating steps I-3 and I-4, measuring the amount of the reaction product produced in step (I), and determining the amount of amino acid based on the measured amount of the reaction product; step (II) comprising determining
A method for quantifying amino acids in a sample comprising
A method for quantifying amino acids , wherein the amino acid regeneration reagent used in the step (I) is a nucleotide and an alkaline compound . 2. The method for quantifying amino acids according to claim 1, wherein the amount of tRNA used in step (I) is 0.25 times or more the molar amount of AARS. 3. The method for quantifying amino acids according to claim 1 or 2 , wherein the amount of the reaction product produced in step (I) is measured by measuring absorbance change by an absorbance method. 4. The method for quantifying amino acids according to any one of claims 1 to 3 , wherein at least one of pyrophosphate and hydrogen ions is measured as the reaction product generated in step (I). The amino acid according to any one of claims 1 to 4 , wherein the number of moles of the reaction product produced in step (I) is greater than the number of moles of tRNA and/or amino acid contained in the reaction solution. Quantification method. An amino acid quantification kit for performing the amino acid quantification method according to any one of claims 1 to 5 , comprising ATP, an amino acid regeneration reagent, and AARS and tRNA corresponding to the amino acid. Kit for.

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