JPS61242594A - Reagent kit for determination of enzymatic activity - Google Patents

Abstract

PURPOSE:To provide a reagent kit for the determination of enzymatic activity, consisting of the first reagent containing a specific substrate and a coupler and the second reagent containing an oxidase, capable of determining the activity under mild reaction condition in excellent operability, simplicity and accuracy, having high storage stability, and applicable to initial rate process. CONSTITUTION:The objective reagent kit for the determination of enzymatic activity consists of the first reagent and the second reagent. The second reagent contains an oxidase. The first reagent contains the substrate of formula (Xm is peptide residue composed of m amino acid residues bonded together wherein the C-terminal amino acid forms amide bond with the amino group bonded to the aromatic group; A is group protecting the N-terminal of the peptide residue or H atom; m is 1-10; Z is H, halogen, lower alkyl, etc.; n is 1-5) and a coupler and/or a chelating agent. The pH of the first reagent is <=5.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a reagent kit for measuring enzyme activity.

The enzymes targeted by the present invention are peptide bond hydrolases, such as various (amino)peptidases such as leucine aminopeptidase, γ-glutamyl transpeptidase, cystine aminopeptidase, trypsin,
α-Chymotrypsin.

Examples include various proteases such as plasmin, thrombin, kallikrein, urokinase, endotoxin, factor Xa, collagenase, elastase, and cathepsin. Measurement of the activity of various hydrolytic enzymes such as Ton et al. in body fluids is of great clinical significance as it provides useful diagnostic information.

(Prior art) Conventional methods for measuring these hydrolytic enzymes include, for example, γ
- A method of colorimetric determination by oxidative condensation of an amine compound with a suitable coupler using metaperiodic acid, which is often used to measure glutamyl transpeptidase (r-GTP) activity (for example, Toshiyuki Takabayashi et al., Clinical Pathology (Supplementary)) 29, p5
38 (1979)), or a method in which the amine compound found in the measurement of leucine aminopeptidase (LAP) activity is diazotized with nitrite and coupled with an appropriate coupler, followed by colorimetric determination (e.g. J, A, Gol
dbarg, Ca-ncer II, p 28
There are colorimetric methods using chemical methods such as 3 (1958)). However, in chemical methods using diazo coupling methods using chemical oxidizing agents (e.g., potassium hepterythyanide, metaperiodic acid) and nitrite, the desired enzyme activity cannot be detected according to the reaction conditions of the desired enzyme. The oxidation reaction and diazo coupling reaction conditions used in the detection reaction are different, and the oxidizing agent and diazotization agent used in the detection reaction inhibit the target enzyme. Since detection is difficult, there are many operating steps, the measurement time is long, and the overall measurement is complicated.

Further, there are problems in that harmful chemicals such as potassium ferricyanide are used and harsh reaction conditions are required such as the diazotization reaction under acidic conditions with hydrochloric acid.

Recently, an enzymatic method (Japanese Unexamined Patent Publication No. 59-88099) has been proposed. In other words, using an aniline derivative as a substrate for LAP or γ-GTP, the aniline compound liberated by the action of an enzyme is colored with an oxidizing enzyme such as laccase, ascorbate oxidase, or tyrosinase in the presence of a coupler, and the change in absorbance is This method measures hydrolytic enzyme activity, and is an excellent method for measuring enzyme activity since it eliminates the drawback of conventional chemical methods, which cannot measure initial velocity. However, in this method, the stability of the reaction solution containing the substrate, coupler, and oxidase is poor. Moreover, there are restrictions on the couplers, substrates, and oxidizing enzymes that can be used for this purpose, which inevitably limits the range of practical applications. For example, compounds with relatively low oxidation reactions must be selected as couplers and substrates to prevent deterioration of storage stability.

As a result, there is a drawback that sufficient sensitivity required for measurement cannot be obtained. There is also a restriction that an oxidizing enzyme having a relatively low oxidative condensation reaction ability may cause deterioration of the coupler, and therefore an oxidizing enzyme having a high oxidizing reaction ability cannot be used.

(Means for Solving the Problem) The present inventors have solved such conventional drawbacks, and have developed a modified initial velocity method that is easy to operate, easy to use, highly accurate, and has good storage stability under mild reaction conditions. The present invention was achieved as a result of various intensive studies to find a method for measuring enzyme activity that would allow the following. That is, the present invention includes a substrate and a coupler represented by the following general formula [I], and further has a pH of 5 or less, and/
Or, a reagent for enzyme activity measurement, CHILA (
In the formula, Xm represents a peptide residue in which m pieces of the same or different amino acids are bonded, and the C-terminal amino acid forms an amide bond with the amino group bonded to the aromatic group. A represents a group or hydrogen atom that protects the N-terminus of the peptide residue. m=1 to 10. 2 is a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxyl group, an amine group,
It represents a hydroxyl group, a carbonyl group, a sulfonic acid group, or a substituted amino group, and n=1 to 5. A plurality of Z may be the same or different, and may be a group forming a ring at the ortho position and meta position of the substituent -NH-. ) In the present invention, the general formula [■
] As a result of the enzymatic reaction, the substrate represented by the following general formula ('
A free aniline derivative represented by 10 is produced, and this free aniline derivative is reacted with an oxidase in the presence of a coupler to obtain an oxidized condensate, and the change in spectroscopic absorbance thereof is measured or consumed by the oxidase. Enzyme activity is measured by measuring the oxygen produced using an oxygen electrode, etc. (in the formula,
Z represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxyl group, an amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, or a substituted amino group, and is n-1 to n-5.

A plurality of 2's may be the same or different, and a substituent -
It may also be a group that forms a ring at the ortho and meta positions of NHL. ) The first reagent of the present invention contains a substrate represented by general formula [I] and a coupler, and further has a pH of 5 or less or contains a chelating agent, or has a pH of 5 or less and contains a chelating agent.

The substrate represented by the general formula [I] is a compound having the above-mentioned free α-IJ derivative [■] as a constituent, and on which peptide bond hydrolases such as various peptidases and various proteases act. Any substrate may be used as long as it is a substrate. For example, the group A-Xm of the substrate [■] is L-, which is the substrate of leucine aminopeptidase.
leucyl group, a γ-L-glutamyl group that is a substrate for γ-glutamyl transpeptidase, an S-benzyl-L-cystenyl group that is a substrate for cystine aminopeptidase,
Glycyl-L-prolyl group serving as a substrate for X-prolyl-dipeptidyl-aminopeptidase, Bz-phe-Val-Arg-(Bz: benzoyl group) Tos-Gly-Pro-Arg serving as a substrate for thrombin
(Tos: ) syl group) Boa -Val -
Pro -Arg - (BoCst-butoxycarbonyl group) HD -Pro -Arg -, HD-Phe-P
ip -Arg -Tos -017-Pro-Arg -Boc -Gl, which is a substrate for plasmin
u -Lys -Ly8-Boc -Vat -Leu
-Ly8-H-D-Val -Leu -Lys -Bz -Val -Gly -Arg -Pyro - which is a substrate of urokinase
Glu-GlyArg-G1utaryl-Gl
y-Arg -Bz-Pro-Phe-Arg-H-D-P, which is a substrate for kallikrein
ro -Phe -Arg -Z -Phe -Arg
-(Z: carpobenzoyl group)HD-Val-
Leu-Arg -pro -Phe -Arg - Bz -11e -Glu -Gly-Arg--Bo, which is a substrate for factor Xa
c-11e-Glu-Gly-Arg-Boc-Leu-Boc-Vat-Leu-Boc-8er-Boc-Vat-8er-Bz-Leu-Bz-Val-Lyeu as a substrate used for detection of endotoxin - Bz-8er- Bz-Val-8er - etc.

The coupler to be used next uses the compound represented by the general formula [■] as a substrate and undergoes oxidative condensation with the compound represented by the free aniline derivative [11] generated as a result of the enzymatic reaction, resulting in a new absorption property. Any substance may be used as long as it forms a compound.

For example, p-xylenol, p-chlorophenol 3.5
- Phenol derivatives such as dichlorophenolsulfonic acid, 3-hydroxy-2,4,6-)ryodobenzoic acid, N,N-dimethylaniline, N,N-10-diethylaniline, N,N-diethyl -m-) Leucine, N-ethyl-N-(3-sulfopropyl) -m-7
Nisidine, N-ethyl-N-(2-human gastric xy-3-sulfopropyl)-m-toluidine, 3-methyl-N-ethyl-N-(2-hydroxyethyl)-aniline, N-
Ethyl-N-(3-tylphenyl)-N-succinylethylenediamine, N-ethyl-N-(3-methyltubonyl)-N-acetylethylenediamine, N-ethyl-
N-sulfopropyl-m-toluidine, N-ethyl-N
Examples include aniline derivatives such as -(β-methylsulfonamidoethyl)-m-)leucine and naphthol derivatives such as 1-naphthol-2-sulfonic acid. Next, as free aniline derivatives [■], various hydrolases such as various peptidases and various proteases act to liberate the compound CID, which is a component of the compound that becomes the substrate m of the above-mentioned hydrolase. A compound that is a river [any compound is fine, even if it is a river]. For example, 4-amino-N-ethyl-N-(β
-methanesulfonamidoethyl)-m-toluidine, 4
-amino-3-methyl-N-(β-hydroxyethyl)
-N-ethylaniline, p-N-ethyl-N-hydroxyethylaniline, 2-amino-5-diethylaminotoluene, p-morpholinoaniline, p-N, N-disulfopropylaminoaniline, p-N, Compounds such as N-diethylamine aniline, β-naphthylamine, α-naphthylamine, 3-carboxy-4-hydroxyaniline, 3-carboxy-4-N, and N-di-n-propylaminoaniline are mentioned.

As the chelating agent used in the present invention, a general metal chelating agent is used, and the type is not limited as long as it can capture and inactivate metal ions, but a typical example is ethylenediamine tetra. Examples include acetic acid and its salts.

The second reagent of the invention contains an oxidase.

The oxidase used in the present invention is not particularly limited as long as it consumes at least oxygen in the reaction and produces an oxidized condensate that exhibits new absorption. Such enzymes include, for example, laccase, bilirubin oxidase, polyfusunol oxidase, tyrosinase,
Examples include ascorbic acid oxidase and amine phenol oxidase. Its origin and L4 enzymes, such as laccase, are found in sumac and Coriolus.
) (Japanese Unexamined Patent Publication No. 58-47489), and lupine oxidase derived from the genus Myrocesium (M)'rothecium (Agri).
c, Biol, Chern.

45, 2383 (1981)), and examples of ascorbic acid oxidase include those derived from kapocha and cucumber (Japanese Unexamined Patent Publication No. 56-88793).

The first reagent and second reagent of the present invention contain necessary substances such as amino acids and peptides depending on the type of enzyme to be measured. When the enzyme to be measured is, for example, γ-glutamyl transpeptidase, the first reagent or the second reagent contains an appropriate amount of glycylglycine.

The reagent kit of the present invention includes a substrate and a coupler represented by the general formula CI), and further has a pH of 5 or less, or contains a chelating agent, or has a reagent that has a pH of 5 or less and contains a chelating agent as a first reagent, A reagent containing an oxidizing enzyme is used as a second reagent.

If the first reagent does not contain a coupler and the second reagent contains a coupler, the blank value will increase, which is not preferable. Furthermore, even if the first reagent contains a coupler, if the pH is 5 or less or if it does not contain a chelating agent, the blank value will increase.

The reagent kit for enzyme activity measurement of the present invention consists of the above-mentioned first reagent and the above-mentioned second reagent, and in order to measure the tetratease activity, the second reagent is added to a test liquid such as serum or urine, and approximately The first reagent may be prewarmed to 37° C. and then the first reagent added, or the second reagent may be added after the first reagent is added. Alternatively, the first reagent and the second reagent may be added to the test liquid at the same time. Furthermore, a second reagent and a test liquid may be added to the first reagent of the present invention.

There are no particular limitations on the conditions under which the above reagents are allowed to act, but
A buffer solution is often used that maintains the pH conditions under which the hydrolase in the test solution acts. 4 Usually pH 5.0-10
．． A buffer solution that maintains the temperature at 0 is used.

Next, the amount of oxidase used is not particularly limited as long as it is sufficient to monitor the hydrolase reaction. For example, 0.1 to 100 units per reaction solution is sufficient. The concentration of the coupler may be at least equimolar to the liberated aniline derivative [■], and in some cases, the upper limit may be an amount that does not inhibit the desired hydrolase activity.

Next, the substrate concentration may be any concentration that sufficiently exhibits the desired hydrolase activity, and in some cases, the upper limit may be such that the hydrolase is not inhibited by the substrate. Next, the concentration of the metal chelating agent is not particularly limited, but usually a concentration of about 0.1 to 100 mM is sufficient.

Next, the method for maintaining the substrate and coupler or the reagent containing the oxidizing enzyme and coupler at I) H5,0 or lower may be any composition and method as long as it does not affect the stability of the constituent components.

The reaction temperature is the temperature normally used for clinical chemistry analysis.
For example, it may be carried out at a temperature of 20 to 50°C.

In the present invention, the change in spectroscopic absorbance of the oxidized condensate produced after the action of an oxidase is measured, or
Alternatively, the corresponding hydrolase activity is determined by measuring the oxygen consumed by the oxidase using an oxygen electrode or the like.

(Effect of the invention) Using the reagent kit of the present invention, it is easy to operate under mild conditions.
It is possible to determine the hydrolase activity using compound [I] as a substrate, which is simple and highly accurate, and allows for a modified initial velocity method with good storage stability, and is extremely meaningful for the diagnostic field of clinical testing. Become something.

(Example) The present invention will be explained below with reference to Examples.

Example 1 A reagent blank and γ-glutamyl transfer
The results are shown in Table 2.

Substrate; L-γ-glutamyl-p-diethylanoanilide (Wako Tsumugi Pharmaceutical Co., Ltd. 5mM Gly-Gly; glycylglycine (Gyudon Chemical Co., Ltd.) 100mM coupler; diethylaniline (Wako Tsumugi Co., Ltd.) 1mM chelating agent; ED
TA-2Na buffer; Tris-i (CA buffer (pH 7,5) 10
0mM Table 1 B, Assay method Add 2.0ml of reagent R1 from Table 1 to 50μl of the sample. Add Od,
After prewarming at 37° C. and subsequently adding 0.5 μm of reagent R3, the increase in absorbance per minute at a wavelength of 745 was measured and defined as γ-GTP activity (ΔOD/min). Also, add 50 μl of water instead of the temple, add reagent R1 in the same manner as above, and measure the absorbance at a wavelength of 745 cm, which is used as a blank value.

As is clear from Table 2, reagent Kira) Al, 2 has a high absorbance immediately after preparation and is not practical, and A3゜4 can be used when it is manufactured, but the reagent blank increases during storage. , r-
The GTP activity value also showed a low value, indicating deterioration of the reagent. However, A5*6e7 can be used without any problem.

Example 2 FIG. 1 shows the results of analyzing a sample with a known concentration using a reagent kit for measuring γ-GTP having the following reagent composition.

A, Reagent composition R, 0.1M) Lis-hydrochloric acid buffer (pH 7.5) Glycylglycine 100mM Laccase 51m/d RI HCA 10mM EDTA
2Na 1mML-γ-glutamylp
-Diethyl 5mM aminoanetodiethylaniline 1mM B, 2. After adding Od and prewarming at 37°C, then adding reagent R, 0,54, wavelength 74
5. Measure the change in absorbance of the tooth for 1 minute during the linear part of the reaction.

[Brief explanation of drawings]

FIG. 1 shows a calibration curve when γ-GTP was measured using the reagent kit of the present invention.

Claims (1)

[Claims] A first reagent containing a substrate and a coupler represented by the following general formula [I], having a pH of 5 or less, and/or containing a chelating agent and a second reagent containing an oxidizing enzyme. A reagent kit for measuring enzyme activity. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] (In the formula, X_m indicates a peptide residue in which m of the same or different amino acids are bonded, and the C-terminal amino acid is an amino group bonded to an aromatic group. Forms an amide bond.A
represents a group or hydrogen atom that protects the N-terminus of the above peptide residue. m=1 to 10. Z represents a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxyl group, an amino group, a hydroxyl group, a carbonyl group, a sulfonic acid group, or a substituted amino group, and n=1 to 5. A plurality of Z may be the same or different, and may be a group forming a ring at the ortho position and meta position of the substituent -NH-. )