JP2007111045A - Reagent containing 6-phosphogluconic acid dehydrogenase and method for stabilizing 6-phosphogluconic acid dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent containing 6PGDH and a 6PGDH stabilizing agent and provide a method for stabilizing 6PGDH by using a 6PGDH stabilizing agent. <P>SOLUTION: The 6PGDH-containing reagent contains 6-phosphogluconic acid dehydrogenase (6PGDH), at least one coenzyme selected from nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and at least one compound selected from a water-soluble compound, an oxyacid having reducing power and containing sulfur atom as the center atom, a thioic acid of the oxyacid, salts of the oxyacid or the thioic acid and aminoethylaminoethanol. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reagent containing 6-phosphogluconate dehydrogenase (hereinafter referred to as 6PGDH) and a method for stabilizing 6PGDH in the reagent.

  6PGDH dehydrates 6-phosphogluconic acid (hereinafter referred to as 6PG) in the presence of coenzymes such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), and reduces reduced NAD ( NADH) or an enzyme that catalyzes a reaction that produces reduced NADP (NADPH). Utilizing this catalytic action, 6PGDH is used as a reagent for clinical examinations (for example, Patent Document 1).

  However, 6PGDH in the reagent is unstable, and the activity as an enzyme is significantly reduced during storage of the reagent. For this reason, development of the technique which can suppress the deactivation of 6PGDH in a reagent and can be stabilized is desired.

JP-A-7-250698

  An object of the present invention is to provide a reagent containing 6PGDH and a substance that stabilizes 6PGDH (hereinafter referred to as 6PGDH stabilizer), and a method of stabilizing 6PGDH using the 6PGDH stabilizer. That is.

The present invention comprises 6-phosphogluconate dehydrogenase (6PGDH), at least one coenzyme selected from the group consisting of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), Reagents containing at least one 6PGDH stabilizer selected from the following group are provided:
Formula (I)

（Ｒ１は水素原子、ＣＨ_３、またはＳＯ_３Ｈ；Ｒ２は、ＳＨ、ＣＨ_２ＳＨ、またはＮＨ_２であり、Ｒ３はＳＨ、ＣＨ_２ＳＨ、ＮＨ_２、ＮＨＣＯＮＨ_２、またはＮＨＣ（ＮＨ）ＮＨ_２であり、Ｒ４は水素原子、ＯＨ、ＣＨ_３、ＮＨ_２、ＯＣＨ_３、ＣＯＯＨ、ＣＯＯＣＨ_３、またはＣＯＯＣ_２Ｈ_５である。ただし、Ｒ２がＳＨまたはＣＨ_２ＳＨの場合、Ｒ３はＮＨ_２、ＮＨＣＯＮＨ_２、またはＮＨＣ（ＮＨ）ＮＨ_２であり、Ｒ２がＮＨ_２の場合、Ｒ３はＳＨ、またはＣＨ_２ＳＨである）で表される水溶性化合物；
下記式（ＩＩ）

(R1 is a hydrogen atom, CH ₃ or _SO 3 H,; R2 is, SH, a _CH 2 SH or NH _2,, R3 is _{SH, CH 2 SH, NH 2} , NHCONH 2 or NHC (NH) NH _2, in it, R4 is _{hydrogen, OH, CH 3, NH 2} , OCH 3, COOH, COOCH 3, or COOC ₂ H _5. However, if R2 is SH or _CH 2 SH, R3 is _NH 2, NHCONH ₂ or NHC (NH) NH ₂ and when R 2 is NH ₂ , R 3 is SH or CH ₂ SH);
The following formula (II)

（Ｒ５およびＲ６は同一または異なって、水素原子、NH_２、低級アルキル、シクロアルキル、低級アルケニル、アラルキル、アリール、低級アルカノイル、アロイル、シンナミル、またはシンナモイルである）で表される水溶性化合物；
下記式（ＩＩＩ）

(R5 and R6 are the same or different and each represents a hydrogen atom, NH ₂ , lower alkyl, cycloalkyl, lower alkenyl, aralkyl, aryl, lower alkanoyl, aroyl, cinnamyl, or cinnamoyl);
Formula (III) below

（Ｒ７およびＲ８は同一または異なって、水素原子、低級アルキル、アリル、ＣＨＯ、Ｃ（ＮＨ）ＮＨＮＨ_２、ＣOＣＨ_３、ＣＨ_３ＯＣＯ、Ｃ_２Ｈ_５ＯＣＯまたは下記式（ＩＶ）

(R7 and R8 are the same or different, a hydrogen atom, a lower alkyl, _{allyl, CHO, C (NH) NHNH} 2, COCH 3, CH 3 OCO, C 2 H 5 OCO or formula (IV)

（R９は硫黄原子、酸素原子、NHまたはNNH_２であり、R１０は低級アルキル、NH_２、NHNH_２、CONH_２、またはCONHNH_２である）で表される基である）で表される水溶性化合物；
還元性を有し且つ硫黄原子を中心原子とする酸素酸；
前記酸素酸のチオ酸；
前記酸素酸または前記チオ酸の塩；および
アミノエチルアミノエタノール。

(R9 sulfur atom, an oxygen atom, NH or NNH _2, R10 is lower _{alkyl, NH 2, NHNH 2, CONH} 2, or CONHNH ₂ a is) a water-soluble represented by a group represented by) Compound;
Oxygen acids having reducibility and having a sulfur atom as a central atom;
A thioacid of the oxygen acid;
Said oxygen acid or salt of said thioacid; and aminoethylaminoethanol.

  Further, the present invention relates to 6-phosphogluconate dehydrogenase (6PGDH), cyclohexyldiaminetetraacetic acid (CyDTA), Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-Acetamido ) iminodiacetic acid (ADA), and at least one 6PGDH stabilizer selected from the group consisting of compounds having SH groups, and containing nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) 6PGDH-containing reagent substantially free of).

  In addition, in the presence of at least one coenzyme selected from the group consisting of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), 6-phosphogluconate dehydrogenase (6PGDH) and A 6PGDH stabilization method for stabilizing 6PGDH by coexisting with at least one of the above 6PGDH stabilizers is provided.

  The present invention also relates to 6-phosphogluconate dehydrogenase (6PGDH) and cyclohexyldiaminetetraacetic acid in the substantial absence of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). (CyDTA), Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-Acetamido) iminodiacetic acid (ADA), and at least one selected from the group consisting of compounds having an SH group The 6PGDH stabilization method which stabilizes 6PGDH by coexisting is provided.

  According to the present invention, a reagent containing 6PGDH and a 6PGDH stabilizer can be provided. Moreover, the method of stabilizing 6PGDH in a reagent can be provided.

  The 6PGDH-containing reagent of this embodiment contains 6PGDH and a 6PGDH stabilizer. The 6PGDH stabilizer refers to a substance that can suppress the deactivation of 6PGDH and improve storage stability by coexisting with 6PGDH in a 6PGDH-containing reagent. As used herein, “6PGDH stabilizer” includes salts of 6PGDH stabilizer and the like. As salts of 6PGDH stabilizer, inorganic acid salts (hydrochloride, hydrobromide, sulfate, nitrate, nitrite, phosphate, etc.), organic acid salts (oxalate, acetate, succinate) , Fumarate, maleate, tartrate, citrate, etc.) and base addition salts (sodium salt, potassium salt, calcium salt, magnesium salt, etc.).

  6PGDH requires a coenzyme such as NAD or NADP in the enzyme reaction. The coenzyme may be contained in a 6PGDH-containing reagent, or may be accommodated in a separate container from the 6PGDH-containing reagent.

When 6PGDH coexists with a coenzyme such as NAD or NADP, the coenzyme destabilizes 6PGDH. In the reagent solution, NAD and NADP are decomposed to generate ADP ribose. A part of the produced ADP ribose is oxidized to an aldehyde, and this aldehyde group is considered to destabilize 6PGDH. Since this coenzyme has a strong destabilizing effect, a compound that reduces the destabilizing effect of the coenzyme can be used to stabilize 6PGDH in the presence of the coenzyme. In order to reduce the destabilizing effect of the coenzyme, a substance that can reduce the influence of the aldehyde generated by the oxidation of alcohol is preferably used. An example of such a substance is a compound having a reducing property. In particular, oxygen acids having reducibility and having a sulfur atom as a central atom, thioacids of this oxyacid, salts of this oxyacid or thioacid, compounds having an amino group, and the like are effective. Examples of the compound having an amino group include compounds having the structures of the above formulas (I) and (III). A molecule having a structure (NH ₂ —N, NH—O— or the like) in which elements such as nitrogen, oxygen, and sulfur having high electronegativity are connected in the molecule is also effective. Examples of such substances include compounds having the structures of the above formulas (II) and (III).

Examples of the oxygen acid having a reducing property and having a sulfur atom as a central atom and its thioacid include sulfoxylic acid [H ₂ SO ₂ ], sulfurous acid [H ₂ SO ₃ ], thiosulfurous acid [H ₂ S ₂ O ₂ ], Thiosulfuric acid [H ₂ S ₂ O ₃ ], dithionic acid [H ₂ S ₂ O ₄ ], disulfurous acid [H ₂ S ₂ O ₅ ], dithionic acid [H ₂ S ₂ O ₆ ], disulfuric acid [H ₂ S ₂ O ₇ ], polythioic acid [H ₂ S _X O ₄ (X is an integer of 3 or more)] and the like.

Specific examples of the oxygen acid salt and the thioacid salt include ammonium sulfite [(NH ₄ ) ₂ SO ₃ ], potassium sulfite [K ₂ SO ₃ ], calcium sulfite [CaSO ₃ ], and ammonium hydrogen sulfite [NH ₄ HSO ₃ ], sodium bisulfite [NaHSO ₃ ], sodium sulfite [Na ₂ SO ₃ ], barium sulfite [BaSO ₃ ], bismuth sulfite [Bi ₂ (SO ₃ ) ₃ ], potassium bisulfite [KHSO ₃ ], thio Ammonium sulfate [(NH ₄ ) ₂ S ₂ O ₃ ], sodium thiosulfate [Na ₂ S ₂ O ₃ ], barium thiosulfate [BaS ₂ O ₃ ], magnesium thiosulfate [MgS ₂ O ₃ ], calcium thiosulfate [CaS ₂ O ₃ ], potassium thiosulfate [K ₂ S ₂ O ₃ ], sodium dithionite [Na ₂ S ₂ O ₄ ], potassium dithionite [K ₂ S ₂ O ₄ ], calcium dithionite [CaS ₂ O ₄ ], sodium disulfite [Na ₂ S ₂ O ₅ ], potassium disulfite [K ₂ S ₂ O ₅ ], magnesium disulfite [MgS ₂ O ₅ ], Calcium disulfite [CaS ₂ O ₅ ], sodium dithionate [Na ₂ S ₂ O ₆ ], potassium disulfate [K ₂ S ₂ O ₇ ], sodium disulfate [Na ₂ S ₂ O ₇ ], sodium tetrathionate [Na ₂ S ₄ O ₆ ] and the like are exemplified.

  Further, water-soluble compounds represented by the following formulas (I) to (III) are also preferably used.

（Ｒ１は水素原子、ＣＨ_３、またはＳＯ_３Ｈ；Ｒ２は、ＳＨ、ＣＨ_２ＳＨ、またはＮＨ_２であり、Ｒ３はＳＨ、ＣＨ_２ＳＨ、ＮＨ_２、ＮＨＣＯＮＨ_２、またはＮＨＣ（ＮＨ）ＮＨ_２であり、Ｒ４は水素原子、ＯＨ、ＣＨ_３、ＮＨ_２、ＯＣＨ_３、ＣＯＯＨ、ＣＯＯＣＨ_３、またはＣＯＯＣ_２Ｈ_５である。ただし、Ｒ２がＳＨまたはＣＨ_２ＳＨの場合、Ｒ３はＮＨ_２、ＮＨＣＯＮＨ_２、またはＮＨＣ（ＮＨ）ＮＨ_２であり、Ｒ２がＮＨ_２の場合、Ｒ３はＳＨ、またはＣＨ_２ＳＨである）

(R1 is a hydrogen atom, CH ₃ or _SO 3 H,; R2 is, SH, a _CH 2 SH or NH _2,, R3 is _{SH, CH 2 SH, NH 2} , NHCONH 2 or NHC (NH) NH _2, in it, R4 is _{hydrogen, OH, CH 3, NH 2} , OCH 3, COOH, COOCH 3, or COOC ₂ H _5. However, if R2 is SH or _CH 2 SH, R3 is _NH 2, NHCONH ₂ or NHC (NH) NH ₂ and when R 2 is NH ₂ , R 3 is SH or CH ₂ SH)

（Ｒ５およびＲ６は同一または異なって、水素原子、NH_２、低級アルキル、シクロアルキル、低級アルケニル、アラルキル、アリール、低級アルカノイル、アロイル、シンナミル、またはシンナモイルである）

(R5 and R6 each independently represent a hydrogen atom, NH _2, lower alkyl, cycloalkyl, lower alkenyl, aralkyl, aryl, lower alkanoyl, aroyl, cinnamyl or cinnamoyl)

（Ｒ７およびＲ８は同一または異なって、水素原子、低級アルキル、アリル、ＣＨＯ、Ｃ（ＮＨ）ＮＨＮＨ_２、ＣOＣＨ_３、ＣＨ_３ＯＣＯ、Ｃ_２Ｈ_５ＯＣＯまたは下記式（ＩＶ）

(R7 and R8 are the same or different, a hydrogen atom, a lower alkyl, _{allyl, CHO, C (NH) NHNH} 2, COCH 3, CH 3 OCO, C 2 H 5 OCO or formula (IV)

（R９は硫黄原子、酸素原子、NHまたはNNH_２であり、R１０は低級アルキル、NH_２、NHNH_２、CONH_２、またはCONHNH_２である）で表される基である）

(R9 sulfur atom, an oxygen atom, NH or NNH _2, R10 is lower alkyl, NH _2, NHNH _2, CONH _2, or CONHNH ₂₎ a group represented by)

Here, the lower alkyl in the above formulas (II) and (III) is linear or branched alkyl having 1 to 6 carbon atoms (for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, etc.), which may have a substituent.
The lower alkenyl in the above formula (II) is a linear or branched alkenyl having 2 to 6 carbon atoms (for example, vinyl, allyl, isopropenyl, 4-pentenyl, 5-hexenyl, etc.) having a substituent. You may do it.
The lower alkanoyl in the above formula (II) is a linear or branched alkanoyl having 1 to 6 carbon atoms (for example, formyl, acetyl, propionyl, butyryl, valeryl, pivaloyl, pentanoyl, etc.) and has a substituent. It may be.
Examples of the substituent include hydroxy, carboxy, halogen, amino and the like.

In the compound represented by the above formula (I), it is presumed that NH _{2 of} R 2 or R 3 reduces the destabilizing effect of the coenzyme by the aldehyde. Moreover, since this compound has SH in R2 or R3, it is considered that this also has an effect of directly stabilizing 6PGDH. .

  Examples of the compound represented by the formula (I) include compounds listed in Table 1 below.

  Examples of the compound represented by the formula (II) include compounds listed in Table 2 below.

  Examples of the compound represented by the formula (III) include compounds listed in Table 3 below.

  In addition to the above, aminoethylaminoethanol can also reduce the destabilizing effect of coenzyme on 6PGDH.

  When 6PGDH and coenzyme are housed in separate containers, 6PGDH does not become unstable by the coenzyme. However, since 6PGDH originally is an unstable enzyme, the enzyme activity decreases even in a solution that does not substantially contain a coenzyme. In the substantial absence of a coenzyme, a substance that stabilizes 6PGDH in the presence of a coenzyme as described above cannot achieve a satisfactory 6PGDH stabilizing effect. For this reason, in order to stabilize 6PGDH, the substance which acts directly on 6PGDH and can suppress the deactivation of 6PGDH can be used. Examples of the substance having such an action include compounds having an SH group (hereinafter referred to as SH compounds). Since 6PGDH is oxidized in a solution and its activity decreases, it is considered that deactivation can be suppressed by acting a reducing SH compound.

  The SH compound capable of stabilizing 6PGDH in the substantial absence of a coenzyme is not particularly limited as long as it has an SH group. For example, N-acetyl-L-cysteine (NAC), thioglycerol, β-mercaptopropionic acid, cysteine, dithiothreitol (DTT), 2-mercaptoethanol (2ME) and the like can be mentioned.

  In addition to the above substances, chelating agents such as cyclohexyldiaminetetraacetic acid (CyDTA) and buffering agents such as PIPES and ADA can also stabilize 6PGDH. If metal ions are contained as impurities in the solution, 6PGDH may become unstable due to this, and thus the substance having the chelating effect as described above is also considered effective.

  The above 6PGDH stabilizers may be used alone or in combination.

  The concentration of the 6PGDH stabilizer in the reagent is not particularly limited as long as it is a concentration that can stabilize 6PGDH.

  The 6PGDH that can be used in the present embodiment may be derived from bacteria, yeast, animals or plants, or may be generated using a gene recombination technique.

  The pH of the 6PGDH-containing reagent in the solution state is not particularly limited as long as the pH does not destabilize 6PGDH, but it is preferably 5-8. In order to maintain this pH, the reagent preferably contains a buffer. Examples of the buffer include Tris-HCl buffer, imidazole-acetate buffer, phosphate buffer, citrate buffer, malate buffer, oxalate buffer, phthalate buffer, glycine buffer, acetate buffer. Succinic acid buffer, boric acid buffer, carbonic acid buffer, Good buffer, and the like can be used.

The 6PGDH-containing reagent of this embodiment can be used for clinical examination. A 6PGDH-containing reagent is mixed with a sample collected from a living body or a sample pretreated from the sample, and a change in absorbance or the like is monitored to quantify a specific substance in the sample or measure an activity of a specific enzyme.
Examples of the sample include serum, plasma, blood, cerebrospinal fluid, urine, semen and the like, but it is preferable to use plasma or serum.

  The substance that can be quantitatively or activity-measured using the 6PGDH-containing reagent is not particularly limited as long as 6PG, which is a 6PGDH substrate, is used in the reaction system. Examples of substances that can be quantified include neutral fat, inorganic phosphate, lactose, maltose, glycogen, starch, sucrose, glucose, fructose, mannose, creatine phosphate, G6P, fructose-6-phosphate, maltose-6. -Phosphate, fructose-2-phosphate, L-sorbose-6-phosphate, glucose-1-phosphate, NAD, NADP, uridine triphosphate, adenosine triphosphate (ATP), uridine diphosphate glucose, etc. Is mentioned. Examples of substances whose activity can be measured include creatine kinase (CK), CK isozymes (eg, CKMB), β-galactosidase, α-galactosidase, amyloglucosidase, invertase, transaldolase, galactose-1-phosphate. Enzymes such as uridyltransferase, phosphoglucose isomerase, and fructose diphosphatase can be mentioned.

Among the above, the 6PGDH-containing reagent used for measuring the activity of CK will be described below.
CK is a dimeric phosphorylase consisting of two subunits. There are two types of CK subunits, B type (brain type) and M type (muscle type). CK has three types of isozymes (CKMM, CKMB, and CKBB) by combining two types of subunits. CKMM is contained in skeletal muscle, CKMB is contained in heart muscle, and CKBB is contained in brain. It is. Since the CK isozyme present in the disease-causing site deviates into the blood due to a disease such as myocardial infarction or muscular dystrophy, the activity value of CK isozyme contained in a sample such as serum is important in diagnosing the disease in clinical tests. It becomes an indicator. As a sample used for measurement of CK activity, serum, plasma and the like can be used.

  In addition to 6PGDH and 6PGDH stabilizers, CK activity measurement reagents include buffers, SH compounds, NAD or NADP, hexokinase (HK) or glucokinase (GK), glucose, G6PDH, adenosine diphosphate (ADP) It is preferable to contain magnesium ion and creatine phosphate.

  When the sample and the reagent for measuring CK activity are mixed, CK contained in the sample is activated by the SH compound (the SH compound not only stabilizes 6PGDH in the absence of a coenzyme, but also activates CK. Have). Since CK is inactivated when it deviates into the blood, in order to measure the activity of CK, it is necessary to first activate CK with an SH compound. The SH compound is not particularly limited as long as it can activate CK. For example, NAC, cysteamine, DTT, cysteine, glutathione, β-mercaptopropionic acid, 2ME and thioglycerol can be used. These compounds may be used in combination of two or more kinds, or may be used alone. The concentration of the compound having an SH group in the reagent is 5 to 250 mM, preferably 5 to 100 mM, more preferably 10 to 40 mM.

  A reaction system as shown in FIG. 1 is constructed by adding a reagent for measuring CK activity containing the above components to a sample containing CK. In FIG. 1, CK activated by an SH compound catalyzes a reaction for producing creatine and ATP from creatine phosphate and ADP (reaction 1). HK or GK contained in the reagent produces G6P and ADP from glucose contained in the reagent and ATP produced in Reaction 1 (Reaction 2). Furthermore, G6PDH contained in the reagent generates 6-phosphogluconic acid (6PG) and NADH or NADPH from NAD or NADP and G6P generated in Reaction 2 (Reaction 3). Next, 6PGDH contained in the reagent generates carbon dioxide, ribulose-5-phosphate, and NADH or NADPH from NAD or NADP and 6PG generated in Reaction 3 (Reaction 4). When NADH or NADPH is generated, the absorbance in the vicinity of a wavelength of 340 nm of the mixed solution of the sample and the 6PGDH-containing reagent increases. By monitoring this increase in absorbance, the activity of CK in the sample can be measured.

  The reagent for measuring CK activity is preferably a reagent kit comprising two or more kinds of reagents. A reagent kit for measuring CK activity comprising a first reagent and a second reagent comprises a buffer, ADP, glucose, NAD or NADP, HK or GK, G6PDH, SH compound, 6PGDH and 6PGDH stabilizer, It preferably comprises a second reagent containing creatine phosphate.

  A reagent kit for measuring CK activity comprising a first reagent, a second reagent, and a third reagent comprises a buffer, ADP, glucose, NAD or NADP, HK or GK, G6PDH, and a first reagent containing an SH compound, creatine phosphate And a third reagent containing 6PGDH and a 6PGDH stabilizer.

  In addition to the above components, preservatives, chelating agents and the like may be added to the reagent as appropriate. As a preservative, for example, sodium azide can be used. The chelating agent is used for suppressing inhibition of CK activity by metal ions in the sample, and specifically, EDTA or the like can be used.

  In addition, a compound having a surface active action may be added to the reagent. For example, nonionic surfactants, cationic surfactants, zwitterionic surfactants, albumin and the like can be used. Specifically, Tritons (registered trademark of Union Carbide Chemicals and Plastics Co.), Emulgens ( Kao Corporation registered trademark), bovine serum albumin (BSA), and the like can be used.

  The sample may contain adenylate kinase. Adenylate kinase is particularly abundant in hemolyzed samples and adversely affects CK activity measurement. In order to avoid this adverse effect, it is preferable to add an inhibitor that inhibits the action of adenylate kinase to the reagent. The type of inhibitor is not particularly limited as long as it inhibits the action of adenylate kinase. For example, adenosine monophosphate (AMP), P1P5 diadenosine-5′-pentaphosphate (AP5A), or the like is used. it can.

  Furthermore, by measuring the activity by the double kinetic method, it is possible to avoid the adverse effects of enzymes that are not measurement targets such as adenylate kinase. In the double kinetic method, first, the activity of an enzyme such as adenylate kinase is measured, and then creatine phosphate is added to initiate the enzymatic reaction by CK to activate the activity of the kinase contained in the sample (CK activity and adenylate). Measure the sum of the activity of enzymes such as rate kinase). The difference between these measurement results is the CK activity value.

  By adding an antibody that specifically recognizes the M-type subunit of CK (hereinafter referred to as anti-CK-M antibody) to any of the reagents constituting the above-described reagent kit for measuring CK activity, a sample is obtained. It is possible to measure the activity of CKMB in the medium (Wurzburg et al., 1977, J. Clin. Chem. Clin. Biochem., 15: 131-135). When the anti-CK-M antibody is contained in the reagent, the anti-CK-M antibody binds to the M-type subunit of CKMM among the CK isozymes in the sample, and the CKMM activity is completely inhibited. Also, the activity of CKBB in the sample is negligibly small. The anti-CK-M antibody binds to the CKMB M-type subunit, but does not bind to the B-type subunit. Therefore, the activity of the CKMB B-type subunit is measured and doubled. Can be calculated. The anti-CK-M antibody is preferably contained in the first reagent. The anti-CK-M antibody may be a polyclonal antibody or a monoclonal antibody as long as it is an antibody that specifically recognizes the M-type subunit, or a mixture of these may be used. Antibody fragments and derivatives thereof can also be used. Specific examples of antibody fragments and derivatives thereof include Fab, Fab ′, F (ab) 2 and sFv fragments (Blazar et al., 1997, J. Immunol., 159: 5821-5833 and Bird et al. , 1988, Science, 242: 423-426). The subclass of the antibody is not limited to IgG, and may be IgM.

Hereinafter, the 6PGDH stabilizing effect of the 6PGDH stabilizer was analyzed by showing Examples and Comparative Examples. In the following examples and comparative examples, 6PGDH activity was measured by adding 4.5 μl of a reagent containing 6PGDH to 270 μl of a substrate solution (0.7 mg / ml magnesium chloride hexahydrate, 0.3 mg / ml NADP and 0 mg). .9 mg / ml 6PG) and the absorbance at 340 nm was measured.

(Comparative Example 1)
First to third reagents for measuring CK activity including the following compositions were prepared.
<First reagent>
Imidazole buffer 144 mM (pH 6.6)
NAC 20 mM
EDTA 2.5 mM
Magnesium acetate 12.5 mM
ADP 2.5 mM
AMP 6.25 mM
AP5A 12.5μM
Glucose 25 mM
NADP 2.5 mM
G6PDH 1875U / l
Hexokinase 3750U / l
<Second reagent>
Creatine phosphate 150 mM
<Third reagent>
6PGDH 200U / l
Imidazole buffer 144 mM (pH 6.6)

  In this example, among the above, since the stabilization of 6PGDH contained in the third reagent was evaluated, only the third reagent was used. In Comparative Example 1 and Example 1, this third reagent was used as a control reagent. This control reagent was stored at 4 ° C. for 3 days and the activity of 6PGDH was measured.

  Next, it was left to stand at 37 ° C. for 3 days against the control reagent, and was subjected to a temperature load. After temperature loading, the residual activity of 6PGDH was measured.

In addition, one of the following substances was added to the third reagent as a control reagent to prepare a reagent.
20 mM aminoguanidine 20 mM acetohydrazine 20 mM hydrazine 20 mM hydroxylamine 20 mM semicarbazide 1.2 g / L βNADP
2.4 g / L βNADP
3.6 g / L βNADP

  Next, a temperature load was applied to the above reagent by allowing it to stand at 37 ° C. for 3 days. After temperature loading, the residual activity of 6PGDH was measured.

Example 1
One of the following substances was added as a 6PGDH stabilizer to the control reagent prepared in Comparative Example 1 to prepare a reagent.
42.5 mM thioglycerol 85 mM thioglycerol 20 mM β-mercaptopropionic acid 20 mM cysteine 20 mM cysteine and 10 mM thioglycerol 20 mM DTT
20 mM 2ME
2 mM CyDTA
In addition, a reagent was prepared in the same manner as the control reagent of Comparative Example 1 except that 144 mM PIPES was used as a 6PGDH stabilizer instead of 144 mM imidazole buffer.
A reagent was prepared in the same manner as the control reagent of Comparative Example 1 except that 144 mM ADA was used as a 6PGDH stabilizer instead of 144 mM imidazole buffer.

  After leaving these reagents at 4 ° C. for 3 days, 6PGDH activity was measured. Next, these reagents were allowed to stand at 37 ° C. for 3 days and subjected to a temperature load. After temperature loading, 6PGDH residual activity was measured.

  Table 4 shows the residual activities measured in Comparative Example 1 and Example 1. The residual activity is expressed as a percentage when the 6PGDH activity measured after standing at 4 ° C. for 3 days is defined as 100%.

From Table 1, in the case of the control reagent (no additive), 6PGDH activity decreased to 76.1% after temperature loading compared to before temperature loading.
In addition, when NADP was added to the control reagent, 6PGDH activity significantly decreased after temperature loading. This confirmed that NADP has a strong 6PGDH destabilizing effect.

  The additive of Comparative Example 1, which is a substance that suppresses the destabilizing effect of NADP and stabilizes 6PGDH, did not show an effect of stabilizing 6PGDH in a control reagent not containing NADP.

When the 6PGDH stabilizer used in Example 1 was added, the 6PGDH residual activity was close to 100% even after temperature loading.
From the above, it was found that these additives (SH compound, CyDTA, PIPES and ADA) can improve the storage stability of 6PGDH in the substantial absence of NADP.

(Comparative Example 2)
A control reagent was prepared containing 200 U / l 6PGDH, 144 mM imidazole buffer (pH 6.6), and 1.2 g / l NADP. This reagent was allowed to stand at 4 ° C. for 3 days, and 6PGDH activity was measured. Next, this reagent was allowed to stand at 37 ° C. for 3 days and subjected to a temperature load. After temperature loading, the residual activity of 6PGDH was measured.

In addition, any of the following substances was added to the control reagent of this comparative example to prepare a reagent.
20 mM NAC
40 mM NAC
42.5 mM thioglycerol 85 mM thioglycerol 20 mM β-mercaptopropionic acid 20 mM DTT
20 mM 2ME

  Next, a temperature load was applied to the above reagent by allowing it to stand at 37 ° C. for 3 days. After temperature loading, the residual activity of 6PGDH was measured.

(Example 2)
One of the following substances was added to the control reagent prepared in Comparative Example 2 to prepare a reagent.
20 mM hydroxylamine 20 mM cysteine 20 mM cysteine and 10 mM thioglycerol

  Next, a temperature load was applied to the reagent as in Comparative Example 2. After temperature loading, the residual activity of 6PGDH was measured.

  Table 6 shows the 6PGDH residual activity measured in Comparative Example 2 and Example 2. The residual activity is expressed as a percentage when the 6PGDH activity measured after leaving the control reagent at 4 ° C. for 3 days is defined as 100%.

From Table 5, the activity of 6PGDH was reduced even when the SH compound of Comparative Example 2 was added in the presence of NADP, and almost no 6PGDH stabilizing effect was obtained. However, even in the case of the SH compound as in Example 2, it was found that when cysteine having an amino group was added, high residual activity was obtained and 6PGDH could be stabilized. It was also found that hydroxylamine, which is one of the compounds represented by the above formula (II), has a higher residual activity than when cysteine is added and can further improve the storage stability of 6PGDH.

(Comparative Example 3)
A first reagent and a second reagent for CK activity measurement including the following composition were prepared.
<First reagent>
Imidazole buffer 144 mM (pH 6.6)
EDTA 2.5 mM
Magnesium acetate 12.5 mM
ADP 2.5 mM
AMP 6.25 mM
AP5A 12.5μM
Glucose 25 mM
NADP 2.5 mM
G6PDH 1875U / l
Hexokinase 3750U / l
6PGDH 200U / l
<Second reagent>
Creatine phosphate 150 mM

  Here, among the above, since the stabilization of 6PGDH contained in the first reagent was evaluated, only the first reagent was used. In Comparative Example 3 and Example 3, this first reagent was used as a control reagent.

  This control reagent was allowed to stand at 4 ° C. for 3 days, and then 6PGDH activity was measured. Next, this reagent was allowed to stand at 37 ° C. for 3 days, and a temperature load was applied. After temperature loading, the residual activity of 6PGDH was measured.

In addition, any of the following substances was added to the control reagent of this comparative example to prepare a reagent.
20 mM N, N-diethylhydroxylamine 40 mM N, N-diethylhydroxylamine 20 mM mercaptoethanol 40 mM mercaptoethanol 20 mM cysteic acid 40 mM cysteic acid

  Next, a temperature load was applied to the above reagent by allowing it to stand at 37 ° C. for 3 days. After temperature loading, the residual activity of 6PGDH was measured.

(Example 3)
One of the following substances was added as a 6PGDH stabilizer to the control reagent prepared in Comparative Example 3 to prepare a reagent.
20 mM aminoguanidine 30 mM aminoguanidine 40 mM aminoguanidine 20 mM cysteine 40 mM cysteine 20 mM hydroxylamine 40 mM hydroxylamine 20 mM N-methylhydroxylamine 40 mM N-methylhydroxylamine 20 mM O-methylhydroxylamine 40 mM O-methylhydroxylamine 20 mM carboxymethoxylamine 40 mM carboxymethoxy Ruamine 20 mM Acetohydrazide 40 mM Acetohydrazide 20 mM Semicarbazide 40 mM Semicarbazide 20 mM Hydrazine 40 mM Hydrazine 20 mM Cysteamine 40 mM Cysteamine 20 mM Sodium sulfite 40 mM Sodium sulfite 20 mM Aminoethylaminoethanol 40 mM Aminoethylamino ethanol

  These reagents were allowed to stand at 4 ° C. for 3 days to measure 6PGDH activity. Next, a temperature load was applied to these reagents in the same manner as in Comparative Example 3, and the residual activity of 6PGDH was measured after the temperature load.

  Table 6 shows the residual activities measured in Comparative Example 3 and Example 3. The residual activity is expressed as a percentage when the 6PGDH activity measured after standing at 4 ° C. for 3 days is defined as 100%.

  From Table 6, when the 6PGDH stabilizer of Example 3 represented by any one of the above formulas (I) to (III) was added, the residual activity was higher than the 6PGDH residual activity of the control reagent. High residual activity was also exhibited when aminoethylaminoethanol or sodium sulfite, a salt of oxo acid having a reducing property and having a sulfur atom as a central atom, was used. From the above, it was found that these substances can improve the storage stability of 6PGDH in the reagent.

  When any of N, N-diethylhydroxylamine, 2ME, and cysteic acid in Comparative Example 3 was added, the residual activity was almost the same as the residual activity of the control reagent. From the above, these substances could not stabilize 6PGDH.

N, N-diethylhydroxylamine is similar to the structural formula of the above formula (II), but has a structure in which a hydrogen atom bonded to N in the formula is substituted with an ethyl group. From this, in the compound having the structure of the formula (II), it is considered that -NH-O- suppresses the 6PGDH destabilizing effect of NADP and stabilizes 6PGDH.
Since 2ME is an SH compound, it has a 6PGDH stabilizing effect in the absence of NADP (see Example 1), but as can be seen from Table 6, 6PGDH could not be stabilized in the presence of NADP. 2ME is similar to the structural formula of formula (I) above, but differs in that it does not have an amino group. Cysteinic acid is similar to the structural formula of the above formula (I), but differs in that it does not have SH.
From this, in the compound having the structure of the formula (I), it is considered that SH and amino group in the molecule suppress the 6PGDH destabilizing effect of NADP and stabilize 6PGDH.

It is the schematic diagram which showed the reaction system of CK activity measurement.

Claims (9)

6-phosphogluconate dehydrogenase (6PGDH);
At least one coenzyme selected from the group consisting of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP);
Formula (I)

(R1 is a hydrogen atom, CH ₃ or _SO 3 H,; R2 is, SH, a _CH 2 SH or NH _2,, R3 is _{SH, CH 2 SH, NH 2} , NHCONH 2 or NHC (NH) NH _2, in it, R4 is _{hydrogen, OH, CH 3, NH 2} , OCH 3, COOH, COOCH 3, or COOC ₂ H _5. However, if R2 is SH or _CH 2 SH, R3 is _NH 2, NHCONH ₂ or NHC (NH) NH ₂ and when R 2 is NH ₂ , R 3 is SH or CH ₂ SH),
The following formula (II)

(R5 and R6 are the same or different and each represents a hydrogen atom, NH ₂ , lower alkyl, cycloalkyl, lower alkenyl, aralkyl, aryl, lower alkanoyl, aroyl, cinnamil, or cinnamoyl),
Formula (III) below

(R7 and R8 are the same or different, a hydrogen atom, a lower alkyl, allyl, CHO, C (NH) NHNH2 , COCH3, CH 3 OCO, C 2 H 5 OCO or formula (IV)

(R9 sulfur atom, an oxygen atom, NH or NNH _2, R10 is lower _{alkyl, NH 2, NHNH 2, CONH} 2, or CONHNH ₂ a is) a water-soluble represented by a group represented by) Compound,
An oxyacid having a reducing property and having a sulfur atom as a central atom,
A thioacid of the oxygen acid,
A 6PGDH-containing reagent containing at least one selected from the group consisting of the oxygen acid or the salt of the thioacid and aminoethylaminoethanol. The water-soluble compound represented by the formula (I) is cysteine or cysteamine, and the water-soluble compound represented by the formula (II) is hydroxylamine, N-methylhydroxylamine, O-methylhydroxylamine, or The reagent according to claim 1, which is carboxymethoxylamine, wherein the compound represented by the formula (III) is aminoguanidine, acetohydrazide, semicarbazide, hydrazine, or hydroxylamine, and the oxygen acid is sulfurous acid. The 6PGDH-containing reagent according to claim 1 or 2, further comprising hexokinase or glucokinase, glucose and adenosine diphosphate (ADP). A 6PGDH-containing reagent according to any one of claims 1 to 3,
A reagent kit comprising a reagent containing creatine phosphate. 6-phosphogluconate dehydrogenase (6PGDH);
Selected from the group consisting of cyclohexyldiaminetetraacetic acid (CyDTA), Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-Acetamido) iminodiacetic acid (ADA), and a compound having an SH group And at least one of
Substantially free of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP),
Reagent containing 6PGDH. The compound having an SH group is at least one selected from the group consisting of N-acetyl-L-cysteine, thioglycerol, β-mercaptopropionic acid, cysteine, dithiothreitol (DTT), and 2-mercaptoethanol. The reagent according to claim 5, wherein A reagent comprising an SH group, a nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), hexokinase or glucokinase, glucose, and adenosine diphosphate (ADP);
A reagent containing creatine phosphate;
A reagent kit comprising the reagent according to claim 5 or 6. In the presence of at least one coenzyme selected from the group consisting of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP),
6-phosphogluconate dehydrogenase (6PGDH);
Formula (I)

(R1 is a hydrogen atom, CH ₃ or _SO 3 H,; R2 is, SH, a _CH 2 SH or NH _2,, R3 is _{SH, CH 2 SH, NH 2} , NHCONH 2 or NHC (NH) NH _2, in it, R4 is _{hydrogen, OH, CH 3, NH 2} , OCH 3, COOH, COOCH 3, or COOC ₂ H _5. However, if R2 is SH or _CH 2 SH, R3 is _NH 2, NHCONH ₂ or NHC (NH) NH ₂ and when R 2 is NH ₂ , R 3 is SH or CH ₂ SH),
The following formula (II)

(R5 and R6 are the same or different and each represents a hydrogen atom, NH ₂ , lower alkyl, cycloalkyl, lower alkenyl, aralkyl, aryl, lower alkanoyl, aroyl, cinnamil, or cinnamoyl),
Formula (III) below

(R7 and R8 are the same or different, a hydrogen atom, a lower alkyl, _{allyl, CHO, C (NH) NHNH} 2, COCH 3, CH 3 OCO, C 2 H 5 OCO or formula (IV)

(R9 sulfur atom, an oxygen atom, NH or NNH _2, R10 is lower _{alkyl, NH 2, NHNH 2, CONH} 2, or CONHNH ₂ a is) a water-soluble represented by a group represented by) Compound,
An oxyacid having a reducing property and having a sulfur atom as a central atom,
A thioacid of the oxygen acid,
6PGDH stabilization method which stabilizes said 6PGDH by coexisting with at least one selected from the group which consists of said oxygen acid or the salt of said thioacid, and aminoethylaminoethanol. In the substantial absence of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP),
6-phosphogluconate dehydrogenase (6PGDH);
Selected from the group consisting of cyclohexyldiaminetetraacetic acid (CyDTA), Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-Acetamido) iminodiacetic acid (ADA), and a compound having an SH group. 6PGDH stabilization method which stabilizes said 6PGDH by making it coexist with at least one.

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