JPH0542273B2 - - Google Patents
Info
- Publication number
- JPH0542273B2 JPH0542273B2 JP19246785A JP19246785A JPH0542273B2 JP H0542273 B2 JPH0542273 B2 JP H0542273B2 JP 19246785 A JP19246785 A JP 19246785A JP 19246785 A JP19246785 A JP 19246785A JP H0542273 B2 JPH0542273 B2 JP H0542273B2
- Authority
- JP
- Japan
- Prior art keywords
- acid
- hydroxy
- reagent
- acetyl
- substrate
- 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.)
- Expired - Lifetime
Links
- 239000003153 chemical reaction reagent Substances 0.000 claims description 30
- 239000000758 substrate Substances 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 20
- 102000007478 beta-N-Acetylhexosaminidases Human genes 0.000 claims description 8
- 108010085377 beta-N-Acetylhexosaminidases Proteins 0.000 claims description 8
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 5
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- 125000005843 halogen group Chemical group 0.000 claims description 4
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical group CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 claims description 3
- 229910052783 alkali metal Inorganic materials 0.000 claims description 3
- 150000001340 alkali metals Chemical class 0.000 claims description 3
- 239000002585 base Substances 0.000 claims description 3
- 125000002843 carboxylic acid group Chemical group 0.000 claims description 3
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 3
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 3
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- -1 2-nitro-4-chloro-5-hydroxybenzenesulfonic acid Chemical compound 0.000 description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 238000000034 method Methods 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000011088 calibration curve Methods 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000007979 citrate buffer Substances 0.000 description 6
- 239000012085 test solution Substances 0.000 description 6
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 4
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 4
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical group OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 3
- 210000001124 body fluid Anatomy 0.000 description 3
- 239000010839 body fluid Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004040 coloring Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-BKJPEWSUSA-N N-acetyl-D-hexosamine Chemical compound CC(=O)NC1C(O)O[C@H](CO)C(O)C1O OVRNDRQMDRJTHS-BKJPEWSUSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000001491 aromatic compounds Chemical class 0.000 description 2
- 230000000850 deacetylating effect Effects 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- KTKATRLKMZQBCL-UHFFFAOYSA-N 2-chloro-3-hydroxy-6-nitrobenzenesulfonic acid Chemical compound OC1=CC=C([N+]([O-])=O)C(S(O)(=O)=O)=C1Cl KTKATRLKMZQBCL-UHFFFAOYSA-N 0.000 description 1
- SLAMLWHELXOEJZ-UHFFFAOYSA-N 2-nitrobenzoic acid Chemical compound OC(=O)C1=CC=CC=C1[N+]([O-])=O SLAMLWHELXOEJZ-UHFFFAOYSA-N 0.000 description 1
- GHJLCCLHTCSRCE-UHFFFAOYSA-N 3-bromo-2-hydroxy-5-nitrobenzoic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC(Br)=C1O GHJLCCLHTCSRCE-UHFFFAOYSA-N 0.000 description 1
- NKALRCUMHHXJRN-UHFFFAOYSA-N 3-chloro-2-hydroxy-5-nitrobenzoic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC(Cl)=C1O NKALRCUMHHXJRN-UHFFFAOYSA-N 0.000 description 1
- YXRDRTMMQXNDIW-UHFFFAOYSA-N 4-bromo-5-hydroxy-2-nitrobenzoic acid Chemical compound OC(=O)c1cc(O)c(Br)cc1[N+]([O-])=O YXRDRTMMQXNDIW-UHFFFAOYSA-N 0.000 description 1
- QCHIKMGLJXCQCR-UHFFFAOYSA-N 4-chloro-5-hydroxy-2-nitrobenzoic acid Chemical compound OC(=O)C1=CC(O)=C(Cl)C=C1[N+]([O-])=O QCHIKMGLJXCQCR-UHFFFAOYSA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- BWMLOHZXNRULPK-UHFFFAOYSA-N C1=CC(=C(C(=C1[N+](=O)[O-])C(=O)O)Cl)O Chemical compound C1=CC(=C(C(=C1[N+](=O)[O-])C(=O)O)Cl)O BWMLOHZXNRULPK-UHFFFAOYSA-N 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 206010018366 Glomerulonephritis acute Diseases 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- YLZXCBDYLUZTPV-UHFFFAOYSA-N OC(=O)c1c(Br)c(O)ccc1[N+]([O-])=O Chemical compound OC(=O)c1c(Br)c(O)ccc1[N+]([O-])=O YLZXCBDYLUZTPV-UHFFFAOYSA-N 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 101100020289 Xenopus laevis koza gene Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical class OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 230000002140 halogenating effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明はβ−Ｎ−アセチル−Ｄ−ヘキソサミニ
ダーゼ活性測定試薬に関するものである。体液中
のβ−Ｎ−アセチル−Ｄ−ヘキソサミニダーゼ活
性の測定は、腎移植後の拒絶反応の早期診断、急
性腎不全、糸球体腎炎等の各種腎疾患の診断及び
経過観察、薬物の腎毒性等に有用な情報を与える
ものとして臨床的意義が高い。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) 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 is useful for early diagnosis of rejection after kidney transplantation, diagnosis and follow-up of various renal diseases such as acute renal failure and glomerulonephritis, and drug administration. It has great clinical significance as it provides useful information on nephrotoxicity, etc.
（従来の技術）
従来、β−Ｎ−アセチル−Ｄ−ヘキソサミニダ
ーゼ（以下NAGと称する）活性は、Ｎ−アセチ
ル−Ｄ−グルコサンの還元末端にｐ−ニトロフエ
ノールを結合させた基質を用いてNAGを作用さ
せ、遊離してくるｐ−ニトロフエノールをアルカ
リ性下で比色する方法が一般的である（Metods
Engymol.、28、702（1972））。(Prior art) Conventionally, β-N-acetyl-D-hexosaminidase (hereinafter referred to as NAG) activity has been developed using a substrate in which p-nitrophenol is bound to the reducing end of N-acetyl-D-glucosane. A common method is to colorimetrically measure the liberated p-nitrophenol under alkaline conditions (Metods
Engymol., 28 , 702 (1972)).
ところがこ方法では目的とする酸素NAGの至
適PH（PH４〜5.5）と発色用であるｐ−ニトロフ
エノールの発色PH（PH９以上）とが異なる為に
NAG活性を測定する為には酵素反応と発色反応
を別々に行なう必要があり、その為に試薬数及び
操作ステツ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
（レートアツセイ）法が出来ない欠点がある。 However, in this method, the optimum pH of the target oxygen 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.
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（特開発54−
60997号）、Ｎ−アセチルグルコサミンにｍ−クレ
ゾールスルホフタレインを結合させた基質を用い
る方法（Clin.Chem.29、1713（1983））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。 In addition, a method using a substrate in which phenol is bound to N-acetylglucosamine (Special Development 54-
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.
（発明の解決しようとする問題点）
本発明の目的は定量性に優れたNAGのレート
アツセイが可能となるNAG活性測定試薬を提供
することである。(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.
（問題点を解決する為の手段）
本発明者らは、上記目的を達成するために種々
鋭意検討したところ、一般式〔〕で示される基
質を用いることにより体液中のNAG活性を短時
間に正確簡単にレートアツセイ出来ることを見い
出し本発明に到達した。(Means for Solving the Problems) In order to achieve the above object, the present inventors conducted various intensive studies and found that by using a substrate represented by the general formula [], NAG activity in body fluids can be reduced in a short time. We have discovered that rate assay can be done accurately and easily, and have arrived at the present invention.
すなわち本発明は基質として下記一般式〔〕
で示される化合物を含有することを特徴とするβ
−Ｎ−アセチル−Ｄ−ヘキソサミニダーゼ活性測
定試薬である。 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.
（式中、Ａは還元性末端でβ−結合しているＮ−
アセチルグルコサミン又はＮ−アセチルガラクト
サミン残基である。Ｘはニトロ基を示す。R1〜
R4のうち、少なくとも１つはハロゲン原子を示
し、かつR1〜R4のうち、少なくとも１つはスル
ホン酸基又はカルボン酸基又はこれらのアルカリ
金属塩基を示し、他の基は水素原子を示す。）
本発明に用いる基質としては一般式〔〕で示
される化合物、すなわちＮ−アセチルグルコサミ
ン又はＮ−アセチルガラクトサミンがその還元性
末端で置換芳香族基とβ−結合したものである。
置換芳香族基とは解裂したアグリコンとして基質
とは異なつたスペクトル吸収を示す置換芳香族基
である。解裂したアグリコンとは具体的には次の
一般式
（Ｘ、R1〜R4は前記のものと同じ）で示される
フエノール誘導体である。 (In the formula, A is β-bonded N-
It is an acetylglucosamine or N-acetylgalactosamine residue. X represents a nitro group. R1〜
At least one of R 4 represents a halogen atom, at least one of R 1 to R 4 represents a sulfonic acid group or a carboxylic acid group, or an alkali metal base thereof, and the other groups represent a hydrogen atom. show. ) The substrate used in the present invention is a compound represented by the general formula [ ], that is, N-acetylglucosamine or N-acetylgalactosamine, which is β-bonded with a substituted aromatic group at its reducing end.
A substituted aromatic group is a substituted aromatic group that exhibits a different spectral absorption from the substrate as a cleaved aglycone. Specifically, the cleaved aglycon has the following general formula: It is a phenol derivative represented by (X, R 1 to R 4 are the same as above).
例えば２，４−ジクロロ−３−ヒドロキシ−６
−ニトロンベンゼンスルホン酸、２−クロロ−３
−ヒドロキシ−６−ニトロベンゼンスルホン酸、
２−ニトロ−４−クロロ−５−ヒドロキシベンゼ
ンスルホン酸、２，４，５−トリクロロ−３−ヒ
ドロキシ−６−ニトロベンゼンスルホン酸、２，
４−ジブロモ−３−ヒドロキシ−６−ニトロベン
ゼンスルホン酸、２−ブロモ−３−ヒドロキシ−
６−ニトロベンゼンスルホン酸、２−ニトロ−４
−ブロモ−５−ヒドロキシベンゼンスルホン酸、
２，４，５−トリブロモ−３−ヒドロキシ−６−
ニトロベンゼンスルホン酸、２，４−ジヨード−
３−ヒドロキシ−６−ニトロベンゼンスルホン
酸、２−ヨード−３−ヒドロキシ−６−ニトロベ
ンゼンスルホン酸、２−ニトロ−４−ヨード−５
−ヒドロキシベンゼンスルホン酸、２，４，５−
トリヨード−３−ヒドロキシ−６−ニトロベンゼ
ンスルホン酸、２−ヒドロキシ−３−クロロ−５
−ニトロベンゼンスルホン酸、３，４−ジクロロ
−２−ヒドロキシ−５−ニトロベンゼンスルホン
酸、３，４，６−トリクロロ−２−ヒドロキシ−
５−ニトロベンゼンスルホン酸、２−ヒドロキシ
−３−ブロモ−５−ニトロベンゼンスルホン酸、
３，４−ジブロモ−２−ヒドロキシ−５−ニトロ
ベンゼンスルホン酸、３，４，６−トリブロモ−
２−ヒドロキシ−５−ニトロベンゼンスルホン
酸、２−ヒドロキシ−３−ヨード−５−ニトロベ
ンゼンスルホン酸、３，４−ジヨード−２−ヒド
ロキシ−５−ニトロベンゼンスルホン酸、３，
４，６−トリヨード−２−ヒドロキシ−５−ニト
ロベンゼンスルホン酸、２，４−ジクロロ−３−
ヒドロキシ−６−ニトロベンゼンカルボン酸、２
−クロロ−３−ヒドロキシ−６−ニトロベンゼン
カルボン酸、２−ニトロ−４−クロロ−５−ヒド
ロキシベンゼンカルボン酸、２，４，５−トリク
ロロ−３−ヒドロキシ−６−ニトロベンゼンカル
ボン酸、２，４−ジブロモ−３−ヒドロキシ−６
−ニトロベンゼンカルボン酸、２−ブロモ−３−
ヒドロキシ−６−ニトロベンゼンカルボン酸、２
−ニトロ−４−ブロモ−５−ヒドロキシベンゼン
カルボン酸、２，４，５−トリブロモ−３−ヒド
ロキシ−６−ニトロベンゼンカルボン酸、２，４
−ジヨード−３−ヒドロキシ−６−ニトロベンゼ
ンカルボン酸、２−ヨード−３−ヒドロキシ−６
−ニトロベンゼンカルボン酸、２−ニトロ−４−
ヨード−５−ヒドロキシベンゼンカルボン酸、
２，４，５−トリヨード−３−ヒドロキシ−６−
ニトロベンゼンカボン酸、２−ヒドロキシ−３−
クロロ−５−ニトロベンゼンカルボン酸、３，４
−ジクロロ−２−ヒドロキシ−５−ニトロベンゼ
ンカルボン酸、３，４，６−トリクロロ−２−ヒ
ドロキシ−５−ニトベンゼンカルボン酸、２−ヒ
ドロキシ−３−ブロモ−５−ニトロベンゼンカル
ボン酸、３，４−ジブロモ−２−ヒドロキシ−５
−ニトロベンゼンカルボン酸、３，４，６−トリ
ブロモ−２−ヒドロキシ−５−ニトロベンゼンカ
ルボン酸、３，４−ジブロモ−２−ヒドロキシ−
５−ニトロベンゼンカルボン酸、３，４，６−ト
リブロモ−２−ヒドロキシ−５−ニトロベンゼン
カルボン酸、２−ヒドロキシ３−ヨード−５−ニ
トロベンゼンカルボン酸、３，４−ジヨード−２
−ヒドロキシ−５−ニトロベンゼンカルボン酸、
３，４，６−トリヨード−２−ヒドロキシ−５−
ニトロベンゼンカルボン酸またはそれらのベンゼ
ンカルボン酸とこれらのNa塩またはＫ塩等があ
げられる。 For example 2,4-dichloro-3-hydroxy-6
-nitronebenzenesulfonic acid, 2-chloro-3
-hydroxy-6-nitrobenzenesulfonic acid,
2-nitro-4-chloro-5-hydroxybenzenesulfonic acid, 2,4,5-trichloro-3-hydroxy-6-nitrobenzenesulfonic acid, 2,
4-dibromo-3-hydroxy-6-nitrobenzenesulfonic acid, 2-bromo-3-hydroxy-
6-nitrobenzenesulfonic acid, 2-nitro-4
-bromo-5-hydroxybenzenesulfonic acid,
2,4,5-tribromo-3-hydroxy-6-
Nitrobenzenesulfonic acid, 2,4-diiodo-
3-hydroxy-6-nitrobenzenesulfonic acid, 2-iodo-3-hydroxy-6-nitrobenzenesulfonic acid, 2-nitro-4-iodo-5
-Hydroxybenzenesulfonic acid, 2,4,5-
Triiodo-3-hydroxy-6-nitrobenzenesulfonic acid, 2-hydroxy-3-chloro-5
-Nitrobenzenesulfonic acid, 3,4-dichloro-2-hydroxy-5-nitrobenzenesulfonic acid, 3,4,6-trichloro-2-hydroxy-
5-nitrobenzenesulfonic acid, 2-hydroxy-3-bromo-5-nitrobenzenesulfonic acid,
3,4-dibromo-2-hydroxy-5-nitrobenzenesulfonic acid, 3,4,6-tribromo-
2-Hydroxy-5-nitrobenzenesulfonic acid, 2-hydroxy-3-iodo-5-nitrobenzenesulfonic acid, 3,4-diiodo-2-hydroxy-5-nitrobenzenesulfonic acid, 3,
4,6-triiodo-2-hydroxy-5-nitrobenzenesulfonic acid, 2,4-dichloro-3-
Hydroxy-6-nitrobenzenecarboxylic acid, 2
-Chloro-3-hydroxy-6-nitrobenzenecarboxylic acid, 2-nitro-4-chloro-5-hydroxybenzenecarboxylic acid, 2,4,5-trichloro-3-hydroxy-6-nitrobenzenecarboxylic acid, 2,4- dibromo-3-hydroxy-6
-nitrobenzenecarboxylic acid, 2-bromo-3-
Hydroxy-6-nitrobenzenecarboxylic acid, 2
-Nitro-4-bromo-5-hydroxybenzenecarboxylic acid, 2,4,5-tribromo-3-hydroxy-6-nitrobenzenecarboxylic acid, 2,4
-diiodo-3-hydroxy-6-nitrobenzenecarboxylic acid, 2-iodo-3-hydroxy-6
-nitrobenzenecarboxylic acid, 2-nitro-4-
iodo-5-hydroxybenzenecarboxylic acid,
2,4,5-triiodo-3-hydroxy-6-
Nitrobenzenecarboxylic acid, 2-hydroxy-3-
Chloro-5-nitrobenzenecarboxylic acid, 3,4
-dichloro-2-hydroxy-5-nitrobenzenecarboxylic acid, 3,4,6-trichloro-2-hydroxy-5-nitrobenzenecarboxylic acid, 2-hydroxy-3-bromo-5-nitrobenzenecarboxylic acid, 3,4- dibromo-2-hydroxy-5
-Nitrobenzenecarboxylic acid, 3,4,6-tribromo-2-hydroxy-5-nitrobenzenecarboxylic acid, 3,4-dibromo-2-hydroxy-
5-nitrobenzenecarboxylic acid, 3,4,6-tribromo-2-hydroxy-5-nitrobenzenecarboxylic acid, 2-hydroxy 3-iodo-5-nitrobenzenecarboxylic acid, 3,4-diiodo-2
-hydroxy-5-nitrobenzenecarboxylic acid,
3,4,6-triiodo-2-hydroxy-5-
Examples include nitrobenzenecarboxylic acid and their benzenecarboxylic acids and their Na salts or K salts.
これら基質の合成方法はＮ−アセチルヘキソサ
ミンをアセチル化し、このアセチル化されたＮ−
アセチルヘキソサミンと置換芳香族化合物、アグ
リコンを結合させた後、脱アセチルすることによ
り合成するか（実験化学講座第24巻第304頁、
1958年）、又はアセチル化されたＮ−アセチルヘ
キソサミンをハロゲン化し、次いでそのハロゲン
化物と置換芳香族化合物、アグリコンをエーテル
結合させたあと、脱アセチルすることにより合成
することが出来る（Methods in Carbohydrate
Chemistry 、第334頁）。 The method for synthesizing these substrates is to acetylate N-acetylhexosamine, and this acetylated N-
Is it synthesized by combining acetylhexosamine with a substituted aromatic compound or aglycone and then deacetylating it? (Jikken Kagaku Koza Vol. 24, p. 304,
(1958), or by halogenating acetylated N-acetylhexosamine, then ether bonding the halide with a substituted aromatic compound or aglycone, and then deacetylating it (Methods in Carbohydrate).
Chemistry, p. 334).
基質の置換芳香族基においてR1〜R4がハロゲ
ン原子のみで置換されている場合には、水に対す
る溶解性が悪く、基質必要量の溶解が不可能であ
る。 When R 1 to R 4 in the substituted aromatic group of the substrate are substituted only with halogen atoms, the solubility in water is poor and it is impossible to dissolve the required amount of the substrate.
本発明のβ−Ｎ−アセチル−Ｄ−ヘキソサミニ
ダーゼ活性測定試薬は、ハロゲン置換のニトロフ
エノールに−SO3H、−COOH、−SO3Na、−
COONa、−SO3K、−COOK等を置換し、水溶性
に優れた基質を使用する。 The reagent for measuring β-N-acetyl-D-hexosaminidase activity of the present invention is a halogen-substituted nitrophenol containing -SO 3 H, -COOH, -SO 3 Na, -
Substitute COONa, -SO 3 K, -COOK, etc. and use a substrate with excellent water solubility.
試薬のPHは体液中のNAGの至適PHであるPH4.0
〜5.5を保つ緩衝液であれば、いかなるものでも
良い。例えばクエン酸緩衝液やその他有機酸緩衝
液、例えば酢酸、コハク酸、フタル酸等の緩衝液
があげられる。 The pH of the reagent is PH4.0, which is the optimum pH for NAG in body fluids.
Any buffer solution that maintains ~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.
基質濃度としては特に制限がないが、好ましく
は最大のNAGの酵素活性を示す濃度が適当であ
る。例えば１ｍＭ以上である。 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.
本発明の試薬は必要により界面活性剤、防腐
剤、塩化ナトリウム、シクロデキストリン、安定
化剤等を含有してもよい。 The reagent of the present invention may contain a surfactant, preservative, sodium chloride, cyclodextrin, stabilizer, etc., if necessary.
本発明のβ−Ｎ−アセチル−Ｄ−ヘキソサミニ
ダーゼ活性測定試薬を用いて、NAG活性を測定
する方法としては、試料を該試薬と反応させて生
成するアグリコン、フエノール誘導体の吸光度変
化を直接分光光度計を用いて比色定量する方法で
ある。 A method for measuring NAG activity using the reagent for measuring β-N-acetyl-D-hexosaminidase activity of the present invention is to directly measure the change in absorbance of aglycone and phenol derivatives produced by reacting a sample with the reagent. This is a method of colorimetric determination using a spectrophotometer.
（発明の効果）
本発明のβ−Ｎ−アセチル−Ｄ−ヘキソサミニ
ダーゼ活性測定試薬において、一般式〔〕で示
される化合物を基質として用いることにより、体
液中のβ−Ｎ−アセチル−Ｄ−ヘキソサミニダー
ゼ活性を短時間に正確、かつ簡単にレートアツセ
イすることができる。基質に結合したフエノール
誘導体がハロゲン原子、ニトロ基の他に、スルホ
ン酸基又はカルボン酸基あるいはこれらのアルカ
リ金属塩基を有することにより、基質自体の水溶
性が向上する。(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. When the phenol derivative bonded to the substrate has a sulfonic acid group, a carboxylic acid group, or an alkali metal base thereof in addition to a halogen atom and a nitro group, the water solubility of the substrate itself is improved.
（実施例） 以下、本発明を実施例により詳細に説明する。(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.
実施例 １
被検液中のNAG活性量を下記試薬を用いて下
記方法により測定した。Example 1 The amount of NAG activity in the test solution was measured using the following reagent and the following method.
１ 試薬
２−クロロ−４−ニトロ−６−スルホフエニル
−Ｎ−アセチル−β−Ｄ−グルコサミニド
２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
２ 測定方法
NAG含有被検液50μに上記試薬２ｍを
加えて37℃で反応させ、その吸光度を波長
400nｍで測定して発色速度を求めた。1 Reagent 2-chloro-4-nitro-6-sulfophenyl-N-acetyl-β-D-glucosaminide
2mM sodium chloride 200mM citrate buffer 0.05M PH4, 5 2 Measurement method Add 2m of the above reagent to 50μ of the NAG-containing test solution, react at 37°C, and measure the absorbance at the wavelength.
The color development rate was determined by measurement at 400 nm.
反応曲線を第１図に示す。検量線を第２図に
示す。第１図および第２図から明らかなよう
に、水溶性基質を用いた本発明の試薬では、短
時間に正確かつ簡単にレートアツセイすること
ができる。 The reaction curve is shown in FIG. The calibration curve is shown in Figure 2. As is clear from FIGS. 1 and 2, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
実施例 ２
被液中のNAG活性量を下記試薬を用いて実施
例１と同じ方法により測定した。Example 2 The amount of NAG activity in the liquid was measured in the same manner as in Example 1 using the following reagents.
１ 試薬
２，６−ジクロロ−４−ニトロ−５−スルホ
フエニル−Ｎ−アセチル−β−Ｄ−グルコサミ
ニド ２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
反応曲線を第３図に示す。検量線を第４図に示
す。第３図および第４図から明らかなように、水
溶性基質を用いた本発明の試薬では、短時間に正
確かつ簡単にレートアツセイすることができる。1 Reagents 2,6-dichloro-4-nitro-5-sulfophenyl-N-acetyl-β-D-glucosaminide 2mM Sodium chloride 200mM Citrate buffer 0.05M PH4,5 The reaction curve is shown in FIG. The calibration curve is shown in Figure 4. As is clear from FIGS. 3 and 4, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
実施例 ３
被検液中のNAG活性量を下記試薬を用いて実
施例１と同じ方法により測定した。Example 3 The amount of NAG activity in the test solution was measured in the same manner as in Example 1 using the following reagents.
１ 試薬
２−ブロモ−４−ニトロ−６−スルホフエニル
−Ｎ−アセチル−β−Ｄ−グルコサミニド
２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
反応曲線を第５図に示す。検量線を第６図に示
す。第５図および第６図から明らかなように、水
溶性基質を用いた本発明の試薬では、短時間に正
確かつ簡単にレートアツセイすることができる。1 Reagent 2-bromo-4-nitro-6-sulfophenyl-N-acetyl-β-D-glucosaminide
2mM sodium chloride 200mM citrate buffer 0.05M PH4,5 The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is clear from FIGS. 5 and 6, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
実施例 ４
被検液中のNAG活性量を下記試薬を用いて実
施例１と同じ方法により測定した。Example 4 The amount of NAG activity in the test solution was measured in the same manner as in Example 1 using the following reagents.
１ 試薬
２，６−ジブロモ−４−ニトロ−５−スルホフ
エニル−Ｎ−アセチル−β−Ｄ−グルコサミニ
ド ２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
反応曲線を第７図に示す。検量線を第８図に示
す。第７図および第８図から明らかなように、水
溶性基質を用いた本発明の試薬では、短時間に正
確かつ簡単にレートアツセイすることができる。1 Reagents 2,6-dibromo-4-nitro-5-sulfophenyl-N-acetyl-β-D-glucosaminide 2mM Sodium chloride 200mM Citrate buffer 0.05M PH4,5 The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is clear from FIGS. 7 and 8, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
実施例 ５
被検液中のNAG活性量を下記試薬を用いて実
施例１と同じ方法により測定した。Example 5 The amount of NAG activity in the test solution was measured in the same manner as in Example 1 using the following reagents.
１ 試薬
２，３，６−トリクロロ−４−ニトロ−５−
スルホフエニル−Ｎ−アセチル−β−Ｄ−グル
コサミニド ２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
反応曲線を第９図に示す。検量線を第１０図に
示す。第９図および第１０図から明らかなよう
に、水溶性基質を用いた本発明の試薬では、短時
間に正確かつ簡単にレートアツセイすることがで
きる。1 Reagent 2,3,6-trichloro-4-nitro-5-
Sulfophenyl-N-acetyl-β-D-glucosaminide 2mM Sodium chloride 200mM Citrate buffer 0.05M PH4,5 The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is clear from FIGS. 9 and 10, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
実施例 ６
被検液中のNAG活性量を下記試薬を用いて実
施例１と同じ方法により測定した。Example 6 The amount of NAG activity in the test solution was measured in the same manner as in Example 1 using the following reagents.
１ 試薬
２，３，６−トリブロモ−４−ニトロ−５−ス
ルホフエニル−Ｎ−アセチル−β−Ｄ−グルコ
サミド ２ｍＭ
塩化ナトリウム 200ｍＭ
クエン酸緩衝液 0.05M
PH４、５
反応曲線を第１１図に示す。検量線を第１２図
に示す。第１１図および第１２図から明らかなよ
うに、水溶性基質を用いた本発明の試薬では、短
時間に正確かつ簡単にレートアツセイすることが
できる。1 Reagent 2,3,6-tribromo-4-nitro-5-sulfophenyl-N-acetyl-β-D-glucosamide 2mM Sodium chloride 200mM Citrate buffer 0.05M PH4,5 The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is clear from FIGS. 11 and 12, the reagent of the present invention using a water-soluble substrate allows rate assay to be performed accurately and easily in a short period of time.
第１図、第３図、第５図、第７図、第９図およ
び第１１図は本発明実施例の反応曲線を示す。第
２図、第４図、第６図、第８図、第１０図および
第１２図は本発明実施例の検量線を示す。
FIGS. 1, 3, 5, 7, 9, and 11 show reaction curves of examples of the present invention. FIG. 2, FIG. 4, FIG. 6, FIG. 8, FIG. 10, and FIG. 12 show calibration curves of Examples of the present invention.
Claims (1)
物を含有することを特徴とするβ−Ｎ−アセチル
−Ｄ−ヘキソサミニダーゼ活性測定試薬。 （式中、Ａは還元性末端でβ−結合しているＮ−
アセチルグルコサミン又はＮ−アセチルガラクト
サミン残基である。Ｘはニトロ基を示す。R1〜
R4のうち、少なくとも１つはハロゲン原子を示
し、かつR1〜R4のうち、少なくとも１つはスル
ホン酸基又はカルボン酸基又はこれらのアルカリ
金属塩基を示し、他の基は水素原子を示す。）[Scope of Claims] 1. A reagent for measuring β-N-acetyl-D-hexosaminidase activity, which contains a compound represented by the following general formula [] as a substrate. (In the formula, A is β-bonded N-
It is an acetylglucosamine or N-acetylgalactosamine residue. X represents a nitro group. R1 ~
At least one of R 4 represents a halogen atom, at least one of R 1 to R 4 represents a sulfonic acid group or a carboxylic acid group, or an alkali metal base thereof, and the other groups represent a hydrogen atom. show. )
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19246785A JPS6252000A (en) | 1985-08-30 | 1985-08-30 | Reagent for measuring beta-n-acetyl-d-hexosaminidase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19246785A JPS6252000A (en) | 1985-08-30 | 1985-08-30 | Reagent for measuring beta-n-acetyl-d-hexosaminidase activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6252000A JPS6252000A (en) | 1987-03-06 |
| JPH0542273B2 true JPH0542273B2 (en) | 1993-06-28 |
Family
ID=16291778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP19246785A Granted JPS6252000A (en) | 1985-08-30 | 1985-08-30 | Reagent for measuring beta-n-acetyl-d-hexosaminidase activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6252000A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113252587B (en) * | 2021-05-11 | 2022-01-07 | 上海奥普生物医药股份有限公司 | A kind of NAG detection kit and its application |
-
1985
- 1985-08-30 JP JP19246785A patent/JPS6252000A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6252000A (en) | 1987-03-06 |
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