JPH0815430B2 - Method for stabilizing ascorbate oxidase - Google Patents
Method for stabilizing ascorbate oxidaseInfo
- Publication number
- JPH0815430B2 JPH0815430B2 JP61191972A JP19197286A JPH0815430B2 JP H0815430 B2 JPH0815430 B2 JP H0815430B2 JP 61191972 A JP61191972 A JP 61191972A JP 19197286 A JP19197286 A JP 19197286A JP H0815430 B2 JPH0815430 B2 JP H0815430B2
- Authority
- JP
- Japan
- Prior art keywords
- aso
- acid
- solution
- peroxidase
- stabilizing
- 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
- 238000000034 method Methods 0.000 title claims description 15
- 230000000087 stabilizing effect Effects 0.000 title claims description 7
- 108010024957 Ascorbate Oxidase Proteins 0.000 title claims description 4
- 102000003992 Peroxidases Human genes 0.000 claims description 14
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 14
- 102000016938 Catalase Human genes 0.000 claims description 10
- 108010053835 Catalase Proteins 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 239000002184 metal Substances 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 4
- 230000006641 stabilisation Effects 0.000 claims description 3
- 238000011105 stabilization Methods 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 239000000460 chlorine Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 2
- 229910052744 lithium Inorganic materials 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- 229910052749 magnesium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 21
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 8
- 235000010323 ascorbic acid Nutrition 0.000 description 7
- 239000011668 ascorbic acid Substances 0.000 description 7
- 229960005070 ascorbic acid Drugs 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 239000003381 stabilizer Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 235000019800 disodium phosphate Nutrition 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 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
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 239000000852 hydrogen donor Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- ZNJHFNUEQDVFCJ-UHFFFAOYSA-M sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCS(O)(=O)=O)CC1 ZNJHFNUEQDVFCJ-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- QZTKDVCDBIDYMD-UHFFFAOYSA-N 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid Chemical compound NC(=O)CN(CC(O)=O)CC(O)=O QZTKDVCDBIDYMD-UHFFFAOYSA-N 0.000 description 1
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-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
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 description 1
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- ZTQGWROHRVYSPW-UHFFFAOYSA-N 3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 ZTQGWROHRVYSPW-UHFFFAOYSA-N 0.000 description 1
- BMUDPLZKKRQECS-UHFFFAOYSA-K 3-[18-(2-carboxyethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethylporphyrin-21,24-diid-2-yl]propanoic acid iron(3+) hydroxide Chemical compound [OH-].[Fe+3].[N-]1C2=C(C)C(CCC(O)=O)=C1C=C([N-]1)C(CCC(O)=O)=C(C)C1=CC(C(C)=C1C=C)=NC1=CC(C(C)=C1C=C)=NC1=C2 BMUDPLZKKRQECS-UHFFFAOYSA-K 0.000 description 1
- 239000007991 ACES buffer Substances 0.000 description 1
- 239000007988 ADA buffer Substances 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- 102000004539 Acyl-CoA Oxidase Human genes 0.000 description 1
- 108020001558 Acyl-CoA oxidase Proteins 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 239000007992 BES buffer Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 102000005870 Coenzyme A Ligases Human genes 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 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 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 108010061951 Methemoglobin Proteins 0.000 description 1
- DBXNUXBLKRLWFA-UHFFFAOYSA-N N-(2-acetamido)-2-aminoethanesulfonic acid Chemical compound NC(=O)CNCCS(O)(=O)=O DBXNUXBLKRLWFA-UHFFFAOYSA-N 0.000 description 1
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- 239000007994 TES buffer Substances 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 229940093530 coenzyme a Drugs 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- -1 generally Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 229940109738 hematin Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明はアスコルビン酸オキシダーゼ（以下ASOと略
する）の安定化法に関し、より詳しくはASOを含有する
溶液に適当な塩を主として使用することによりASOを安
定化する方法に関するものである。TECHNICAL FIELD The present invention relates to a method for stabilizing ascorbate oxidase (hereinafter abbreviated as ASO), and more specifically, it mainly uses a salt suitable for a solution containing ASO. It relates to a method of stabilizing ASO by.
臨床化学分析における生体成分を酵素的に測定する方
法は数多く開発されている。この方法には、ウリカー
ゼ、アシルコエンザイムＡオキシダーゼ、グルコースオ
キシダーゼ、コレステロールオキシダーゼ、グリセロ−
３−リン酸オキシダーゼなどのオキシダーゼが広く用い
られている。これらのオキシダーゼの作用で生成された
過酸化水素はパーオキシダーゼと水素供与体の発色系を
用いる方法で比色定量されるのが一般的である。Many methods for enzymatically measuring biological components in clinical chemistry analysis have been developed. This method includes uricase, acyl coenzyme A oxidase, glucose oxidase, cholesterol oxidase, glycero-
Oxidases such as 3-phosphate oxidase are widely used. Hydrogen peroxide produced by the action of these oxidases is generally colorimetrically determined by a method using a color development system of peroxidase and a hydrogen donor.
この場合、パーオキシダーゼを用いる反応系において
は、生体試料中に存在するアスコルビン酸の干渉を著し
く受け易い。そこで、この解決策のひとつとして測定試
薬中にASOを共存させることによってアスコルビン酸を
酸化分解して、その干渉を排除することが一般的に行な
われている。In this case, the reaction system using peroxidase is extremely susceptible to the interference of ascorbic acid present in the biological sample. Therefore, as one of the solutions, it is common practice to coexist ASO in the measurement reagent to oxidatively decompose ascorbic acid and eliminate the interference.
しかしながら、溶液中のASOは極めて不安定であるこ
とから、試薬保存中にASOの失活が起こる。However, since ASO in the solution is extremely unstable, deactivation of ASO occurs during storage of the reagent.
従来、ASOの失活防止にカタラーゼ、ゼラチン、グロ
ブリン、パーオキシダーゼ、メトヘモグロビン又はヘマ
チン等を添加すると有効であることが報告されている
〔化学：第17巻、第10号、第844頁及び第６回臨床化学
分析談話会夏期セミナー講演予講集：第106頁〕。しか
し、これらの報告にみられるカタラーゼ又はパーオキシ
ダーゼ等は、ASO安定化効果のうえで不十分なものであ
る。It has been previously reported that it is effective to add catalase, gelatin, globulin, peroxidase, methemoglobin, hematin or the like to prevent the inactivation of ASO [Chemical: Volume 17, No. 10, 844 and 844]. 6th clinical chemistry analysis discourse summer seminar lecture lecture collection: p. 106]. However, catalase, peroxidase and the like found in these reports are insufficient in terms of ASO stabilizing effect.
本発明者らは、溶液中に含有されるASOの安定性を向
上する目的で、種々検討を行った結果、塩を使用するこ
とにより、ASOの失活防止が可能になることを見い出
し、本発明を完成するに至った。The present inventors have conducted various studies for the purpose of improving the stability of ASO contained in a solution, and found that the use of a salt makes it possible to prevent deactivation of ASO. The invention was completed.
即ち、本発明はASOを含有する溶液に、金属または陽
性の塩基性基と陰性の酸基とから成る化合物、もしくは
該化合物とカタラーゼ及び／又はパーオキシダーゼと酸
化性色素カップラーを併用して添加することを特徴とす
るASOの安定化法である。That is, in the present invention, a compound containing a metal or a positive basic group and a negative acid group, or the compound and catalase and / or peroxidase and an oxidative dye coupler are used in combination in a solution containing ASO. It is a stabilizing method of ASO characterized by the following.
本発明の化合物とは、適当な塩を意味し、金属はナト
リウム、カリウム、リチウム、カルシウム、マグネシウ
ム、ストロンチウム、マンガン、銅、ニッケル、コバル
ト及び鉄等から選ばれる陽イオンであり、また陽性の塩
基性基はアンモニウムであり、陰性の酸基は塩素、臭
素、ヨウ素、硫酸、酢酸及びリン酸から選ばれる陰イオ
ンとから成る安定化剤である。The compound of the present invention means a suitable salt, and the metal is a cation selected from sodium, potassium, lithium, calcium, magnesium, strontium, manganese, copper, nickel, cobalt and iron, and a positive base. The functional group is ammonium and the negative acid group is a stabilizer consisting of an anion selected from chlorine, bromine, iodine, sulfuric acid, acetic acid and phosphoric acid.
カタラーゼとパーオキシダーゼは、上記安定化剤と併
用して使用する。この場合、両酵素は別々に、或いは一
緒に、通常0.01U/ml以上を使用する。パーオキシダーゼ
と一緒に用いる酸化性色素カップラーは、アニリン系、
フェノール系等公知のものを使用する。Catalase and peroxidase are used in combination with the above stabilizer. In this case, both enzymes are usually used at 0.01 U / ml or more separately or together. The oxidative dye coupler used with peroxidase is an aniline type,
A known one such as phenol is used.
本発明のASOを含有する溶液とは、ASOと緩衝液を溶解
させた、PH4〜9.5の溶液を意味する。この溶液の中に安
定化剤を1mM以上を、場合によりカタラーゼ及び／又は
パーオキシダーゼと酸化性色素カップラーを併用して添
加する。The solution containing ASO of the present invention means a solution of PH4 to 9.5 in which ASO and a buffer solution are dissolved. Stabilizers of 1 mM or more are added to this solution, optionally in combination with catalase and / or peroxidase and an oxidizing dye coupler.
ASO含有溶液は、例えば尿酸、遊離脂肪酸、グルコー
ス、中性脂肪、コレステロール、リン脂質、乳酸等の測
定を目的とする酵素的測定法における各々の基質とオキ
シダーゼが作用して過酸化水素を生成させる酵素反応
系、及び過酸化水素をパーオキシダーゼと水素供与体の
発色系により比色定量を行なうに必要な測定試薬を含
む。通常、この試薬形態は１試薬系タイプか、または２
試薬系タイプにまとめられているので、後者の場合には
ASOを含有する試薬中に安定化剤を添加する。しかし、
酵素反応系で使用する共役酵素が金属を必要とする場合
には、その金属使用量は通常1mM〜10mM程度で酵素触媒
作用が行われており、それ以上の濃度を添加して本発明
の目的とすることはできない。The ASO-containing solution produces hydrogen peroxide by the action of each substrate and oxidase in the enzymatic measurement method for measuring uric acid, free fatty acid, glucose, neutral fat, cholesterol, phospholipid, lactic acid, etc. It contains an enzyme reaction system and a measurement reagent necessary for colorimetric determination of hydrogen peroxide with a peroxidase and a hydrogen donor color development system. Usually, this reagent form is one reagent type or two
In the latter case, it is
Stabilizer is added into the reagent containing ASO. But,
When the coupled enzyme used in the enzyme reaction system requires a metal, the amount of the metal used is usually about 1 mM to 10 mM for enzyme catalysis, and the object of the present invention is to add a higher concentration. Can not be.
緩衝液は、一般的にPH4〜9.5の範囲で維持できるリン
酸塩、ビス−トリス、ADA、PIPES、ACES、イミダゾー
ル、BES、MOPS、TES、HEPES等を用いる。As the buffer solution, generally, phosphate, bis-tris, ADA, PIPES, ACES, imidazole, BES, MOPS, TES, HEPES and the like which can be maintained in the range of PH4 to 9.5 are used.
ASOを含有する溶液に、本発明の安定化剤を添加する
ことにより無添加に比べ、ASOの安定化が極めて大き
い。By adding the stabilizer of the present invention to the solution containing ASO, the stabilization of ASO is extremely large as compared with the case where the stabilizer is not added.
なお、溶液中のASOの酵素活性は以下の方法で測定す
る。The enzyme activity of ASO in the solution is measured by the following method.
1.反応液の組成 （１） 0.2Mリン酸カリウム及び1mMエチレンジアミン
テトラ酢酸・２ナトリウム塩を含む1mMアスコルビン酸
溶液。1. Composition of reaction solution (1) A 1 mM ascorbic acid solution containing 0.2 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid disodium salt.
（２） 10mMリン酸２ナトリウム溶液。(2) 10 mM disodium phosphate solution.
（３） 1U/mlASOを含有する溶液（ASOは使用時0.08〜
0.35U/mlに0.05％BSA含有10mMリン酸２ナトリウムで希
釈する）。(3) Solution containing 1U / ml ASO (ASO is 0.08-
Dilute to 0.35 U / ml with 10 mM disodium phosphate containing 0.05% BSA).
2.反応条件及びASOの力価 試験管にアスコルビン酸溶液0.5mlとリン酸２ナトリ
ウム溶液0.5mlを加え、30℃で25分間予備加温する。次
に、ASOを含有する溶液0.1mlを加え、30℃で５分間反応
させた後、0.2N塩酸溶液を3.0ml加えて反応を停止させ
る。この時、分解されるアスコルビン酸量を245nmの吸
光度の減少量から求める。この際のアスコルビン酸のミ
リモル吸光係数はε＝10〔mmol-1×１×cm-1〕を用い、
ASOの力価を算出する。2. Reaction conditions and ASO titer Add 0.5 ml of ascorbic acid solution and 0.5 ml of disodium phosphate solution to a test tube and preheat at 30 ° C for 25 minutes. Next, 0.1 ml of a solution containing ASO is added and reacted at 30 ° C. for 5 minutes, and then 3.0 ml of 0.2N hydrochloric acid solution is added to stop the reaction. At this time, the amount of ascorbic acid decomposed is determined from the amount of decrease in absorbance at 245 nm. The mmol extinction coefficient of ascorbic acid at this time was ε = 10 [mmol −1 × 1 × cm −1 ],
Calculate the ASO titer.
ASO力価の表示は、上記条件下で１分間に１μモルの
アスコルビン酸が分解されるASO量を１単位として行
う。The ASO titer is displayed with 1 unit of the amount of ASO at which 1 μmol of ascorbic acid is decomposed in 1 minute under the above conditions.
以下、実施例により本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail with reference to examples.
実施例 １ （１） 方 法 2U/mlASO〔ベーリンガー・マンハイム社製〕を含有す
る50mMHEPES−水酸化ナトリウム緩衝液（PH7.0）を100m
l調整した。これに、第１表に示される塩類を50mMずつ
添加して、25℃で12日間保存した場合のASO活性の残存
率を測定した。この結果を第１表に示す。Example 1 (1) Method 50mM HEPES-sodium hydroxide buffer (PH7.0) containing 2U / ml ASO (manufactured by Boehringer Mannheim) was added to 100m.
l adjusted. 50 mM of each of the salts shown in Table 1 was added thereto, and the residual rate of ASO activity was measured when the salt was stored at 25 ° C for 12 days. The results are shown in Table 1.
（２） 結 果 第１表から明らかなように、いずれのASO溶液も無添
加に比べ、本発明の塩類の添加で、ASOの保存安定性が
著しく向上した。(2) Results As is clear from Table 1, the addition of the salts of the present invention markedly improved the storage stability of ASO as compared with the case where no ASO solution was added.
実施例 ２ （１） 方 法 2U/mlASO及び50mM塩化カリウムを含有する次の緩衝液
を各々100ml調整した。これらに、第２表に示される酵
素、100U/mlカタラーゼ及び／または1U/mlパーオキシダ
ーゼと3mMN−エチル−Ｎ−（２−ヒドロキシ−３−スル
ホプロピル）−ｍ−トルイジンを含有させてASO活性の
残存率を測定した。この結果を第２表に示す。 Example 2 (1) Method 100 ml each of the following buffer containing 2 U / ml ASO and 50 mM potassium chloride was prepared. The enzymes shown in Table 2, 100 U / ml catalase and / or 1 U / ml peroxidase, and 3 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine were added to these to obtain ASO activity. The residual rate of was measured. The results are shown in Table 2.
50mMリン酸カリウム緩衝液（PH7.0）。 50 mM potassium phosphate buffer (PH7.0).
50mMHEPES−水酸化ナトリウム緩衝液（PH7.0）。 50 mM HEPES-sodium hydroxide buffer (PH7.0).
50mMビス−トリス−塩酸緩衝液（PH7.0）。 50 mM Bis-Tris-hydrochloric acid buffer (PH7.0).
（２） 結 果 第２表から明らかなように、いずれの緩衝液も無添加
に比べ、本発明の塩類と酵素カタラーゼ及び／又はパー
オキシダーゼを併用して添加することで、ASOの保存安
定性が向上した。(2) Results As is clear from Table 2, the storage stability of ASO is improved by adding the salt of the present invention and the enzyme catalase and / or peroxidase in combination as compared with the case where no buffer solution is added. Has improved.
実施例 ３ （１） 方 法 遊離脂肪酸測定試薬（２試薬系）のうち、ASOを含有
する方の試薬、即ち0.3mMコエンザイムＡ、1.5mMアデノ
シントリフォスフェート、0.4U/mlアシルコエンザイム
Ａシンセターゼ、1mM塩化マグネシウム、2mM4−アミノ
アンチピリン1U/mlASOを含有する50mMリン酸カリウム緩
衝液（PH7.0）を100ml調製した。これに50ml塩化カリウ
ムまたは100U/mlカタラーゼと10U/mパーオキシダーゼを
添加して、２〜８℃で７日間保存した場合のASO活性の
残存率を測定した。この結果を第３表に示す。 Example 3 (1) Method Among the free fatty acid measurement reagents (two-reagent system), the reagent containing ASO, that is, 0.3 mM coenzyme A, 1.5 mM adenosine triphosphate, 0.4 U / ml acyl coenzyme A synthetase, 100 ml of 50 mM potassium phosphate buffer solution (PH7.0) containing 1 mM magnesium chloride and 2 mM 4-aminoantipyrine 1 U / ml ASO was prepared. To this, 50 ml of potassium chloride or 100 U / ml catalase and 10 U / m peroxidase were added, and the residual rate of ASO activity when stored at 2 to 8 ° C for 7 days was measured. The results are shown in Table 3.
（２） 結 果 第３表から明らかなように、ASOを含有した生体成分
を測定する試薬系においても無添加に比べ、塩及びカタ
ラーゼ及び／又はパーオキシダーゼの添加で、ASOの保
存安定性が著しく向上した。(2) Results As is clear from Table 3, even in the reagent system for measuring a biological component containing ASO, the storage stability of ASO was improved by the addition of salt and catalase and / or peroxidase, as compared with the case of no addition. Significantly improved.
Claims (4)
液に、金属または陽性の塩基性基と陰性の酸基とから成
る化合物、もしくは該化合物とカタラーゼ及び／又はパ
ーオキシダーゼと酸化性色素カップラーを併用して添加
することを特徴とするアスコルビン酸オキシダーゼの安
定化法。1. A solution containing ascorbate oxidase, which comprises a compound comprising a metal or a positive basic group and a negative acid group, or the compound, catalase and / or peroxidase, and an oxidative dye coupler. A method for stabilizing ascorbate oxidase, which comprises adding.
たはマグネシウムである、特許請求の範囲第１項記載の
安定化法。2. The stabilizing method according to claim 1, wherein the metal is sodium, potassium, lithium or magnesium.
許請求の範囲第１項の安定化法。3. The stabilization method according to claim 1, wherein the positive basic group is ammonium.
酢酸またはリン酸である、特許請求の範囲第１項記載の
安定化法。4. The negative acid group is chlorine, bromine, iodine, sulfuric acid,
The stabilization method according to claim 1, which is acetic acid or phosphoric acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61191972A JPH0815430B2 (en) | 1986-08-19 | 1986-08-19 | Method for stabilizing ascorbate oxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61191972A JPH0815430B2 (en) | 1986-08-19 | 1986-08-19 | Method for stabilizing ascorbate oxidase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6349081A JPS6349081A (en) | 1988-03-01 |
| JPH0815430B2 true JPH0815430B2 (en) | 1996-02-21 |
Family
ID=16283502
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61191972A Expired - Lifetime JPH0815430B2 (en) | 1986-08-19 | 1986-08-19 | Method for stabilizing ascorbate oxidase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0815430B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07102133B2 (en) * | 1990-07-05 | 1995-11-08 | サンスター株式会社 | Method for stabilizing ascorbate oxidase and stabilized enzyme composition |
| JP2521097Y2 (en) * | 1990-09-14 | 1996-12-25 | シャープ株式会社 | Liquid crystal display |
| JP2838866B2 (en) * | 1995-05-10 | 1998-12-16 | 株式会社同仁化学研究所 | Method for suppressing the activity of reducing substances in oxidative colorimetric analysis |
| JP6132837B2 (en) * | 2012-05-25 | 2017-05-24 | 協和メデックス株式会社 | Method for stabilizing ascorbate oxidase |
| JP7425459B2 (en) * | 2019-10-11 | 2024-01-31 | 株式会社シノテスト | Stabilized HMGB1-containing solution |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS52143285A (en) * | 1976-05-26 | 1977-11-29 | Kikkoman Corp | Stabilization of cholesterol-oxydase |
| ATE4328T1 (en) * | 1980-09-19 | 1983-08-15 | Boehringer Mannheim Gmbh | METHODS AND REAGENTS FOR THE DETERMINATION OF GLYCERIN. |
-
1986
- 1986-08-19 JP JP61191972A patent/JPH0815430B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6349081A (en) | 1988-03-01 |
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