JPH05317094A - Method for determining activity of n-acetylhexosaminidase - Google Patents

Method for determining activity of n-acetylhexosaminidase

Info

Publication number
JPH05317094A
JPH05317094A JP34048292A JP34048292A JPH05317094A JP H05317094 A JPH05317094 A JP H05317094A JP 34048292 A JP34048292 A JP 34048292A JP 34048292 A JP34048292 A JP 34048292A JP H05317094 A JPH05317094 A JP H05317094A
Authority
JP
Japan
Prior art keywords
acetylhexosaminidase
hydrogen peroxide
acetylhexosamine
nag
nahod
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP34048292A
Other languages
Japanese (ja)
Other versions
JPH0710237B2 (en
Inventor
Yuzo Hayashi
勇蔵 林
Shigenori Aisui
重典 愛水
Sachiko Yamaguchi
佐知子 山口
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toyobo Co Ltd
Original Assignee
Toyobo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyobo Co Ltd filed Critical Toyobo Co Ltd
Priority to JP34048292A priority Critical patent/JPH0710237B2/en
Publication of JPH05317094A publication Critical patent/JPH05317094A/en
Publication of JPH0710237B2 publication Critical patent/JPH0710237B2/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

PURPOSE:To sensitively and accurately determine the subject enzyme in a simple operation and high specificity without being influenced by a coexisting substance and contribute to early discover of a renal disease by a specific method using N-acetylhexosaminin oxidase. CONSTITUTION:N-Acetylhexosaminide of the formula (R is alkyl, phenyl, etc.) is made to react with a sample (e.g. urine or serum) containing N- acetylhexosaminidase(NAG) to produce N-acetylhexosamine. Further, the N- acetylhexosamine is made to react with N-acetylhexosamineoxydase(NAHOD) and the produced hydrogen peroxide or the consumed oxygen is measured. When NAG or NAHOD reacts, a buffer capable of keeping pH to 4-10 is preferably together used and the reaction temperature is preferably kept to 20-50 deg.C. Furthermore, hydrogen peroxide is measured by e.g. hydrogen peroxide electrode.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【産業上の利用分野】本発明は試料、特に水性液中のN
−アセチルヘキソサミニダーゼ活性の定量法に関するも
のである。近年臨床医学の分野において、体液中の各種
化学物質、酵素活性の定量は、有用な診断情報を与える
ものとして臨床的意義の重要性が高まってきている。特
に体液中のN−アセチルヘキソサミンの酵素的定量は多
くの糖関連化合物及び酵素活性についての情報を与える
ものである。これら対象となる化学物質、酵素として
は、例えばN−アセチルヘキソサミニダーゼ、ムラミダ
ーゼ、β−グルクロニダーゼ、シアル酸、シアリダー
ゼ、ムコ多糖等が挙げられる。これらの臨床的意義とし
ては、N−アセチルヘキソサミニダーゼ活性の測定は腎
疾患、特に近位尿細管障害、腎移植拒絶反応等の早期発
見に、ムラミダーゼ活性の測定は急性単球性白血病、非
典型的結核症等の診断に、β−グルクロニダーゼ活性の
測定は、尿路悪性腫瘍、膀胱癌等の診断に、シアル酸の
定量は炎症的疾患等の診断に、シアリダーゼ活性の測定
は細菌感染症等の診断に、ムコ多糖の測定はムコ多糖症
の診断等に有効な情報を与える。
BACKGROUND OF THE INVENTION The present invention is directed to samples, especially N in aqueous liquids.
-A method for quantifying acetylhexosaminidase activity. BACKGROUND ART In recent years, in the field of clinical medicine, the quantification of various chemical substances and enzyme activities in body fluids has become more important in clinical significance as they provide useful diagnostic information. In particular, the enzymatic determination of N-acetylhexosamine in body fluids provides information on many sugar-related compounds and enzyme activity. Examples of these target chemical substances and enzymes include N-acetylhexosaminidase, muramidase, β-glucuronidase, sialic acid, sialidase, and mucopolysaccharide. The clinical significance of these is that the measurement of N-acetylhexosaminidase activity is for the early detection of renal diseases, especially proximal tubular injury, renal transplant rejection, and the measurement of muramidase activity is for acute monocytic leukemia. For the diagnosis of atypical tuberculosis, etc., the measurement of β-glucuronidase activity is for the diagnosis of urinary tract malignant tumor, bladder cancer, etc., the determination of sialic acid is for the diagnosis of inflammatory diseases, etc., and the measurement of sialidase activity is for bacterial infection. The measurement of mucopolysaccharides provides useful information for the diagnosis of mucopolysaccharidosis.

【0002】[0002]

【従来の技術】従来、N−アセチルヘキソサミニダーゼ
(β−N−アセチル−D−ヘキソサミニダーゼ、以下N
AGと略する)活性は、N−アセチル−D−グルコサミ
ンの還元末端にp−ニトロフエノール等の発色団を結合
したクロモジェニックな基質を用いてNAGを作用さ
せ、遊離していくるp−ニトロフエノール等の発色団を
アルカリ性下で比色する方法が一般的である(Methods
Enzymol., 28, 702 (1971), Clinca Chimica et Acta 6
9(1), 85-91 (1976))。ところがこの方法では目的とす
る酵素、NAGの至適pH(pH4〜5.5)と発色団
であるp−ニトロフエノールの発色pH(pH9以上)
とが異なるために、NAG活性を測定するには酵素反応
と発色反応を別々に行う必要があり、そのため、試薬数
及び操作ステップが多く必要となり、酵素活性を求める
場合に一番適当であるといわれている速度分析(レート
アッセイ)法が出来ない欠点がある。その他、N−アセ
チルグルコサミンにフエノールを結合させた基質を用い
る方法(特開昭54−60997号)、N−アセチルグ
ルコサミンにm−クレゾールスルホイソフタレインを結
合させた基質を用いる方法(Clin. Chem., 29, 1713(19
83)) も上記p−ニトロフエノールを用いる場合と同様
の欠点がある。
Conventionally, N-acetylhexosaminidase (β-N-acetyl-D-hexosaminidase, hereinafter referred to as N-acetylhexosaminidase
The activity (abbreviated as AG) is that p-nitrophenol is released by allowing NAG to act using a chromogenic substrate in which a chromophore such as p-nitrophenol is bound to the reducing end of N-acetyl-D-glucosamine. It is common to compare the chromophores such as
Enzymol., 28, 702 (1971), Clinca Chimica et Acta 6
9 (1), 85-91 (1976)). However, in this method, the optimum pH of the target enzyme NAG (pH 4 to 5.5) and the color development pH of the chromophore p-nitrophenol (pH 9 or higher).
Therefore, in order to measure NAG activity, it is necessary to carry out the enzyme reaction and the color reaction separately. Therefore, the number of reagents and the number of operating steps are required, and it is the most suitable for obtaining the enzyme activity. There is a drawback that the so-called rate analysis method cannot be used. In addition, a method using a substrate in which phenol is bound to N-acetylglucosamine (JP-A-54-60997), a method in which a substrate in which m-cresolsulfoisophthalein is bound to N-acetylglucosamine is used (Clin. Chem. ., 29, 1713 (19
83)) also has the same drawbacks as in the case of using p-nitrophenol.

【0003】[0003]

【発明が解決しようとする課題】本発明者等はこれらの
事情を考慮して、正確で簡便、迅速な糖関連酵素の定量
法を見出すために、種々鋭意検討したところ、N−アセ
チルヘキソサミニンオキシダーゼ(以下NAHODと略
する)を用いるN−アセチルヘキソサミニダーゼ活性の
定量法が上記目的を達成することを見出し本発明に到達
した。
In consideration of these circumstances, the present inventors have made various studies in order to find an accurate, simple and rapid method for quantifying sugar-related enzymes, and as a result, N-acetylhexosa The present inventors have found that a method for quantifying N-acetylhexosaminidase activity using minin oxidase (hereinafter abbreviated as NAHOD) achieves the above object, and thus arrived at the present invention.

【0004】[0004]

【課題を解決するための手段】すなわち本発明は下記式
(I)で示されるN−アセチルヘキソサミニドに、N−
アセチルヘキソダミニダーゼを含有する試料を作用さ
せ、生成するN−アセチルヘキソサミンにN−アセチル
ヘキソサミンオキダーゼを作用させ、生成する過酸化水
素または消費される酸素を測定することを特徴とするN
−アセチルヘキソサミニダーゼ活性の定量法である。
That is, the present invention provides N-acetylhexosaminide represented by the following formula (I) with N-acetylhexosaminide.
N-characterized by reacting a sample containing acetylhexodaminidase, reacting N-acetylhexosamine produced with N-acetylhexosamine oxidase, and measuring hydrogen peroxide produced or oxygen consumed
-A method for quantifying acetylhexosaminidase activity.

【0005】[0005]

【化2】 (式中、Rはアルキル基、フェニル基、ナフチル基また
は置換基としてニトロ基を有するこれらの基を示す。)
[Chemical 2] (In the formula, R represents an alkyl group, a phenyl group, a naphthyl group, or a group having a nitro group as a substituent.)

【0006】本発明の測定原理は下記に示す通りであ
る。
The measurement principle of the present invention is as follows.

【化3】 [Chemical 3]

【0007】本発明における上記式(化1)で示される
N−アセチルヘキソサミニドとしては、例えばメチル−
N−アセチル−β─D−グルコサミニド、フェニル−N
−アセチル−β─D−グルコサミニド、p−ニトロフェ
ニル−N−アセチル−β─D−グルコサミニド、メチル
−N−アセチル−β─D−ガラクトアミニド、フェニル
−N−アセチル−β─D−ガラクトサミニド、p−ニト
ロフェニル−N−アセチル−β─D−ガラクトサミニド
などが挙げられる。
Examples of the N-acetylhexosaminide represented by the above formula (Formula 1) in the present invention include methyl-
N-acetyl-β-D-glucosaminide, phenyl-N
-Acetyl-β-D-glucosaminide, p-nitrophenyl-N-acetyl-β-D-glucosaminide, methyl-N-acetyl-β-D-galactoaminide, phenyl-N-acetyl-β-D-galactosaminide, p- Examples thereof include nitrophenyl-N-acetyl-β-D-galactosaminide.

【0008】本発明におけるN−アセチルヘキソダミニ
ダーゼ(NAG)を含有する試料とは尿、血清などの体
液を含有するものであり、本発明におけるN−アセチル
ヘキソサミンは上記式で示される基質、N−アセチルヘ
キソサミニドにNAGを含有する試料を作用させて生成
したN−アセチルヘキソサミンである。
The sample containing N-acetylhexodaminidase (NAG) in the present invention is a sample containing body fluids such as urine and serum, and N-acetylhexosamine in the present invention is a substrate represented by the above formula, It is N-acetylhexosamine produced by allowing a sample containing NAG to act on N-acetylhexosaminide.

【0009】本発明において使用するNAHODはいか
なる起源のものでもよいが、例えばシュードモナス属に
属する微生物が産生するNAHODがある。NAHOD
の精製と性質については、日本農芸化学会 昭和58年
度大会において発表されている。
The NAHOD used in the present invention may be of any origin, for example, NAHOD produced by a microorganism belonging to the genus Pseudomonas. NAHOD
The refining and properties of sucrose have been announced at the Japan Society for Agricultural Chemistry 1983 conference.

【0010】NAGを含有する試料に上記N−アセチル
ヘキソサミニドを作用させ、生成するN−アセチルヘキ
ソサミンにNAHODを作用させる条件としては、特に
限定はないが、pH4.0〜10.0を保つ適当な緩衝
液を用い、反応温度20〜50℃が適当である。緩衝液
の種類は特に制限されない。使用pHに最適な緩衝液を
選択すればよい。例えば中性域ではリン酸緩衝液、酸性
域では酢酸緩衝液、アルカリ域ではトリス塩酸緩衝液な
どがある。緩衝液の濃度は特に限定されないが、通常
0.01M〜1M程度である。反応方法としてはN−ア
セチルヘキソサミニダーゼ反応とNAHOD反応を同時
に行うワンステップ法でも、N−アセチルヘキソサミニ
ダーゼ反応をまず行い、続いてNAHOD反応を行うツ
ーステップ法でも良い。また反応系には必要に応じて界
面活性剤等を添加してもよい。
The conditions for causing the N-acetylhexosaminide to act on the sample containing NAG and allowing the resulting N-acetylhexosamine to act on NAHOD are not particularly limited, but pH 4.0 to 10.0 is used. A reaction temperature of 20 to 50 ° C. is suitable, using a suitable buffer to maintain. The type of buffer solution is not particularly limited. The optimum buffer solution for the pH used may be selected. For example, there are a phosphate buffer solution in the neutral range, an acetate buffer solution in the acidic range, and a Tris-HCl buffer solution in the alkaline range. The concentration of the buffer solution is not particularly limited, but is usually about 0.01M to 1M. The reaction method may be a one-step method in which the N-acetylhexosaminidase reaction and NAHOD reaction are carried out simultaneously, or a two-step method in which the N-acetylhexosaminidase reaction is carried out first and then the NAHOD reaction is carried out. If necessary, a surfactant and the like may be added to the reaction system.

【0011】本発明では生成した過酸化水素を例えばペ
ルオキシダーゼあるいはカタラーゼと共役させて測定す
るか、または直接過酸化水素電極により測定する。ペル
オシダーゼと共役する測定系では、一般的には4−アミ
ノアンチピリンとフェノール誘導体又はアニリン誘導体
を使用する系が好ましい。
In the present invention, the produced hydrogen peroxide is measured by coupling it with, for example, peroxidase or catalase, or directly by a hydrogen peroxide electrode. In the measurement system coupled with perosidase, a system using 4-aminoantipyrine and a phenol derivative or an aniline derivative is generally preferable.

【0012】過酸化水素の定量を妨害する試料中のアス
コルビン酸はアスコルビン酸オキシダーゼ(以下ASO
と略記する)を作用させることによって除去することが
好ましい。この場合、ASOは他の酵素反応に先立って
作用させても、また他の酵素反応と共役させて同時に作
用させて除去してもよい。ASOは微生物由来のものま
たは植物由来のものでもいが、特にカボチャ、キュウリ
等の植物由来のものが好ましい。HAHODの反応系で
消費する酸素の量は、例えば酸素電極などにより測定す
ることができる。
Ascorbic acid in the sample that interferes with the determination of hydrogen peroxide is ascorbic acid oxidase (hereinafter referred to as ASO).
Abbreviated). In this case, ASO may be allowed to act prior to the other enzymatic reaction, or may be conjugated with the other enzymatic reaction to act simultaneously and removed. The ASO may be of microbial origin or plant origin, with plant origin such as pumpkin and cucumber being particularly preferred. The amount of oxygen consumed in the HAHOD reaction system can be measured by, for example, an oxygen electrode.

【0013】[0013]

【発明の効果】本発明に従えば、簡単な操作で感度よく
共存物質の影響受けずに特異性高く正確に試料中のN−
アセチルヘキソサミニダーゼ(NAG)活性を定量する
ことが可能となり、臨床検査の診断分野において極めて
有意義である。
INDUSTRIAL APPLICABILITY According to the present invention, N-type in a sample can be accurately and accurately obtained by a simple operation with high sensitivity and without being influenced by coexisting substances.
It becomes possible to quantify the acetylhexosaminidase (NAG) activity, which is extremely significant in the diagnostic field of clinical examination.

【0014】[0014]

【実施例】次に本発明を実施例により説明する。実施例
中の略号は以下のものを示す。 NAHOD(N−アセチルヘキソサミンオキシダーゼ) 4−AA (4−アミノアンチピリン) TOOS (N−エチル−N−(2−ヒドロキシ−3−
スルホプロピル)−m−トルイジン・ナトリウム)
EXAMPLES Next, the present invention will be described with reference to examples. The abbreviations in the examples indicate the following. NAHOD (N-acetylhexosamine oxidase) 4-AA (4-aminoantipyrine) TOOS (N-ethyl-N- (2-hydroxy-3-)
Sulfopropyl) -m-toluidine sodium)

【0015】[0015]

【実施例】被検液中のN−アセチルヘキソサミニダーゼ
活性量を下記試薬を用いて下記方法により測定した。 1.試薬 p−ニトロフェニル−N−アセチル−β−D−グルコサミニド 1mM 塩化ナトリウム 200mM N−アセチルヘキソサミンオキシダーゼ 50U/ml ペルオキシダーゼ 30U/ml TOOS 1mM 4−AA 0.5mM 0.05Mクエン酸緩衝液(pH5.5) 2.測定方法 NAG含有被検液50μlに上記試薬2mlを加え、3
7℃で反応させ、その吸光度を波長550nmで測定し
て発色速度を求めた。その反応曲線を図1に示す。また
検量線を図2に示す。図1および図2から明らかなよう
に、本発明方法では短時間に正確にかつ簡単にNAGを
レートアッセイすることができる。
Example The amount of N-acetylhexosaminidase activity in a test solution was measured by the following method using the following reagents. 1. Reagents p-nitrophenyl-N-acetyl-β-D-glucosaminide 1 mM sodium chloride 200 mM N-acetylhexosamine oxidase 50 U / ml peroxidase 30 U / ml TOOS 1 mM 4-AA 0.5 mM 0.05M citrate buffer (pH 5.5). ) 2. Measurement method 2 ml of the above reagent was added to 50 μl of NAG-containing test solution, and 3
The reaction was carried out at 7 ° C., and the absorbance was measured at a wavelength of 550 nm to obtain the color development rate. The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is apparent from FIGS. 1 and 2, the method of the present invention can accurately and easily perform NAG rate assay in a short time.

【図面の簡単な説明】[Brief description of drawings]

【図1】実施例1における反応曲線を示す。1 shows a reaction curve in Example 1. FIG.

【図2】実施例1における検量線を示す。FIG. 2 shows a calibration curve in Example 1.

Claims (1)

【特許請求の範囲】[Claims] 【請求項1】 下記式(I)で示されるN−アセチルヘ
キソサミニドに、N−アセチルヘキソダミニダーゼを含
有する試料を作用させ、生成するN−アセチルヘキソサ
ミンにN−アセチルヘキソサミンオキダーゼを作用さ
せ、生成する過酸化水素または消費される酸素を測定す
ることを特徴とするN−アセチルヘキソサミニダーゼ活
性定量法。 【化1】 (式中、Rはアルキル基、フェニル基、ナフチル基また
は置換基としてニトロ基を有するこれらの基を示す。)
1. A sample containing N-acetylhexosaminidase is allowed to act on N-acetylhexosaminide represented by the following formula (I), and the N-acetylhexosamine thus produced is subjected to N-acetylhexosamine oxidase. A method for quantifying N-acetylhexosaminidase activity, which comprises: reacting with hydrogen peroxide to measure hydrogen peroxide produced or oxygen consumed. [Chemical 1] (In the formula, R represents an alkyl group, a phenyl group, a naphthyl group, or a group having a nitro group as a substituent.)
JP34048292A 1992-12-21 1992-12-21 N-acetylhexosaminidase activity assay method Expired - Lifetime JPH0710237B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP34048292A JPH0710237B2 (en) 1992-12-21 1992-12-21 N-acetylhexosaminidase activity assay method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP34048292A JPH0710237B2 (en) 1992-12-21 1992-12-21 N-acetylhexosaminidase activity assay method

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
JP4265284A Division JPS60186300A (en) 1984-03-05 1984-03-05 Determination of n-acetylhexosamine

Publications (2)

Publication Number Publication Date
JPH05317094A true JPH05317094A (en) 1993-12-03
JPH0710237B2 JPH0710237B2 (en) 1995-02-08

Family

ID=18337389

Family Applications (1)

Application Number Title Priority Date Filing Date
JP34048292A Expired - Lifetime JPH0710237B2 (en) 1992-12-21 1992-12-21 N-acetylhexosaminidase activity assay method

Country Status (1)

Country Link
JP (1) JPH0710237B2 (en)

Also Published As

Publication number Publication date
JPH0710237B2 (en) 1995-02-08

Similar Documents

Publication Publication Date Title
Gochman et al. Application of a new peroxide indicator reaction to the specific, automated determination of glucose with glucose oxidase
Hemalatha et al. Enzymes in clinical medicine: an overview
JPS59125899A (en) Reagent for determination of direct-acting bilirubin by the enzymatic method and its determination procedure
JPH0698032B2 (en) Methods for measuring creatine or creatinine and reagents for these measurements
CN105203533B (en) A kind of high N acetyl β D UNAG detection reagents of sensitivity for analysis
CN107084938B (en) Alkaline phosphatase determination method based on chitosan-platinum mimic oxidase
US3677903A (en) Determination of uricase activity
JPH05317094A (en) Method for determining activity of n-acetylhexosaminidase
JP4217815B2 (en) Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose
US4816394A (en) Quantitative analysis of 3α-hydroxysteroid and reagent useful therefor
US4983512A (en) Reagent for determination of acid phosphatase
US5350678A (en) Method of differential assay for α-amylase isozymes and a kit for the same
RU2316002C2 (en) Method for detection of alkaline sphingomyelinase and kit useful for the same
EP0245528B1 (en) Quantitative analysis of 3 alpha-hydroxysteroid and reagent useful therefor
JP4544598B2 (en) Liquid reagent and storage method
JPS6296099A (en) Reagent for determination of acidic phosphatase activity
US20220017941A1 (en) Electroanalytical determination of leukocyte esterase
Cowell et al. Interference in an electrochemical detection system for peroxidase-linked reactions based on a fluoride ion-selective electrode.
JPH06237795A (en) Determination of 1,5-anhydroglucitol
JPH09168397A (en) Determination of sulfuric acid included bile acid and its kit
Felton et al. Design of glucose analysis suitable for incorporation into a microprocessor controlled regulator of blood glucose in the newborn
JP4073527B2 (en) Isozyme activity assay
JPH0223888A (en) Quantitative determination of lysozyme activity
JPH0549277B2 (en)
JPH0549276B2 (en)