JPH0573398B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a reagent for measuring β-N-acetyl-D-hexosaminidase activity. Measurement of β-N-acetyl-D-hexosaminidase activity in body fluids can be used for early diagnosis of rejection after kidney transplantation,
It has great clinical significance as it provides useful information for the diagnosis and follow-up of various renal diseases such as acute renal failure and glomerulonephritis, and for the nephrotoxicity of drugs. (Prior Art) Conventionally, β-N-acetyl-D-hexosaminidase (hereinafter abbreviated as NAG) activity was achieved by using a substrate in which p-nitrophenol was bound to the reducing end of N-acetyl-D-glucosamine. A common method is to colorimetrically measure p-nitrophenol released under alkaline conditions (Methods Engymol., 28 , 702 (1972)). However, with this method, the optimum pH of the target enzyme NAG (PH4-5.5) is different from the coloring pH of p-nitrophenol (PH9 or higher), which is used for coloring.
In order to measure NAG activity, it is necessary to perform the enzymatic reaction and the coloring reaction separately, which requires a large number of reagents and a large number of operational steps. It has the disadvantage that analysis (rate assay) methods cannot be used. Other methods include a method using a substrate in which phenol is bound to N-acetylglucosamine (Japanese Patent Laid-Open No.
60997), a method using a substrate in which m-cresolsulfophthalein is bound to N-acetylglucosamine (Clin.Chem. 29 , 1713 (1983)) is also described in the above p.
- There are the same disadvantages as when using nitrophenol. (Problems to be Solved by the Invention) An object of the present invention is to provide a reagent for measuring NAG activity that enables rate assay of NAG with excellent quantitative properties. (Means for Solving the Problems) In order to achieve the above object, the present inventors conducted various intensive studies and found that NAG activity in body fluids can be reduced in a short time by using a substrate shown in general color [ ]. We have discovered that rate assay can be done accurately and easily, and have arrived at the present invention. That is, the present invention uses the following general formula [] as a substrate.
β characterized by containing a compound represented by
-N-acetyl-D-hexosaminidase activity measurement reagent.

[Formula, A is N-acetylglucosamine or N-
It is an acetylgalactosamine residue whose reducing end is β-bonded with a substituted aromatic group (a group that exhibits a different spectral absorption than the substrate as a cleaved aglycone). X in the substituted aromatic group represents a nitro group or a hydrogen atom. When X is a hydrogen atom, two or more of the substituents among R ₁ to _{R 4} are nitro groups, and the other groups are hydrogen atoms or halogen atoms. When X is a nitro group, R ₁ and/or R ₄ represent a halogen atom or a nitro group, and the remaining R ₁ to _{R 4} represent either a hydrogen atom, a halogen atom, or a nitro group. ) The substrate used in the present invention is a compound represented by the general formula [ ], that is, N-acetylglucosamine or N-acetylgalactosamine and its reducing end bonded to a substituted aromatic group through a β-bond.
The substituted aromatic group may be any substituted aromatic group that exhibits a different spectral absorption from the substrate as a cleaved aglycone, but specifically,

It is represented by: (X, R ₁ to _{R 4} are the same as above). If the substituted aromatic group is shown in the form of phenol,
For example, 2,6-dichloro-4-nitrophenol, 2,6-dibromo-4-nitrophenol,
2,3,6-trichloro-4-nitrophenol, 2,3,6-tribromo-4-nitrophenol, 2,3,5,6-tetrabromo-4-nitrophenol, 2,3,5,6-tetra Chloro
Examples include 4-nitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2-chloro-4-nitrophenol, 2-bromo-4-nitrophenol, and the like. The method for synthesizing these substrates is to acetylate N-acetylhexosamine, and this acetylated N-
Can it be synthesized by combining acetylhexosamine with a substituted aromatic group or aglycone and then deacetylating it? (Jikken Kagaku Koza Vol. 24, p. 304, 1958
), or by halogenating acetylated N-acetylhexosamine, then linking the halide with a substituted aromatic group or aglycone to an ether bond, followed by deacetylation (Methods in Carbohydrate).
Chemistry, p. 334). When R ₁ or R ₄ in the substituted aromatic group of the substrate is a halogen atom, cyclodextrin is necessary from the viewpoint of solubility of the substrate. The cyclodextrins used in the present invention are known to have polymerization degrees of α, β, and γ, but any cyclodextrin may be used as long as it has an effect on the solubility of the substrate [] or the PH stability of coloration. good. α and β-cyclodextrins are preferred. The reagent for measuring β-N-acetyl-D-hexosaminidase activity of the present invention contains the above substrate, cyclodextrin, and, if necessary, sodium chloride. The pH of the reagent is the optimal pH of NAG in body fluids.
Any buffer solution that maintains the ratio between 4.0 and 5.5 may be used. Examples include citric acid buffers and other organic acid buffers, such as acetic acid, succinic acid, and phthalic acid buffers. There are no particular limitations on the substrate concentration, but a concentration that exhibits the maximum NAG enzymatic activity is preferably appropriate. For example, it is 1 mM or more. Next, the concentration of cyclodextrin is not particularly limited as long as it is effective for the solubility of the substrate or the PH stability of coloration, but it is preferably 0.1% or more. The concentration of sodium chloride is not particularly limited as long as it is effective for activating NAG, but is preferably 1 to 1000 mM. Furthermore, surfactants, preservatives, etc. may be added if necessary. A method for measuring NAG activity using the reagent for measuring β-N-acetyl-D-hexosaminidase activity of the present invention is to directly increase the absorbance of the aglycone produced by reacting the reagent with the reagent. This is a method of colorimetric determination using a spectrophotometer. (Effect of the invention) In the reagent for measuring β-N-acetyl-D-hexosaminidase activity of the present invention, by using the compound represented by the general formula [] as a substrate, β-N-acetyl-D- - Hexosaminidase activity can be accurately and easily rate assayed in a short time. (Example) Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 The amount of NAG activity in the test solution was measured using the following reagent and the following method. 1 Reagent 2-chloro-4-nitrophenyl-N-acetyl β-D-glucosaminide 1mM Sodium chloride 200mM α-cyclodextrin 1% 0.05M citric acid buffer (PH4.5) total volume 10ml 2 Measurement method NAG-containing test solution Add 2 ml of the above reagent to 50μ, react at 37℃, and measure the absorbance at a wavelength of 400n.
The color development rate was determined by measuring in m. The reaction curve is shown in FIG. The calibration wave is shown in Figure 2. As is clear from FIGS. 1 and 2, rate assays can be performed accurately and easily in a short period of time using the reagents of the present invention. Comparative Example 1 1 Reagent p-nitrophenyl-N-acetyl β-D-glucosaminide 1mM Sodium chloride 200mM α-cyclodextrin 1% 0.05M citrate buffer (PH4.5) total volume 10ml 2 Measurement method Same method as Example 1 was carried out. The reaction curve is shown in FIG. Moreover, the calibration curve is shown in FIG. As is clear from FIGS. 3 and 4, when using a reagent with p-nitrophenyl-N-acetyl β-D-glucosaminid as a substrate, the optimal pH for N-acetyl-D-hexosaminidase is 4. It was found that rate assay was not possible at around ~5.5 because p-nitrophenol did not dissociate. Example 2 1 Reagent 2,6-dichloro-4-nitrophenyl-N-
Acetyl β-D-glucosaminide 1mM Sodium chloride 200mM α-cyclodextrin 1% 0.05M citrate buffer (PH4.5) total volume 10ml 2 Measurement method Same as Example 1. The reaction curve is shown in FIG. Further, a calibration curve is shown in FIG. As is clear from FIGS. 5 and 6, rate assays can be performed accurately and easily in a short period of time using the reagents of the present invention.

[Brief explanation of the drawing]

FIG. 1 shows the reaction curve of Example 1 of the present invention. FIG. 2 shows the calibration curve of Example 1 of the present invention. FIG. 3 shows the reaction curve of Comparative Example 1 of the present invention. FIG. 4 shows the calibration curve of Comparative Example 1 of the present invention. FIG. 5 shows the reaction curve of Example 2 of the present invention. Fig. 6 shows the second embodiment of the present invention.
The calibration curve is shown.

Claims (1)

[Scope of Claims] 1. A reagent for measuring β-N-acetyl-D-hexosaminidase activity, which contains a compound represented by the following general formula [] and cyclodextrin as substrates. [Formula, A is N-acetylglucosamine or N-
It is an acetylgalactosamine residue whose reducing end is β-bonded with a substituted aromatic group (a group that exhibits a different spectral absorption than the substrate as a cleaved aglycone). X in the substituted aromatic group represents a nitro group or a hydrogen atom. When X is a hydrogen atom, two or more of the substituents among R ₁ to _{R 4} are nitro groups, and the other groups are hydrogen atoms or halogen atoms. When X is a nitro group, R ₁ and/or R ₄ represent a halogen atom or a nitro group, and the remaining R ₁ to _{R 4} represent either a hydrogen atom, a halogen atom, or a nitro group. ]