JPH01309687A - Stabilized n-acetyl-beta-d-glucosaminidase and controlling drug preparation containing the same enzyme - Google Patents
Stabilized n-acetyl-beta-d-glucosaminidase and controlling drug preparation containing the same enzymeInfo
- Publication number
- JPH01309687A JPH01309687A JP13804388A JP13804388A JPH01309687A JP H01309687 A JPH01309687 A JP H01309687A JP 13804388 A JP13804388 A JP 13804388A JP 13804388 A JP13804388 A JP 13804388A JP H01309687 A JPH01309687 A JP H01309687A
- Authority
- JP
- Japan
- Prior art keywords
- nag
- acetyl
- glucosaminidase
- stabilized
- ammonium salt
- 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.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 108010055851 Acetylglucosaminidase Proteins 0.000 title claims abstract description 11
- 102100030122 Protein O-GlcNAcase Human genes 0.000 title claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 title abstract description 15
- 102000004190 Enzymes Human genes 0.000 title abstract description 15
- 229940079593 drug Drugs 0.000 title abstract description 3
- 239000003814 drug Substances 0.000 title abstract description 3
- 150000003863 ammonium salts Chemical class 0.000 claims abstract description 24
- -1 dialdehyde compound Chemical class 0.000 claims abstract description 23
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims description 20
- 238000004132 cross linking Methods 0.000 abstract description 8
- 238000000108 ultra-filtration Methods 0.000 abstract description 6
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 238000000502 dialysis Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 14
- 239000000203 mixture Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 238000009472 formulation Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 7
- 239000003431 cross linking reagent Substances 0.000 description 6
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 208000017169 kidney disease Diseases 0.000 description 4
- 239000012266 salt solution Substances 0.000 description 4
- OBHRVMZSZIDDEK-UHFFFAOYSA-N urobilinogen Chemical compound CCC1=C(C)C(=O)NC1CC1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(CC3C(=C(CC)C(=O)N3)C)N2)CCC(O)=O)N1 OBHRVMZSZIDDEK-UHFFFAOYSA-N 0.000 description 4
- 108010093096 Immobilized Enzymes Proteins 0.000 description 3
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 3
- 230000009849 deactivation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical class N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 239000002262 Schiff base Substances 0.000 description 2
- 150000004753 Schiff bases Chemical class 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 150000002301 glucosamine derivatives Chemical class 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 229940045136 urea Drugs 0.000 description 2
- 229940116269 uric acid Drugs 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- OMRLTNCLYHKQCK-DHGKCCLASA-N 4-nitrophenyl N-acetyl-beta-D-glucosaminide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 OMRLTNCLYHKQCK-DHGKCCLASA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 206010018366 Glomerulonephritis acute Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical group O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- PCSMJKASWLYICJ-UHFFFAOYSA-N Succinic aldehyde Chemical compound O=CCCC=O PCSMJKASWLYICJ-UHFFFAOYSA-N 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000000909 electrodialysis Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- QCTHLCFVVACBSA-JVNHZCFISA-N n-[(2s,3r,4r,5s,6r)-4,5-dihydroxy-6-(hydroxymethyl)-2-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(C(C)=CC(=O)O2)C2=C1 QCTHLCFVVACBSA-JVNHZCFISA-N 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、安定化されたＮ−アセチル−β−Ｄ−グルコ
サミニダーゼ及びそれを含有し、臨床検査分野で対照（
コントロール）として使用されるコントロール製剤に関
する。Detailed Description of the Invention [Industrial Field of Application] The present invention contains a stabilized N-acetyl-β-D-glucosaminidase and the use of a control (
Concerning the control formulation used as a control).
［従来の技術及び発明が解決しようとする課題］Ｎ−ア
セチルーβ−Ｄ−グルコサミニダーゼ（以下、ＮＡＧと
称する）は、野原細管上皮に多く含まれるリゾソーム中
の酵素の一種で、ムコ多糖類や糖蛋白の分解に関与する
。各種腎疾患や腎臓の術後においては尿中のＮＡＧが上
昇し、また糖尿病においては尿中のみならず血清中のＮ
ＡＧも上昇することが知られており、各種腎疾患の診断
に有用な指標となる酵素である。従って、ＮＡＧ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
性試験等において有用な情報が得らｉ、臨床的意義が大
きいことがら、ＮＡＧの測定が重要視されている。[Prior art and problems to be solved by the invention] N-acetyl-β-D-glucosaminidase (hereinafter referred to as NAG) is a type of enzyme found in lysosomes, which is abundant in field tubule epithelium. Involved in protein breakdown. NAG in the urine increases in various renal diseases and after kidney surgery, and in diabetes, NAG increases not only in the urine but also in the serum.
AG is also known to be elevated, and is an enzyme useful as an indicator for the diagnosis of various kidney diseases. Therefore, measurement of NAG can provide useful information in early diagnosis of rejection after kidney transplantation, diagnosis and follow-up of various renal diseases such as acute renal failure and glomerulonephritis, and nephrotoxicity testing of drugs. Measurement of NAG is considered important because of its great clinical significance.
上記ＮＡＧの活性測定方法としては、例えば、ｐ−ニト
ロフェニル−Ｎ−アセチル−β−Ｄ−グルコサミナイド
［Ｂｉｏｃｈｅｍｌｃａｌ Ｐｒｅｐａｒａｔｉｏｎｓ
。As a method for measuring the activity of NAG, for example, p-nitrophenyl-N-acetyl-β-D-glucosaminide [Biochemical Preparations
.
Ｖｏｌ、１０．１１８ （１９６３）コ、４−メチルウ
ンベリフェリル−Ｎ−アセチル−β−Ｄ−グルコサミナ
イド［Ｃｌ１ｎｉｅａＣｈｆＩａｉｃａ Ａｃｔａ、
Ｖｏｌ、６９ （１）、 ８５−９１ （１９７６）コ
等を用いる方法、特開昭５８−９９４号公報、特開昭８
１−１１２０９２号公報、特開昭８１−１７７９９９号
公報等に開示されるグルコサミン誘導体を使用する方法
等が知られているが、上記方法におけるＮＡＧの活性測
定に際しては、既知量のＮＡＧを含有するコントロール
の測定も併せて行い、測定精度を高め信頼性の向上を図
ることにより、精度管理を行っている。臨床検査におい
ては、一般に多数の検査対象項目を同時に測定すること
が行われ、それら各検査対象についてもコントロールの
測定がなされることから、通常、検査対象に対応する多
数の成分を含有した製剤がコントロールとして繁用され
る。このようなコントロール製剤は、不安定な酵素、蛋
白等を含有するので、保存時の変質を防止し、調製当初
の活性を長期に亘って維持するため、通常、凍結乾燥製
剤の形態で市販され、用時に精製水等で希釈して使用さ
れる。Vol, 10.118 (1963) 4-Methylumbelliferyl-N-acetyl-β-D-glucosaminide [ClnieaChfIaica Acta,
Vol. 69 (1), 85-91 (1976), etc., JP-A-58-994, JP-A-8
Methods using glucosamine derivatives disclosed in JP-A No. 1-112092, JP-A-81-177999, etc. are known, but when measuring the activity of NAG in the above method, it is necessary to use glucosamine derivatives containing a known amount of NAG. Accuracy management is carried out by also conducting control measurements to improve measurement precision and reliability. In clinical tests, a large number of test items are generally measured simultaneously, and control measurements are also performed for each of these test items, so preparations containing many components corresponding to the test items are usually Often used as a control. Since such control preparations contain unstable enzymes, proteins, etc., they are usually commercially available in the form of lyophilized preparations in order to prevent deterioration during storage and maintain the original activity for a long period of time. , diluted with purified water etc. before use.
しかしながら、酵素であるＮＡＧは保存安定性が悪く、
凍結乾燥しても著しく失活するので、長期に亘って保存
できるＮＡＧは知られておらず、未だ安定性に優れたＮ
ＡＧ含有コントロール製剤は提供されていない。そのた
め、従来のＮＡＧ含有コントロール製剤をコントロール
として用いた場合、時間経過に伴う酵素活性の低下によ
り、ＮＡＧの検出精度の信頼性に欠け、ひいては腎疾患
の有無等について誤認を生ずるおそれがある。また検査
のたびごとに、ＮＡＧを溶解してコントロールを調製す
る必要があり、作業が煩雑化するという問題がある。However, the enzyme NAG has poor storage stability;
Since NAG is significantly inactivated even after freeze-drying, there is no known NAG that can be stored for a long period of time.
No AG-containing control formulation is provided. Therefore, when a conventional NAG-containing control preparation is used as a control, there is a risk that the detection accuracy of NAG will be unreliable due to a decrease in enzyme activity over time, leading to misunderstandings regarding the presence or absence of renal disease. Furthermore, it is necessary to prepare a control by dissolving NAG each time a test is performed, which poses a problem of complicating the work.
本発明者らは、ＮＡＧの安定化について鋭意研究の結果
、ジアルデヒド化合物によるＮＡＧの架橋に際してアン
モニウム塩を存在させることによりＮＡＧが極めて安定
化することを見い出し、本発明を完成した。即ち、本発
明は、保存性に優れ且つ長期に亘り酵素活性を維持でき
る安定化されたＮＡＧ及びそれを含有する臨床検査用の
コントロール製剤を提供することを目的とする。As a result of extensive research into the stabilization of NAG, the present inventors have found that NAG is extremely stabilized by the presence of an ammonium salt during crosslinking of NAG with a dialdehyde compound, and has completed the present invention. That is, an object of the present invention is to provide a stabilized NAG that has excellent storage stability and can maintain enzyme activity over a long period of time, and a control preparation for clinical testing containing the same.
なお、酵素を安定化する方法として、グルタルアルデヒ
ド等の架橋剤により酵素を架橋することが知られている
。しかしながら、ＮＡＧをグルタルアルデヒド等の架橋
剤により架橋するとＮＡＧの酵素活性が著しく低下する
ため、上記の方法をＮＡＧに適用することは困難である
。Note that as a method for stabilizing enzymes, it is known to crosslink enzymes with a crosslinking agent such as glutaraldehyde. However, when NAG is crosslinked with a crosslinking agent such as glutaraldehyde, the enzymatic activity of NAG is significantly reduced, so it is difficult to apply the above method to NAG.
また、固定化酵素の分野においては、不溶性担体上に、
グルタルアルデヒド等の架橋剤を用いて酵素を固定化し
て安定化する方法が提案されている（特開昭５９−１３
０１８４号公報等参照）。しかし、コントロール製剤は
液状で使用されるので、不溶性担体を用いた固定化酵素
は適切でない。In addition, in the field of immobilized enzymes, on an insoluble carrier,
A method has been proposed in which enzymes are immobilized and stabilized using a cross-linking agent such as glutaraldehyde (Japanese Patent Laid-Open No. 1986-13).
(See Publication No. 0184, etc.). However, since the control formulation is used in liquid form, immobilized enzymes using insoluble carriers are not suitable.
［課題を解決するための手段］
上記の課題を解決すべくなされた、本発明にかかるＮＡ
Ｇは、ＮＡＧをアンモニウム塩の溶液に溶解し、ジアル
デヒド化合物を添加して架橋させた後、過剰のアンモニ
ウム塩及びジアルデヒド化合物を除去し、凍結乾燥する
ことにより得られたことを特徴とする安定化されたＮＡ
Ｇである。[Means for solving the problems] NA according to the present invention, which was made to solve the above problems
G is characterized by being obtained by dissolving NAG in an ammonium salt solution, adding a dialdehyde compound to crosslink it, removing excess ammonium salt and dialdehyde compound, and freeze-drying. stabilized NA
It is G.
また、本発明にかかるコントロール製剤は、上記のＮＡ
Ｇを少なくとも含有することを特徴とするものである。In addition, the control formulation according to the present invention has the above-mentioned NA
It is characterized by containing at least G.
なお、上記コントロール製剤は、必要に応じて他の検査
対象に対応する成分、例えば、グルコース、ビリルビン
、アルブミン、ウロビリノーゲン、尿素、尿酸、クレア
チニン、へ′モグロビン、アミラーゼ、亜硝酸塩等を適
宜量含有していてもよい。上記のような多数の成分を含
有するコントロール製剤は、多数の検査対象のコントロ
ールとして使用できる利点を有する。The above control preparation may contain appropriate amounts of components corresponding to other test targets, such as glucose, bilirubin, albumin, urobilinogen, urea, uric acid, creatinine, hemoglobin, amylase, nitrite, etc., as necessary. You can leave it there. A control preparation containing a large number of components as described above has the advantage that it can be used as a control for a large number of test objects.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明の安定化されたＮＡＧは、アンモニウム塩の存在
下、ＮＡＧがジアルデヒド化合物により架橋されており
凍結乾燥品の形態を呈する。The stabilized NAG of the present invention is obtained by crosslinking NAG with a dialdehyde compound in the presence of an ammonium salt, and takes the form of a lyophilized product.
上記アンモニウム塩としては、例えば、硫酸アンモニウ
ム、塩化アンモニウム、硝酸アンモニウム、リン酸アン
モニウム、酢酸アンモニウム等の種々のアンモニウム塩
が例示される。上記アンモニウム塩のうち、ＮＡＧの失
活を防止する上で、硫酸アンモニウムが特に好ましい。Examples of the ammonium salt include various ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, and ammonium acetate. Among the above ammonium salts, ammonium sulfate is particularly preferred in terms of preventing deactivation of NAG.
またジアルデヒド化合物としては、固定化酵素の分野に
おいて、酵素の架橋剤として用いられているジアルデヒ
ド化合物の何れも使用でき、例えば、グルタルアルデヒ
ド、スクシンジアルデヒド、アジピンアルデヒド等が例
示される。上記ジアルデヒド化合物のうち、ＮＡＧの失
活度の少ないことからグルタルアルデヒドが特に好まし
い。Further, as the dialdehyde compound, any dialdehyde compound used as a crosslinking agent for enzymes in the field of immobilized enzymes can be used, and examples thereof include glutaraldehyde, succindialdehyde, adipine aldehyde, and the like. Among the dialdehyde compounds mentioned above, glutaraldehyde is particularly preferred because it has a low degree of deactivation of NAG.
上記本発明のＮＡＧは、ＮＡＧを前記アンモニウム塩の
溶液に溶解し、ジアルデヒド化合物を添加して架橋させ
、次いで過剰のアンモニウム塩及びジアルデヒド化合物
を除去し、さらに凍結乾燥することにより得られる。The NAG of the present invention can be obtained by dissolving NAG in a solution of the ammonium salt, adding a dialdehyde compound to crosslink it, removing excess ammonium salt and dialdehyde compound, and further freeze-drying.
ＮＡＧを溶解するアンモニウム塩溶液中の塩濃度は、ア
ンモニウム塩の種類等に応じて適宜選択することができ
、２Ｍ以上、特に２．５〜３．５Ｍの水溶液が好ましい
。アンモニウム塩の濃度が２Ｍ未満であると、ＮＡＧが
失活するおそれがある。またＮＡＧは前記アンモニウム
塩の溶液に適宜の濃度、例えば１〜１０単位／１！程度
溶解させることができる。The salt concentration in the ammonium salt solution for dissolving NAG can be appropriately selected depending on the type of ammonium salt, etc., and an aqueous solution of 2M or more, particularly 2.5 to 3.5M is preferable. If the ammonium salt concentration is less than 2M, NAG may be deactivated. Further, NAG is added to the ammonium salt solution at an appropriate concentration, for example, 1 to 10 units/1! It can be dissolved to some extent.
ＮＡＧを前記アンモニウム塩の溶液に溶解した後、ジア
ルデヒド化合物を添加して架橋させる。After dissolving NAG in the ammonium salt solution, a dialdehyde compound is added to cause crosslinking.
この工程において、ジアルデヒド化合物は適宜の濃度で
使用できるが、反応液中のジアルデヒド化合物の濃度が
１〜１０ （Ｗ／Ｖ）％、好ましくは２〜６ （Ｗ／Ｖ
）％程度となるように調製するのがよい。In this step, the dialdehyde compound can be used at an appropriate concentration, but the concentration of the dialdehyde compound in the reaction solution is 1 to 10 (W/V)%, preferably 2 to 6 (W/V)%.
)%.
ジアルデヒド化合物が１ （Ｗ／Ｖ）５未満であるとＮ
ＡＧを安定化させることが困難であり、また１０（Ｗ／
Ｖ）％を越えるとＮＡＧの活性が低下したり、不溶化す
る場合があるので好ましくない。When the dialdehyde compound is less than 1 (W/V) 5, N
It is difficult to stabilize AG and 10 (W/
If it exceeds V)%, the activity of NAG may decrease or it may become insolubilized, which is not preferable.
また上記架橋工程は、ジアルデヒド化合物の種類等に応
じて適宜の温度で行なうことができ、反応時間は、反応
温度及び使用されるジアルデヒド化合物の反応性等によ
り適宜選択することができるが、ＮＡＧの酵素活性の低
下を抑制するため、低温、例えば２〜８℃で行なうのが
好ましく、通常５〜６０時間、好ましくは１２〜４８時
間程度反応させることにより行なわれる。この架橋工程
において、ジアルデヒド化合物は温和な架橋剤であり、
ＮＡＧと架橋的に結合してシッフ塩基を形成し、ＮＡＧ
の高次構造の変化を抑制し、ＮＡＧを安定化させる。Further, the above crosslinking step can be carried out at an appropriate temperature depending on the type of dialdehyde compound, etc., and the reaction time can be appropriately selected depending on the reaction temperature, reactivity of the dialdehyde compound used, etc. In order to suppress a decrease in the enzymatic activity of NAG, it is preferable to carry out the reaction at a low temperature, for example, 2 to 8°C, and the reaction is usually carried out for about 5 to 60 hours, preferably about 12 to 48 hours. In this crosslinking process, the dialdehyde compound is a mild crosslinking agent,
Forms a Schiff base by cross-linking with NAG, and NAG
It suppresses changes in the higher-order structure of NAG and stabilizes NAG.
上記架橋処理の後、過剰のアンモニウム塩及びジアルデ
ヒド化合物を除去する。この除去工程は、従来慣用の方
法、例えば、透析法、クロマトグラフィー法等の手段に
より行なうことができるが、架橋されたＮＡＧを高純度
かつ効率的に精製するため、限外濾過法によるのが好ま
しい。その際、完全かつ迅速に濾過するため、限外濾過
と電気透析とを同時に行なえる電気限外濾過法を採用す
ることもできる。After the above crosslinking treatment, excess ammonium salt and dialdehyde compound are removed. This removal step can be carried out by conventional methods such as dialysis and chromatography, but in order to efficiently purify cross-linked NAG with high purity, ultrafiltration is preferable. preferable. At this time, in order to perform complete and rapid filtration, it is also possible to employ an electric ultrafiltration method that allows ultrafiltration and electrodialysis to be performed simultaneously.
上記除去工程の後、ＮＡＧ含有溶液を慣用の方法に阜じ
て凍結乾燥することにより、本発明の安定化されたＮＡ
Ｇが得られる。この凍結乾燥は低温で行なうことができ
るので、ＮＡＧの失活を防止できるとともに、得られた
凍結乾燥品は液状ＮＡＧよりも安定性が高い。After the above removal step, the stabilized NA of the present invention is obtained by freeze-drying the NAG-containing solution according to a conventional method.
G is obtained. Since this freeze-drying can be carried out at low temperatures, deactivation of NAG can be prevented, and the resulting freeze-dried product is more stable than liquid NAG.
本発明のコントロール製剤は上記のようにして調製され
たＮＡＧを少なくとも含有するものであり、該製剤は他
の検査対象に対応する成分を併有する製剤であってもよ
い。他の検査対象に対応する成分を併有する製剤は、前
記凍結乾燥品と他の検査対象に対応する成分を混合する
方法、また前記アンモニウム塩及びジアルデヒド化合物
の除去工程を経て得られたＮＡＧ溶液に、他の検査対象
に対応した成分を添加混合し、凍結乾燥する方法等によ
り調製される。また、該製剤は、ＮＡＧの安定性を維持
するため、バイアル等の容器内に密閉した形態で保存す
るのが好ましく、用時に精製水等で希釈して使用される
。The control preparation of the present invention contains at least the NAG prepared as described above, and the preparation may also contain components corresponding to other test targets. Preparations containing components corresponding to other test targets can be prepared using a method of mixing the freeze-dried product with components corresponding to other test targets, or a NAG solution obtained through the step of removing ammonium salts and dialdehyde compounds. It is prepared by adding and mixing other components corresponding to the test target and freeze-drying. In order to maintain the stability of NAG, the preparation is preferably stored in a sealed container such as a vial, and is diluted with purified water or the like before use.
［発明の作用及び効果］
本発明のＮ Ａ Ｇは、単に凍結乾燥されたり架橋剤で
処理された従前のＮＡＧと異なり、アンモニウム塩の存
在下、ジアルデヒド化合物により処理されている。ジア
ルデヒド化合物はＮＡＧ分子中のアミノ基とシッフ塩基
を生成し、架橋構造を形成することにより、ＮＡＧの高
次構造の変形、変性を防止し、ＮＡＧの安定性を向上さ
せる。この際、アンモニウム塩は、ＮＡＧの高次構造の
維持に関与して安定性の向上に寄与しているものと推察
される。また凍結乾燥されているので、ＮＡＧの安定性
はさらに向上する。従って、本発明のＮＡＧは、長期に
亘って保存でき且つ活性を維持することができるという
効果を奏し、特に、可溶性であることから臨床検査のコ
ントロールとして好適に使用できる。[Operations and Effects of the Invention] The NAG of the present invention is treated with a dialdehyde compound in the presence of an ammonium salt, unlike conventional NAG that is simply freeze-dried or treated with a crosslinking agent. The dialdehyde compound forms a Schiff base with the amino group in the NAG molecule to form a crosslinked structure, thereby preventing deformation and denaturation of the higher-order structure of NAG and improving the stability of NAG. At this time, it is presumed that the ammonium salt is involved in maintaining the higher-order structure of NAG and contributes to improving stability. Furthermore, since it is freeze-dried, the stability of NAG is further improved. Therefore, the NAG of the present invention has the advantage of being able to be stored and maintain its activity over a long period of time, and in particular, is soluble, so it can be suitably used as a control for clinical tests.
また、本発明のコントロール製剤は、尿中ＮＡＧの測定
等の臨床検査のコントロールとして有用であり、上記安
定化されたＮＡＧを含有し、調製当初の活性を長期に亘
り維持することができるので、測定結果の信頼性を高め
、精度管理に貢献できるとともにコントロールの調製が
容易となり作業性の向上が図れるという効果を奏する。In addition, the control preparation of the present invention is useful as a control for clinical tests such as the measurement of urinary NAG, and since it contains the above-mentioned stabilized NAG and can maintain the initial activity for a long period of time, This has the effect of increasing the reliability of measurement results, contributing to accuracy control, and making it easier to prepare controls and improving work efficiency.
［実施例］
以下、実施例及び試験例に基づき、本発明をより詳細に
説明するが、本発明はこれら実施例に限定されるもので
はない。[Examples] Hereinafter, the present invention will be explained in more detail based on Examples and Test Examples, but the present invention is not limited to these Examples.
実施例１
３．２Ｍの硫酸アンモニウム水溶液にＮＡＧを３単位／
１１！となるように溶解した後、５０％のグルタルアル
デヒド溶液を４１１１ｄｌ加えた。２〜８℃に冷却し１
５時間撹拌して反応させた。次いで、反応液を展開液（
水酸化リチウムを含有する０、１５ＭＨＥＰＥＳ緩衝液
、ｐＨ８，０）に混和した後、限外濾過に付し、過剰の
硫酸アンモニウム及びグルグルアルデヒドを除去する操
作を２度行なった。残渣液は展開液に混合し、バイアル
に小分けした後、凍結乾燥することにより、ＮＡＧ製剤
を調製した。Example 1 3 units of NAG/3.2M ammonium sulfate aqueous solution
11! After dissolving the mixture, 4111 dl of 50% glutaraldehyde solution was added. Cool to 2-8℃ 1
The reaction mixture was stirred for 5 hours. Next, the reaction solution was diluted with a developing solution (
After mixing with 0.15M HEPES buffer (pH 8.0) containing lithium hydroxide, the mixture was subjected to ultrafiltration to remove excess ammonium sulfate and gluculaldehyde twice. The residual liquid was mixed with a developing solution, divided into vials, and then freeze-dried to prepare a NAG formulation.
実施例２
上記実施例１の方法において、限外濾過して得られたＮ
ＡＧ含有溶液に、グルコース、ビリルビン、アルブミン
、ウロビリノーゲン、尿素、尿酸、クレアチン、ヘモグ
ロビン、アミラーゼを添加した後、凍結乾燥することに
より、上記検査対象項目成分を併有する尿検査用コント
ロール製剤を調製した。Example 2 In the method of Example 1 above, N obtained by ultrafiltration
Glucose, bilirubin, albumin, urobilinogen, urea, uric acid, creatine, hemoglobin, and amylase were added to the AG-containing solution, and then freeze-dried to prepare a control preparation for urine tests containing the above-mentioned components to be tested.
比較例
アンモニウム塩及びグルタルアルデヒドを用いることな
く、ＮＡＧ水溶液を、前記実施例１と同様にして凍結乾
燥することによりＮＡＧ製剤を調製した。Comparative Example A NAG formulation was prepared by freeze-drying an aqueous NAG solution in the same manner as in Example 1 without using ammonium salt and glutaraldehyde.
試験例
前記実施例１及び２で調製した各製剤を、３７℃の温度
で２０日間保存し、調製当初と保存後のＮＡＧの活性を
調べることにより、安定性を調べたところ、残存酵素活
性が９９％であり、殆ど酵素活性が低下していないこと
が判明した。また２〜８℃の条件で６ケ月間保存したと
ころ、ＮＡＧの活性低下は全く認められなかった。Test Example Each of the formulations prepared in Examples 1 and 2 above was stored at a temperature of 37°C for 20 days, and the stability was investigated by examining the NAG activity at the time of preparation and after storage. It was found that the enzyme activity was 99%, indicating that the enzyme activity was hardly decreased. Further, when it was stored for 6 months at 2 to 8°C, no decrease in NAG activity was observed.
これに対して、比較例のＮＡＧ製剤を３７℃の温度で１
週間保存したところ、残存酵素活性は僅か３％にすぎな
いことが判明した。In contrast, the NAG formulation of the comparative example was
After storage for a week, it was found that only 3% of the enzyme activity remained.
Claims (1)
ンモニウム塩を含有する溶液に溶解し、ジアルデヒド化
合物を添加して架橋した後、過剰のアンモニウム塩及び
ジアルデヒド化合物を除去し、凍結乾燥して得られるこ
とを特徴とする安定化されたＮ−アセチル−β−Ｄ−グ
ルコサミニダーゼ。 ２、アンモニウム塩が、硫酸アンモニウムである請求項
１記載の安定化されたＮ−アセチル−β−Ｄ−グルコサ
ミニダーゼ。 ３、ジアルデヒド化合物が、グルタルアルデヒドである
請求項１記載の安定化されたＮ−アセチル−β−Ｄ−グ
ルコサミニダーゼ。 ４、請求項１記載のＮ−アセチル−β−Ｄ−グルコサミ
ニダーゼを少なくとも含有する臨床検査用のコントロー
ル製剤。 ５、他の検査対象に対応する成分を含有する請求項４記
載の臨床検査用のコントロール製剤。[Claims] 1. N-acetyl-β-D-glucosaminidase is dissolved in a solution containing an ammonium salt, crosslinked by adding a dialdehyde compound, and then excess ammonium salt and dialdehyde compound are removed. 1. A stabilized N-acetyl-β-D-glucosaminidase obtained by freeze-drying the stabilized N-acetyl-β-D-glucosaminidase. 2. The stabilized N-acetyl-β-D-glucosaminidase according to claim 1, wherein the ammonium salt is ammonium sulfate. 3. The stabilized N-acetyl-β-D-glucosaminidase according to claim 1, wherein the dialdehyde compound is glutaraldehyde. 4. A control preparation for clinical testing containing at least the N-acetyl-β-D-glucosaminidase according to claim 1. 5. The control preparation for clinical tests according to claim 4, which contains components corresponding to other test objects.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13804388A JPH01309687A (en) | 1988-06-03 | 1988-06-03 | Stabilized n-acetyl-beta-d-glucosaminidase and controlling drug preparation containing the same enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13804388A JPH01309687A (en) | 1988-06-03 | 1988-06-03 | Stabilized n-acetyl-beta-d-glucosaminidase and controlling drug preparation containing the same enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01309687A true JPH01309687A (en) | 1989-12-14 |
Family
ID=15212677
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13804388A Pending JPH01309687A (en) | 1988-06-03 | 1988-06-03 | Stabilized n-acetyl-beta-d-glucosaminidase and controlling drug preparation containing the same enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01309687A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006271257A (en) * | 2005-03-29 | 2006-10-12 | Cci Corp | Polyol dehydrogenase excellent in heat stability and method for producing the same |
| JP2008245559A (en) * | 2007-03-30 | 2008-10-16 | Cci Corp | Method for producing polyol dehydrogenase |
| EP4300105A1 (en) * | 2022-06-29 | 2024-01-03 | ARKRAY, Inc. | Method for stabilizing hemoglobin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5951789A (en) * | 1982-09-18 | 1984-03-26 | Wako Pure Chem Ind Ltd | Stabilized enzyme |
-
1988
- 1988-06-03 JP JP13804388A patent/JPH01309687A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5951789A (en) * | 1982-09-18 | 1984-03-26 | Wako Pure Chem Ind Ltd | Stabilized enzyme |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006271257A (en) * | 2005-03-29 | 2006-10-12 | Cci Corp | Polyol dehydrogenase excellent in heat stability and method for producing the same |
| JP4741270B2 (en) * | 2005-03-29 | 2011-08-03 | シーシーアイ株式会社 | Polyol dehydrogenase with excellent thermal stability and method for producing the same |
| JP2008245559A (en) * | 2007-03-30 | 2008-10-16 | Cci Corp | Method for producing polyol dehydrogenase |
| EP4300105A1 (en) * | 2022-06-29 | 2024-01-03 | ARKRAY, Inc. | Method for stabilizing hemoglobin |
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