JPS6248399A - Reagent for measuring enzymatic activity - Google Patents

Abstract

PURPOSE:The titled reagent for raising solubility (dissolution rate and dissolution concentration) of a substrate and measuring sensitivity, comprising an inclusion compound and a solvent except water. CONSTITUTION:A reagent for measuring activity of N-acetyl-beta-D- glucosaminidase[E.C.3.2.1.30] using 2-chloro-4-nitrophenyl-N-acetyl-beta-D- glucosaminide as a substrate is obtained by dissolving one or more inclusion compounds selected from 13-crown-6,15-crown-5,12-crown-4, (dimethyl-)alpha- cyclodextrin, (dimethyl-)beta-cyclodextrin, (dimethyl-)gamma-cyclodextrin and L-poly-beta- cyclodextrin in one or more solvents selected from aliphatic alcohols (e.g., methanol, etc.,) and glycols (e.g., ethylene glycol) except water.

Description

[Detailed description of the invention] phlegm rat upper a phoenix 肚本 8Jlli 2-90-4-nitrophenyl-N
-acetyl-β-D-glucosaminide (hereinafter 2CNP-
It is abbreviated as NAG. ) with N-acetyl-β-D- as a substrate
When dissolving a substrate in the glucosaminidase [E, C03,2,1,30] (hereinafter abbreviated as NAGase) activity measurement method, the solubility (dissolution rate, The present invention relates to a reagent for measuring NAGase activity, which has the characteristics of increasing the dissolved concentration) and increasing the measurement sensitivity.

NAGase is an enzyme found in lysosomes that is abundant in the prototubular epithelium and is involved in the decomposition of mucopolysaccharides and glycoproteins, but urinary NAGase is found to be elevated in various renal diseases such as tubulonephritis. NAGase measurement is attracting attention in various fields of clinical and animal experiments as an aid to disease diagnosis and treatment, observation guidelines, or as an indicator of drug nephrotoxicity.

Problems to be Solved by the Prior Art and the Invention Many of the substrates for measuring NAGase activity use various synthetic substrates, but among them, 2GNP-NAG does not require the use of a reaction stop solution (for enzymatic reaction). NA at constant time interval in progress
Since it can be measured by the rate method that measures the difference in absorbance of Gase activity, it can be applied to commercially available general-purpose measuring instruments (Japanese Patent Application No. 1987-
233327). However, in this method, the substrate 2CN
P-NAG is poorly soluble in a buffer solution suitable for measuring NAGase activity, and there is a problem that sufficient sensitivity cannot be obtained with the amount of sample measured with some commercially available general-purpose measuring instruments. are doing. As a method to improve the problem of poor solubility in buffer solutions, use of solvents such as DMS○, DMF, etc.
Alternatively, solubility can be improved by using nonionic surfactants such as polyoxyethylene alkyl ether and polyoxyethylene alkyl allyl ether, but these inhibit NAGase activity and are not suitable for this purpose.

In order to increase sensitivity, it is desirable that the absorbance of 2GNP is high, that is, conditions where the molecular extinction coefficient of 2GNP (aglycon) is high are suitable. This aglycon is pH dependent, and its absorbance increases as it becomes more alkaline. But NA
Since the optimum pH for Gase activity is around 4 to 5, measurement at around this level does not provide highly sensitive conditions.

Means for Solving the Problems As a result of efforts by the present inventors to improve the reagent for measuring NAGase activity using 2GNP-NAG, the above problems were solved by combining an clathrate compound with a solvent other than water. We have discovered that this can be done, and have arrived at the present invention.

The reagent for measuring NAGase activity of the present invention is 2GNP-NA.
A reagent for measuring NAGase activity using G or a derivative thereof as a substrate is characterized in that when dissolving the substrate, it is dissolved in a solvent other than the clathrate compound and water, thereby increasing the solubility of the substrate, Sensitivity is improved.

When measuring NAGase, first 2CNP-NA
After G is completely dissolved in one substrate solution containing the clathrate compound and a solvent, the solution is diluted with a buffer or the like to the required concentration to obtain a substrate solution.

The clathrate compound used in the present invention is 18-crown-6,1
Crown ethers such as 5-crown-5,12-crown-4, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dimethyl α-
It includes cyclodextrin and derivatives thereof such as cyclodextrin, dimethyl β-cyclodextrin, dimethyl α-cyclodextrin, and L-poly-β-cyclodextrin, and these can be used alone or in combination of two or more. Krafn ether fabrics are particularly preferred. The amount used is approximately 3 per 2 GNP-NAGI molecules.
The number of molecules is preferably 30 to 150 molecules. As solvents other than water, glycols such as ethylene glycol, diethylene glycol and propylene glycol, and alcohols such as ethanol and methanol are used. Glycols are particularly preferred. These solvents can be used alone or in combination of two or more.

Example 1 15-trilan-5, NA using ethylene glycol
Measurement of Gase activity (1) Preparation of reagent Reagent 1) 5 mg of 2CNP-NAG (substrate) was mixed with 15-crown-5,500 μm and 500 μI of ethylene glycol.
Add to the mixture (dissolution II) and dissolve thoroughly until it becomes transparent. Then 1 m of 0.85% NaCl solution (dissolution solution 11)
1 and mix well to make 2 ml of substrate solution. Reagent 2)
Citric acid (C611,0?・11□0) 0.8618 and sodium citrate (C6HsOtNa -21hO
) 1.

Dissolve 748 in about 48 ml of distilled water and adjust the pH to 5.0 using 0.2M citric acid or sodium citrate solution.
After adjusting to (25°C), add distilled water to make 50+++1. (2) Measurement procedure: Add 0.1 ml of sample to 1.5 ml of the above reagent 2) and
Warm at 37°C for 5 minutes. Next, 0.5 ml of reagent 1), which had been preheated at 37°C, is added and stirred, and the increase in absorbance per unit time at 405 nm is measured using a spectrophotometer. As a control, an enzyme solution having a known activity is measured in the same manner, and the NAGase activity of the unknown sample is determined from the ratio of the absorbances per unit time.

NAGase activity was measured in the same manner as in Example 1, using 12-crown-4 or 18-crown-6 instead of 15-crown-5 and using propylene glycol instead of ethylene glycol. However, since 18-crown-6 is a solid, it is dissolved in ethylene glyfur in advance.

Example 2 Measurement of NAGase activity using α-cyclodextrin and ethylene glycol.

(1) Preparation of reagent Reagent 1) Citric acid (C, 11,07·lI20) 00.
861g and sodium citrate (C, 1150Ja3
-21120) 1, 74g.

Dissolve 0.4 g of α-cyclodextrin in about 48 ml of distilled water, adjust the pH to 5.0 (25°C) using 0.2 M citric acid or sodium citrate solution, and add distilled water to make 50 ml.

Reagent 2) 2CNP NAGlorr (substrate) to 3n+
1 in ethylene glycol (solution I) and dissolve sufficiently until it becomes transparent. Then 0.85% NaCl solution (
Add solution 1) 5a+l and mix well to make 8 ml of substrate solution.

(2) Measurement procedure Sample 0.0ml per 1.0ml of the above reagent 1). Add 1+oi and warm at 37°C for about 1 to 5 minutes. Next, 1 ml of reagent 2) preheated at 37°C is added, and after stirring, the increase in absorbance per unit time at 405 nln is measured using a spectrophotometer. Thereafter, NAGase activity is determined in the same manner as in Example 1.

Using β-cyclodextrin or γ-cyclodextrin instead of α-cyclode or trine, and using propylene glyfur instead of ethylene glycol,
NAGase activity was measured in the same manner as in Example 2.

Example 3 Measurement of NAGase activity using 12-crown-4 and meth-7-al (1) Preparation of reagent Reagent 1) Citric acid (CI, 11.0□・11°O) 0.
861g and sodium citrate (C611sOJa -
Dissolve 2H20) 1, 748 in about 48 ml of distilled water, adjust the pH to 5°0 (25°C) using 0.2M citric acid or sodium citrate solution, and then add distilled water to make 50 ml.

Reagent 2) 2CNP NAG7.8mg (substrate) for 12
-Crown-4.0.4ml, methanol 0.6ml,
Add to the mixture of 1 ml of water and dissolve thoroughly until it becomes transparent.

Mix 31nl of reagent 1) and 2ml of reagent 2) to make 5ml of reaction solution.
Let it be l.

(2) Measurement procedure Add 0.1+nl of the sample to 2ml of the above reaction solution that has been preheated at 37°C, stir, and measure the temperature at 405nm using a spectrophotometer.
The increase in absorbance per unit time is measured. Thereafter, NAGase activity is determined in the same manner as in Example 1.

NAGase activity was measured in the same manner as in Example 3, using 18-crown-6 or 15-crown-5 instead of 12-crown-4, and using ethanol instead of methanol.

Effects of the invention In a buffer solution containing no clathrate and solvent, 2CNP-N
AG has a maximum solubility of 2+++M and takes time to dissolve, whereas a 1:1 mixture of 15-crown-5 and ethylene glycol has a maximum solubility of 2 CN PNA
G can be dissolved up to 27n+M, and 1
About 0+nM dissolves in 1 to 2 minutes. In addition, the NAG activity measured by dissolving the substrate in a mixed solution of 15-crown-5 and ethylene glycol was approximately 2.5 times higher than the activity value measured using an additive-free substrate solution. has value. Therefore, the present invention is considered to be extremely valuable for application to commercially available general purpose measuring instruments.

Claims (1)

[Claims] 1,2-chloro-4-nitrophenyl-N-acetyl-
N-acetyl-β-D-glucosaminidase using β-D-glucosaminid as a substrate [E. C. 3.2.1
．． 30] A reagent for measuring N-acetyl-β-D-glucosaminidase activity, which uses an inclusion compound. 2,2-chloro-4-nitrophenyl-N-acetyl-
N-acetyl-β-D-glucosaminidase using β-D-glucosaminid as a substrate [E. C. 3.2.1
．． 30] A reagent for measuring N-acetyl-β-D-glucosaminidase activity, which uses a clathrate compound and a solvent other than water alone or in combination. 3. As inclusion compounds, 18-crown-6,15-crown-5,12-crown-4, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dimethyl-a-cyclodextrin, dimethyl-β-
The reagent for measuring N-acetyl-β-D-glucosaminidase activity according to claim 2, which uses cyclodextrin, dimethyl-γ-cyclodextrin, and L-poly-β-cyclodextrin. 4. Aliphatic alcohols and/or as solvents
or the reagent for measuring N-acetyl-β-glucosaminidase according to claim 2, which uses glycols.