JPH03210193A - Measuring instrument for hydrolase - Google Patents

Abstract

PURPOSE:To form an instrument for rapidly measuring a hydrolase in a solution with a high sensitivity by holding specific amounts of a phenoxy ester compound expressed by a specific general formula, developer and oxidizing agent on an adsorbent carrier. CONSTITUTION:A phenoxy ester compound expressed by formula I (A represents amino acid residue or peptide group composed of 2 to 5 amino acid residues; B represents nitrogen-protecting group; X represents halogen atom, carboxyl group, sulfone group, etc.; R<1>, R<2>, R<3> and R<4> represent hydrogen atom, halogen atom, alkyl group, etc.), a developer and an oxidizing agent are held on an adsorbent carrier such as filter paper, fabric, etc., to form a measuring instrument for a hydrolase in a solution. The developer in an amount of 100-20 equiv. based on 100 equiv. phenoxy ester compound and the oxidizing agent in an amount of 50-1500 equiv. based on 100 equiv. phenoxy ester compound are preferably blended.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Application of the Invention) The present invention relates to a measuring instrument for hydrolytic enzymes. More specifically, the present invention relates to a measuring instrument used to detect hydrolytic enzymes such as esterase and protease.

BACKGROUND OF THE INVENTION The amount of white blood cells that appear in the urine guides the diagnosis of kidney or urinary tract infections. Additionally, monitoring leukocytes during peritoneal dialysis can guide early detection of peritonitis. Conventionally, detection of these white blood cells was done by microscopic examination, but
This method not only requires expensive equipment, but also has drawbacks such as long measurement times and complicated measurement operations.

In order to solve these problems, detection methods using hydrolytic enzymes such as esterase and protease in leukocytes as guidelines have become widely used. This method has the advantage that measurement results can be obtained with simple operation and in a short time.

In addition, disintegrated white blood cells can also be detected using enzyme activity as a guideline. Already, (A) a method for detecting a proteolytic enzyme containing a phenoxyamino acid ester as an enzyme substrate and a diazonium salt as a color developer (Japanese Patent Publication No. 1983-
45.982), (B) Method for detecting esters and proteolytic enzymes containing indoxyl-amino acid esters and peptide esters as chromogens (Special Publication No. 59
-3,475) and (C) Detection method for hydrolyzed substances using 5-phenylpyrrole ester (Japanese Patent Publication No. 62-
No. 41,475) is known.

However, these methods also have problems such as expensive reagents, lack of color stability, and poor sensitivity.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a novel measuring instrument for hydrolytic enzymes. Another object of the present invention is to use a substrate that has high specificity for the hydrolase to be detected and exhibits excellent coloring properties, and to provide a simple method that does not require any special or expensive equipment. The objective is to provide an instrument for quick and sensitive measurements in operation.

(Means for Solving the Problems) The general formula I (wherein A is an amino acid residue or a peptide group consisting of 2 to 5 amino acid residues, B is a nitrogen protecting group, and X is a Halogen atom, carboxyl group, sulfone group or thioalkyl group, R1, R2, R3 and R4 are hydrogen atoms, halogen atoms, alkyl groups, aryl groups, alkoxy groups,
It is at least one type selected from the group consisting of an acyl group, an amide group, a nitro group, and an alkylthiol group, and R1 and R2 and R3 and R4 may each form a fused aromatic ring. ), a color developer, and an oxidizing agent are retained on an adsorbent carrier.

The present invention also provides 100 to 200 equivalents of color developer and 500 equivalents of oxidizing agent per 100 equivalents of the phenoxy ester compound.
This is a measuring instrument containing ~1,500 equivalents. The present invention further provides that the color developer is 4-aminoantipyrine or 3-aminoantipyrine.
- Methyl-2-benzothiazolinone. In the present invention, the oxidizing agent has the general formula ■ ZY, , 0, (II) (however,
In the formula, Z is an alkali metal, Y is a halogen atom, and m is 1 to
is an integer of 3, and n is an integer of 1 to 6). The present invention is also a measuring device in which the adsorptive carrier is a filter paper or fabric. The present invention further provides a measuring instrument further comprising a surfactant or a wetting agent. The present invention is a measuring instrument containing a buffer having a pH suitable for the reaction of a hydrolytic enzyme, which is a substance to be detected. The present invention also provides a measuring instrument in which the measuring instrument is supported on a support.

(Function) According to the present invention, the phenoxy ester compound represented by the general formula I has high specificity for hydrolytic enzymes such as esterases and proteases, particularly those present in neutrophils and granulocytes (the following formula As shown in Figure 2, the ester bond is decomposed to produce a free phenol represented by the general formula (2).

This free phenol has properties as a chromogen that develops color by being condensed with a color developer. for example,
4-aminoantipyrine, 3-methyl-2 as a color developer
- When a coupler such as benzothiazolinone hydrazone is condensed in the presence of an oxidizing agent, a colored compound is produced. The following formula is an example of condensation with 4-aminoantipyrine.

h The phenoxy ester compound represented by the general formula I of the present invention has a halogen atom (preferably chlorine and bromine atom), a carboxyl group, a sulfone group, or a thioalkyl group (a carbon atom of the alkyl group) at the para position to the ester bond. The number preferably includes a leaving group such as 1 to 6). These substituents play a role in improving the reactivity of the free phenol generated after enzymatic decomposition when it condenses with the above-mentioned color developer. As a result, the color performance is improved compared to unsubstituted phenol.

Furthermore, the electronic effects of these substituents influence the rate of degradation of the ester bond of the substrate. For example, introducing an electron-donating substituent such as halogen or thioalkyl reduces the stability of the ester bond and increases the rate of enzymatic cleavage. As mentioned above, the substitution at the para position of the enzyme substrate is
It is clear that this method is highly effective in increasing sensitivity in the detection of hydrolytic enzymes.

In addition, in general formula I, A is an amino acid residue or a peptide group consisting of 2 to 5 amino acid residues, and the amino acid residues include L-alanyl, L-β-alanyl, L-alanyl,
-valinyl, L-isoleucinyl, L-lysinyl, L-
Examples include phenylalanyl and L-methionyl. B is a nitrogen-protecting group, such as an acyl group, an oxycarbonyl group, a sulfonyl group, a phosphoryl group, a carbamoyl group, a thiocarbonyl group, and the like. R1, R2, R3 and R4 are hydrogen atoms, halogen atoms (preferably chlorine and bromine atoms), alkyl groups (preferably 1 to 10 carbon atoms,
More preferably 1 to 4), aryl group, alkoxy group (
Preferably 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms
), acyl group (preferably the alkyl moiety has 1 to 9 carbon atoms, more preferably 1 to 3 carbon atoms), amide group, nitro group, alkylthiol group (preferably the alkyl moiety has 1 to 10 carbon atoms, more Preferably, there are 1 to 4), etc.
R1 and R2 and R3 and R4 may each form a fused aromatic ring.

Specific examples of the phenoxy ester compound represented by the general formula I used in the present invention are as follows.

(1) 1-(N-p-toluenesulfonyl-L-alanyloxy)-4-chlorobenzene, (2) 1-(N-p-)toluenesulfonyl-L-alanyloxy)-2,4-dichlorobenzene, (3 ) 1-(N-p-toluenesulfonyl-L-alanyloxy)-3,4-dichlorobenzene, (4) 1-(N-p-toluenesulfonyl-l-alanyloxy)-3-methoxy-4-chlorobenzene, ( 5
) 4-(N-p-toluenesulfonyl-l-alanyloxy)-2-chlorotoluene, (6) 1-(N-p-)toluenesulfonyl-L-alanyloxy)-3-acetyl-4-chlorobenzene, (7
) l-<N-p-) luenesulfonyl-L-alanyloxy)-3-nitro-4-chlorobenzene, (8) l-
(N-p-)luenesulfonyl-L-alanyloxy)
-4-chloronaphthalene, (9) l-(N-p-toluenesulfonyl-L-alanyloxy)-4-bromobenzene, (10) 4-(N-p-toluenesulfonyl-L-alanyloxy)benzoic acid, ( 11) 2-methoxy-4-(N-p-toluenesulfonyl-l-alanyloxy)benzoic acid, (12) 1-(N-p-toluenesulfonyl-L-alanyloxy)-4-methylthiobenzene, and the like.

An oxidizing agent is required when the phenoxy ester compound is hydrolyzed by an enzyme for the purpose of detection and the generated free phenol compound is condensed with a color developer.

The oxidizing agent used in the present invention has the general formula: ZYIl, On(II) (wherein, Z is an alkali metal, preferably sodium or potassium, more preferably sodium, Y is a halogen atom, and m is 1 to 3 n is an integer from 1 to
is an integer of 6). - for example, sodium iodate, sodium periodate, sodium bromate, sodium perbromate, sodium bromite, sodium chlorate, sodium perchlorate,
Potassium iodate, potassium periodate, potassium bromate, potassium perbromate, potassium bromite, potassium chlorate, etc.

In the test device according to the present invention, the amount of the color developer to be blended is 100 to 100 equivalents of the phenoxyester compound.
The amount is 200 equivalents, preferably 100 to 150 equivalents, and the amount of the oxidizing agent is 500 to 1,500 equivalents.

A measuring instrument is obtained by impregnating a carrier with a solution of the phenoxy ester compound, a color developer, an oxidizing agent, and other reaction aids if necessary, and then drying the carrier.

Examples of carriers include filter paper, fabrics (eg, woven fabrics, knitted fabrics, nonwoven fabrics, etc.).

Further, a surfactant or a wetting agent is added to the solution as necessary. As surfactants or wetting agents, Triton X-100, sodium lauryl sulfate, Span,
Examples include Tween, polyethylene glycol, higher alcohols, ether alcohols, etc., preferably linear alcohols having 8 to 12 carbon atoms, and 8 to 1 carbon atoms.
A straight chain alcohol containing 5 ether bonds is used.

In addition, a buffer having a pH suitable for the reaction of the hydrolase for detection purposes is added to the solution, if necessary. Examples of such buffers include phosphate buffer, borate buffer, Tris buffer, citrate buffer, and the like.

By supporting the test device on a support, a test device with a handle can be obtained. In this way, as a support,
There are plate-like objects such as plastics, metals, and wood, which are fixed using adhesives.

The sample to be measured is dropped onto the measuring instrument, or after the measuring instrument is immersed in the sample liquid to be measured, the color tone change is visually observed or the reflected absorbance is measured using a spectrophotometer. .

(Example) Next, the present invention will be described in further detail by giving examples.

Synthesis Example 1 Synthesis of l-(N-p-toluenesulfonyl-L-alanyloxy)-4-chlorobenzene p-chlorophenol 1.0 g (7,78 mm o
1), 2.19 g of N-p-toluenesulfonyl-thi-alanine (9, Osmol), 122 mg of 4-N,N-dimethylaminopyridine (1, Ommol) was dissolved in 25 ml of dichloromethane, and the mixture was stirred at 0°C. Dicyclohexylcarbodiimide 1.86° (9, Omm
ol) was added. After stirring for 1 hour at 0°C, stirring is continued for an additional 10 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent to obtain 2.66 g of 1-(N-p)luenesulfonyl-L-alanyloxy)-4-chlorobenzene (yield 96.7).
%) was obtained. This stuff was oily.

Synthesis Example 2 Synthesis of l-(N-p-toluenesulfonyl-shi-alanyloxy)-2,4-dichlorobenzene ■, 3-dichlorophenol 1.0 g (6.13 mmol), N-
p-) Luenesulfonyl-L-alanine 1.79g (
7,46 mmol), 4-N,N-dimethylaminopyridine (122 mg (1,0 mmol)) was dissolved in 25 ml of dichloromethane and stirred at 0°C.
Add 52g (7,36111[1101).

After stirring at 0°C for 1 hour, stirring is continued at room temperature for an additional 10 hours. After the reaction mixture was suction-filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent, and 2.22 g of the target product was obtained.
(yield 93.2%). The melting point of this substance is 156℃
Met.

Synthesis Example 3 Synthesis of l-(N-p-toluenesulfonyl-shi-alanyloxy)-3,4-dichlorobenzene 1.2-dichlorophenol L, Og (6,13 gmol), N-p
-Toluenesulfonyl-L-alanine 1.79g (7
, 46 mmol), 122 mg (1,0 mmol) of 4-N,N-dimethylaminopyridine was dissolved in dichloromethane 251, and D CC1,52 was added with stirring at 0°C.
g (7.36 mmol) is added.

After stirring at 0°C for 1 hour, stirring is continued at room temperature for an additional 10 hours. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an eluent, yielding 2.18 g of the target product.
(yield 91.7%). The melting point of this substance is 144℃
Met.

Synthesis Example 4 Synthesis of l-(N-p-toluenesulfonyl-shi-alanyloxy)-3-methoxy-4-chlorobenzene 3-methoxyphenol 3.0 g (24.17 mmol)
Dissolve in 20 ml of dichloromethane and slowly add 2.0 ml (24.2 mmol) of sulfuryl chloride while stirring at 0°C.

After reacting for 1.5 hours, the reaction mixture was poured into ice water, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with an aqueous sodium hydrogen carbonate solution and an aqueous sodium chloride solution, and then concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 2/n-hexane 8 as an eluent, and 3-methoxy-4-chlorophenol 2.
82 g (yield 73.6%) was obtained.

1-(N-p-)luenesulfonyl-L-alanyloxy)-3-methoxy-4-chlorobenzene 3-methoxy-p-chlorophenol 2.8 g (16.4 mmol
), N-p-toluenesulfonyl-L-alanine 4.3
7 g (18, Ommol), 183 mg (1.5 mmol) of 4-N,N-dimethylaminopyridine were dissolved in 30 ml of dichloromethane, and stirred at 0°C.
Add 71 g (18, Ommol) of CC3. 0℃
After stirring for 1 hour at room temperature, stirring was continued for an additional 10 hours. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 2/n-hexane 8 as an eluent.
-2.02 g of chlorobenzene (yield 83.4%) was obtained. The melting point of this product was 152°C.

Synthesis Example 5 Synthesis of 4-(N-p-toluenesulfonyl-shi-alanyloxy)-2-chlorotoluene 2-chloro-4-hydroxytoluene 1.0 g (7,
01a+mol), N-p-toluenesulfonyl-
Alanine 2.05g (8.42mmol), 4-N,
N-dimethylaminopyridine 122 mg (1, Omm
ol) in 25 ml of dichloromethane, and while stirring at 0°C, 1.52 g (7.36 mmol) of DCC was added.
added. After stirring for 1 hour at 0°C, stirring is continued for an additional 10 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent to obtain 2.46 g (yield 95.4%) of the desired product. The melting point of this product was 168°C.

Synthesis Example 6 Synthesis of l-(N-p-toluenesulfonyl-shi-alanyloxy)-3-acetyl-4-chlorobenzene 2-chloro-5-hydroxyacetophenone 1.0 g (5,
86 mmol), N-p-toluenesulfonyl-di-alanine 1.71 g (7,04111 mol), 4-N
, N-dimethylaminopyridine 122 mg (1,0 m
mol) was dissolved in 25 ml of dichloromethane, and while stirring at 0°C, 1.45 g of DCC (7.04 mmol
) was added. After stirring at 0°C for 1 hour, the mixture was stirred for an additional 10 minutes at room temperature.
Continue stirring for an hour. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product.

This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent to obtain 2.07 g (yield 89.1%) of the target product. The melting point of this product was 193°C.

Synthesis Example 7 Synthesis of l-(N-p-toluenesulfonyl-L-alanyloxy)-3-nitro-4-chlorobenzene 1.0 g (5,76 mm) of 3-nitro-4-chlorophenol
.

1), N-p-toluenesulfonyl-L-alanine 1.
68 g (6.91 mmol) and 122 mg (1.0 mmol) of 4-N,N-dimethylaminopyridine were dissolved in 25 ml of dichloromethane and stirred at 0°C with DC
C1.43g (6.91 mmol) was added. 0°C
After stirring for 1 hour at room temperature, stirring is continued for an additional 20 hours. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent to obtain 2.11 g (yield 91.8%) of the desired product. The melting point of this product was 206°C.

Synthesis Example 8 l-(N-p-toluenesulfonyl-shi-alanineoxy)-4-chloronaphthalene 4-chloronaphthalene 1.0 g (6.15 mmol
), N-p-toluenesulfonyl-cycloalanine 1.
61 g (6.60 mmol) and 122 mg (1.0 mmol) of 4-N,N-dimethylaminopyridine were dissolved in 30 ml of dichloromethane and stirred at 0°C.
Add 1.36 g (6.60 mmol) of CC.

After stirring for 1 hour at 0°C, stirring is continued for an additional 14 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent, and 2.16 g of 1-(N-p-toluenesulfonyl-thi-alanineoxy)-4-chloronaphthalene (yield 90 .6%) was obtained. The melting point of this product was 181°C.

Synthesis Example 9 Synthesis of l-(N-p-)luenesulfonyl-L-alanyloxy)-4-bromobenzene 4-bromophenol 1.0 g (5,78 mmol
), N-p-toluenesulfonyl-L-alanine 1.
Dissolve 89 g (8,94 III layer of), 122 mg (1,0 mol) of 4-N,N-dimethylaminopyridine in 25 ml of dichloromethane, and add 1,43 g' (6,94+ nmol) of DCC while stirring at 0°C. added.

After stirring for 1 hour at 0°C, stirring is continued for an additional 10 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an elution solvent, and the target product 2.20
g (yield 95.4%) was obtained.

This stuff was oily.

Synthesis Example 10 Synthesis of oxazoline protected form of 4-hydroxybenzoic acid 4-hydroxybenzoic acid 2. Og (14,5ml1
ol), 2-amino-2-methyl-1-propatool 5
．． 0 g (56,lll1m.

1), toluene 201 and 1,4-dioxane 2011
The mixture was heated under reflux for 6 hours in a mixed solvent of The reaction was carried out by adding an appropriate amount of molecular sieve to the reaction system to adsorb water generated. After removing the molecular sieve from the reaction mixture by filtration, it was dried under reduced pressure to obtain 2.3 g of a crude product (yield: 83.0%).

Synthesis of 4-(N-p-toluenesulfonyl-L-alanyloxy)benzoic acid oxazoline protected form 4-hydroxybenzoic acid oxazoline protected form 1. .

r (5,23 mmol), N-p-toluenesulfonyl-thi-alanine 1.53 g (6,60++++a
ol), 4-N,N-dimethylaminopyridine 122
mg (1,0 servings of) was dissolved in 20 ml of dichloromethane, and while stirring at 0°C, 1,29 g of DCC (6,
28 mmol) was added. After stirring at 0° C. for 1 hour, stirring is continued for an additional 15 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was dissolved in a mixture of 3.0 ml of 2N hydrochloric acid and 5.0 ml of ethanol, and reacted at room temperature for 15 hours. Water was added to the reaction mixture, extracted with ethyl acetate, concentrated under reduced pressure, and subjected to silica gel flash chromatography using ethyl acetate 4/n-hexane 6 as an eluent to obtain 1.23 g of the desired product (yield 64.8%). ) was obtained. The melting point of this product was 232°C.

Synthesis Example 11 Synthesis of oxazoline protected form of 2-methoxy-4-hydroxybenzoic acid 2-methoxy-4-hydroxybenzoic acid 2. Og(11
4.24 g (47.6 mmol) of 2-amino-2-methyl-1-propatol was heated under reflux for 6 hours in a mixed solvent of 20 ml of toluene and 20 ml of 1,4-dioxane. The reaction was carried out by adding an appropriate amount of molecular sieve to the reaction system to adsorb water generated. After removing the molecular sieve from the reaction mixture by filtration, it was dried under reduced pressure to obtain 2.08 g of a crude product (yield 7).
9.0%).

Synthesis of 2-methoxy-4-(N-p-toluenesulfonyl-s-alanyloxy)benzoic acid oxazoline protected form 2-methoxy-4-hydroxybenzoic acid oxazoline protected form 1.0 g (4,52 mmol), N-p −)
Luenesulfonyl-L-alanine lJ2g (5,43
mmol), 4-N,N-dimethylaminopyridine 12
2 mg (1.0 mmol) in 20 ml of dichloromethane
1.12 g of DCC (5
, 43 mmol) was added. After stirring at 0° C. for 1 hour, stirring is continued for an additional 15 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was dissolved in a mixture of 3.0 ml of 2N hydrochloric acid and 5.0 a+1 ethanol, and reacted at room temperature for 15 hours. Water was added to the reaction mixture, extracted with ethyl acetate, concentrated under reduced pressure, and subjected to silica gel flash chromatography using ethyl acetate 4/n-hexane 6 as an eluent to obtain 1.20 g of the desired product (yield 67.2%). ) was obtained. The melting point of this product was 197°C.

Synthesis Example 12 Synthesis of 1-(N-p-)luenesulfonyl-L-alanyloxy)-4-methylthiobenzene 4-methylthiophenol 1.0 g (7.13 mm
ol), N-p-) luenesulfonyl-L-alanine 2
．． 08 g (8,58 mmol), 122 + ng (1,0 mmol) of 4-N,N-dimethylaminopyridine were dissolved in 25 ml of dichloromethane, and DCC1,76K (8-56 mmol) was added with stirring at 0°C.

After stirring for 1 hour at 0°C, stirring is continued for an additional 10 hours at room temperature. After the reaction mixture was suction filtered to remove the generated urea, the filtrate was concentrated under reduced pressure to obtain a crude product. This was subjected to silica gel flash chromatography using ethyl acetate 3/n-hexane 7 as an eluent, and the target product 2.31
g (yield 88.6%) was obtained.

The melting point of this product was 169°C.

Examples 1 to 12 Filter paper (Atobanchik No. 1650) was
Impregnated in an aqueous solution containing M sodium periodate, 200 mM borax-hydrochloric acid buffer (pH = 8.0),
It was dried with ventilation at 0°C for 50 minutes. Further, add 10 m of this filter paper.
Phenoxy ester compound shown in Table 1 of M, 10mM
It was impregnated with a chloroform solution containing 4-aminoantipyrine and 2% ethylene glycol monohexyl ether, and dried with ventilation at 40°C for 20 minutes. 5% 5mm
Cut into corners, drop a PBS suspension of 200 cells/μm granulocytes, and observe the color change after 1.5 minutes on 500 nm of the surface of the test paper.
The observation was made by measuring the reflected absorbance of m. The measurement was performed using MCPD-200 manufactured by Otsuka Electronics Co., Ltd.

5 (5 1 Table (Effects of the Invention)) As described above, the hydrolytic enzyme measuring instrument according to the present invention contains a phenoxyester compound represented by the general formula I,
Since it is made by holding a color developer and an oxidizing agent on an adsorbent carrier, it has high specificity for the hydrolytic enzyme that is the object to be detected, so it shows good color development and does not require any expensive equipment. It has the advantage of being able to perform measurements quickly, with extremely simple operations, and with high sensitivity, without the need for

Claims (8)

[Claims] (1) General formula I ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, A is an amino acid residue or a peptide group consisting of 2 to 5 amino acid residues, B is a nitrogen protecting group, is a halogen atom, a carboxyl group, a sulfone group or a thioalkyl group, R^1, R^2, R^3 and R^4 are a hydrogen atom, a halogen atom, an alkyl group, an aryl group, an alkoxy group, an acyl group, an amide group, It is at least one type selected from the group consisting of a nitro group and an alkylthiol group, and R^1 and R^2 and R^3 and R^4 may each form a fused aromatic ring. 1. A measuring instrument for a hydrolase in a solution, characterized in that a phenoxy ester compound represented by (), a color developer, and an oxidizing agent are held in an adsorbent carrier. (2) 100 to 20 equivalents of color developer and 500 to 1,50 equivalents of oxidizing agent per 100 equivalents of the phenoxy ester compound
The measuring instrument according to claim 1, wherein 0 equivalent is blended. (3) The measuring instrument according to claim 1, wherein the color developer is 4-aminoantipyrine or 3-methyl-2-benzothiazolinone. (4) The oxidizing agent has the general formula II ZY_mO_n(II) (wherein, Z is an alkali metal, Y is a halogen atom, m is an integer from 1 to 3, and n is an integer from 1 to 6) The measuring instrument according to claim 1, which is a salt represented by: (5) Claims 1 to 4, wherein the adsorbent carrier is filter paper or fabric.
Measuring instruments described in any one of the above. (6) The measuring instrument according to any one of claims 1 to 5, further comprising a surfactant or a wetting agent. (7) P suitable for the reaction of the hydrolase that is the substance to be detected
The measuring instrument according to any one of claims 1 to 6, which contains a buffering agent containing H. (8) A measuring device for a hydrolase in a solution, comprising the measuring device according to any one of claims 1 to 7 supported on a support.