JPH0424999B2 - - Google Patents
Info
- Publication number
- JPH0424999B2 JPH0424999B2 JP24289083A JP24289083A JPH0424999B2 JP H0424999 B2 JPH0424999 B2 JP H0424999B2 JP 24289083 A JP24289083 A JP 24289083A JP 24289083 A JP24289083 A JP 24289083A JP H0424999 B2 JPH0424999 B2 JP H0424999B2
- Authority
- JP
- Japan
- Prior art keywords
- nitrophenyl
- glucosidase
- maltopentaoside
- reagent
- amylase activity
- 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
Links
- 230000000694 effects Effects 0.000 claims description 21
- 239000000758 substrate Substances 0.000 claims description 20
- 102000004139 alpha-Amylases Human genes 0.000 claims description 18
- 108090000637 alpha-Amylases Proteins 0.000 claims description 18
- 229940024171 alpha-amylase Drugs 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 14
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 9
- 102100022624 Glucoamylase Human genes 0.000 claims description 9
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 6
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 6
- 125000005843 halogen group Chemical group 0.000 claims description 5
- 229920001542 oligosaccharide Polymers 0.000 claims description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 4
- 150000002482 oligosaccharides Chemical class 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 3
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- 125000004076 pyridyl group Chemical group 0.000 claims description 2
- 150000002989 phenols Chemical class 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 description 40
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 16
- 108010028144 alpha-Glucosidases Proteins 0.000 description 16
- -1 malt Triose Chemical class 0.000 description 14
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 14
- 238000005259 measurement Methods 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 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 9
- 238000002835 absorbance Methods 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- YXGBAQKCCMQLGH-MYPSSPKESA-N (2r,3r,4s,5s,6r)-2-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-4,5-dihydroxy-2-(hydroxymethyl)-6-(4-nitrophenoxy)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5- Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](OC=5C=CC(=CC=5)[N+]([O-])=O)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O YXGBAQKCCMQLGH-MYPSSPKESA-N 0.000 description 8
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 8
- 102000004366 Glucosidases Human genes 0.000 description 8
- 108010056771 Glucosidases Proteins 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 125000004201 2,4-dichlorophenyl group Chemical group [H]C1=C([H])C(*)=C(Cl)C([H])=C1Cl 0.000 description 5
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 5
- 239000004382 Amylase Substances 0.000 description 5
- 102000013142 Amylases Human genes 0.000 description 5
- 108010065511 Amylases Proteins 0.000 description 5
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 5
- 235000019418 amylase Nutrition 0.000 description 5
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 5
- 238000000691 measurement method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- YXGBAQKCCMQLGH-VQSBMGSQSA-N (2r,3r,4s,5s,6r)-2-[(2r,3s,4r,5r,6r)-6-[(2r,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-6-[(2r,3s,4r,5r,6r)-4,5-dihydroxy-2-(hydroxymethyl)-6-(4-nitrophenoxy)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-4,5-dih Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](OC2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](OC=5C=CC(=CC=5)[N+]([O-])=O)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O YXGBAQKCCMQLGH-VQSBMGSQSA-N 0.000 description 4
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- FTNIPWXXIGNQQF-UHFFFAOYSA-N UNPD130147 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(OC4C(OC(O)C(O)C4O)CO)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O FTNIPWXXIGNQQF-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- FJCUPROCOFFUSR-UHFFFAOYSA-N malto-pentaose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 FJCUPROCOFFUSR-UHFFFAOYSA-N 0.000 description 3
- FJCUPROCOFFUSR-GMMZZHHDSA-N maltopentaose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O[C@@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)[C@@H](CO)O2)O)[C@@H](CO)O1 FJCUPROCOFFUSR-GMMZZHHDSA-N 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 2
- 125000004105 2-pyridyl group Chemical group N1=C([*])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- 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
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical compound CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000009569 Phosphoglucomutase Human genes 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- LUEWUZLMQUOBSB-UHFFFAOYSA-N UNPD55895 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(O)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O LUEWUZLMQUOBSB-UHFFFAOYSA-N 0.000 description 1
- FYGDTMLNYKFZSV-DZOUCCHMSA-N alpha-D-Glcp-(1->4)-alpha-D-Glcp-(1->4)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-DZOUCCHMSA-N 0.000 description 1
- OCIBBXPLUVYKCH-QXVNYKTNSA-N alpha-maltohexaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](O[C@@H]5[C@H](O[C@H](O)[C@H](O)[C@H]5O)CO)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O OCIBBXPLUVYKCH-QXVNYKTNSA-N 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 229960005215 dichloroacetic acid Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 108010046301 glucose peroxidase Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- DJMVHSOAUQHPSN-UHFFFAOYSA-N malto-hexaose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(OC4C(C(O)C(O)C(CO)O4)O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 DJMVHSOAUQHPSN-UHFFFAOYSA-N 0.000 description 1
- UYQJCPNSAVWAFU-UHFFFAOYSA-N malto-tetraose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(CO)O1 UYQJCPNSAVWAFU-UHFFFAOYSA-N 0.000 description 1
- LUEWUZLMQUOBSB-OUBHKODOSA-N maltotetraose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O[C@@H]3[C@@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-OUBHKODOSA-N 0.000 description 1
- 238000006140 methanolysis reaction Methods 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- GEVPUGOOGXGPIO-UHFFFAOYSA-N oxalic acid;dihydrate Chemical compound O.O.OC(=O)C(O)=O GEVPUGOOGXGPIO-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 108091000115 phosphomannomutase Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006227 trimethylsilylation reaction Methods 0.000 description 1
- 150000008501 α-D-glucopyranosides Chemical class 0.000 description 1
Description
〔産業上の利用分野〕
本発明は新規なα−アミラーゼ基質を用いたα
−アミラーゼ活性測定法に関するものである。
〔従来の技術及びその問題点〕
最近、ヒト体液中のアミラーゼ活性の測定用基
質として、構造の明確なマルトオリゴ糖、例えば
マルトテトラオース、マルトペンタオース、マル
トヘキサオースなどが使用される様になりつつあ
る。これらのうち代表例としてマルトペンタオー
スを基質として使用する場合をとりあげて説明す
ると、測定様式は次の様に表わすことができる。
(1) マルトペンタオースアミラーゼ
――――――→
マルトース+マルトトリオース
(2) マルトース+マルトトリオース
α−グルコシダーゼ(EC3.4.1.20、以下同
じ。)
α−グルコシダーゼ(EC3.4.1.20、以下同
じ。)
―――――――――――――――――――――――――
―――――――→
グルコース
ここに生成したグルコースは、公知の方法、例
えばグルコースオキシダーゼ/パーオキシダー
ゼ/色素系、もしくはヘキソキナーゼ/ホスフオ
グルコムターゼ/グルコース−6−ホスフエート
デヒドロゲナーゼ/NADH系等で測定される。
ところがこの測定系には、基質(マルトオリゴ
糖)に対しわずかではあるが、α−グルコシダー
ゼが作用するという欠点があり、ブランク値が上
昇する。
他方、マルトオリゴ糖の還元末端にフエニル
基、ナフチル基或はそれらの類似体を結合した基
質が合成され、次の様なものを基質とするアミラ
ーゼ測定試薬が提案されている。
p−ニトロフエニル マルトペンタオシド(特公
昭57−53079号公報)
p−ニトロフエニル マルトヘキサオシド(特公
昭57−53079号公報)
p−ニトロフエニル マルトヘプタオシド(特公
昭54−51892号公報)
2,4−ジクロロフエニル マルトペンタオシド
(特開昭56−35998号公報)
これらの化合物を基質とするアミラーゼ活性の
測定様式を例示すると、次の様になる。
p−ニトロフエニルα−マルトペンタオシドの場
合
(1) p−ニトロフエニルα−マルトペンタオシド
アミラーゼ
―――――→
p−ニトロフエニル
α−マルトシド+マルトトリオース
(2) p−ニトロフエニルα−マルトシド+マルト
トリオース
α−グルコシダーゼ
――――――――――→
p−ニトロフエノール+グルコース
この系においても、基質のp−ニトロフエニル
α−マルトペンタオシドにα−グリコシダーゼが
作用してブランク値が上昇する。
2,4−ジクロロフエニルβ−マルトペンタオシ
ドの場合
(1) 2,4−ジクロロフエニルβ−マルトペンタ
オシド
アミラーゼ
―――――→
2,4−ジクロロフエニル
β−マルトシド+マルトトリオース
(2−a) 2,4−ジクロロフエニルβ−マル
トシド+マルトトリオース
α−グルコシダーゼ
――――――――――→
2,4−ジクロロフエニル
β−グルコシド+グルコース
(2−b) 2,4−ジクロロフエニルβ−グル
コシド
β−グルコシダーゼ(EC3.4.1.21、以下同じ。)
β−グルコシダーゼ(EC3.4.1.21、以下同じ。)
―――――――――――――――――――――――――
―――→
2,4−ジクロロフエノール+グルコース
(2−b−1) 2,4−ジクロロフエノール+
4−アミノアンチピリン
酸化剤
――――→
キノン色素
この系においても、基質である2,4−ジクロ
ロフエニルβ−マルトペンタオシドにα−グルコ
シダーゼとβ−グルコシダーゼが作用してブラン
クが上昇する。
結局、従来提供されてきた基質を使用する測定
系では、α−グルコシダーゼが基質に作用するこ
とから、試薬ブランク値の上昇が著しいという問
題点がある。さらにα−グルコシダーゼ液と基質
液の一液化は、α−グルコシダーゼの基質分解に
より、試薬の安定性を著しく損うという共通の問
題点があつた。
〔問題点を解決するための手段〕
そこで、本発明者等は種々研究した結果、非還
元末端に置換基を導入することにより、これらの
問題点が解決できることを知り、本発明を完成す
るに至つた。
すなわち本発明は、
一般式():
[式中、R1はハロゲン原子、
[Industrial Application Field] The present invention provides α-amylase using a novel α-amylase substrate.
- This invention relates to a method for measuring amylase activity. [Prior art and its problems] Recently, maltooligosaccharides with well-defined structures, such as maltotetraose, maltopentaose, and maltohexaose, have come to be used as substrates for measuring amylase activity in human body fluids. It's coming. Taking up and explaining the case where maltopentaose is used as a substrate as a typical example of these, the measurement format can be expressed as follows. (1) Maltopentaose amylase――――――→ Maltose + Maltotriose (2) Maltose + Maltotriose α-glucosidase (EC3.4.1.20, the same applies hereinafter) α-glucosidase (EC3.4.1.20 ,same as below.) -------------------------
――――――――→ Glucose The glucose produced here is processed using known methods such as glucose oxidase/peroxidase/dye system or hexokinase/phosphoglucomutase/glucose-6-phosphate dehydrogenase/NADH system. be measured. However, this measurement system has the drawback that α-glucosidase acts, albeit slightly, on the substrate (maltooligosaccharide), which increases the blank value. On the other hand, substrates in which phenyl groups, naphthyl groups, or analogs thereof are bonded to the reducing end of malto-oligosaccharides have been synthesized, and amylase measurement reagents using the following substrates have been proposed. p-Nitrophenyl maltopentaoside (Japanese Patent Publication No. 57-53079) p-Nitrophenyl maltohexaoside (Japanese Patent Publication No. 57-53079) p-Nitrophenyl maltoheptaoside (Japanese Patent Publication No. 54-51892) 2. 4-dichlorophenyl maltopentaoside (JP-A-56-35998) The following is an example of a method for measuring amylase activity using these compounds as substrates. In the case of p-nitrophenyl α-maltopentaoside (1) p-nitrophenyl α-maltopentaoside amylase――――→ p-nitrophenyl α-maltoside + maltotriose (2) p-nitrophenyl α-maltoside + Maltotriose α-glucosidase――――――――――→ p-nitrophenol + glucose In this system as well, α-glycosidase acts on the substrate p-nitrophenyl α-maltopentaoside, resulting in a blank value. Rise. In the case of 2,4-dichlorophenyl β-maltopentaoside (1) 2,4-dichlorophenyl β-maltopentaoside amylase――――→ 2,4-dichlorophenyl β-maltopentaoside + malt Triose (2-a) 2,4-dichlorophenyl β-maltoside + maltotriose α-glucosidase――――――――――→ 2,4-dichlorophenyl β-glucoside + glucose (2- b) 2,4-Dichlorophenyl β-glucoside β-glucosidase (EC3.4.1.21, the same shall apply hereinafter) β-glucosidase (EC3.4.1.21, the same shall apply hereinafter) ―――――――――― ――――――――――――――――
---→ 2,4-dichlorophenol + glucose (2-b-1) 2,4-dichlorophenol +
4-aminoantipyrine oxidizing agent――――→ Quinone dye In this system as well, the blank increases due to the action of α-glucosidase and β-glucosidase on the substrate 2,4-dichlorophenyl β-maltopentaoside. . As a result, in the measurement systems that use conventionally provided substrates, there is a problem in that the reagent blank value increases significantly because α-glucosidase acts on the substrate. Furthermore, the use of a single solution of α-glucosidase and substrate has a common problem in that the stability of the reagent is significantly impaired due to the decomposition of the substrate by α-glucosidase. [Means for solving the problems] As a result of various studies, the present inventors learned that these problems could be solved by introducing a substituent to the non-reducing terminal, and in order to complete the present invention. I've reached it. That is, the present invention has the general formula (): [In the formula, R 1 is a halogen atom,
【式】−O−R3(但し、R3
は炭素数1〜6のアルキル基を示す。)又は−
NH−R4(但し、R4は炭素数1〜6のアルキル
基、フエニル基又はピリジル基を示す。)を示し、
R2は、ハロゲン原子又は/及びニトロ基を置換
基として有していてもよいフエニル基を示し、n
は1〜6の整数を示す。]で表わされる非還元末
端を修飾したオリゴサツカライド誘導体を基質と
して、α−グリコシダーゼ及び/又はグルコアミ
ラーゼ(EC3.2.1.3、以下同じ。)及び必要により
β−グルコシダーゼの共存下に、試料を接触さ
せ、遊離するフエノール系化合物を測定すること
により、試料中のα−アミラーゼ活性を測定する
ことを特徴とするα−アミラーゼ活性測定法であ
る。
本発明の基質はオリゴサツカライド誘導体の非
還元性末端がハロゲン原子、[Formula] -O-R 3 (However, R 3 represents an alkyl group having 1 to 6 carbon atoms.) or -
NH-R 4 (wherein R 4 represents an alkyl group having 1 to 6 carbon atoms, a phenyl group or a pyridyl group),
R 2 represents a phenyl group which may have a halogen atom or/and a nitro group as a substituent, and n
represents an integer from 1 to 6. ] Using an oligosaccharide derivative modified with a non-reducing end represented by the above as a substrate, a sample is prepared in the presence of α-glycosidase and/or glucoamylase (EC3.2.1.3, the same applies hereinafter) and β-glucosidase if necessary. This is an α-amylase activity measurement method characterized by measuring α-amylase activity in a sample by contacting the sample and measuring the liberated phenol compound. In the substrate of the present invention, the non-reducing end of the oligosaccharide derivative is a halogen atom,
実施例 1
4−ニトロフエニルO−6−デオキシ−6−
〔(2−ピリジル)アミノ〕−α−D−グルコピ
ラノシル−(1→4)−O−α−D−グルコピラ
ノシル−(1→4)−O−α−D−グルコピラノ
シル−(1→4)−O−α−D−グルコピラノシ
ル−(1→4)−α−D−グルコピラノシド(ピ
リジルアミノ4−ニトロフエニルα−マルトペ
ンタオシド)の合成
ジメチルスルホキシド200mlに4−ニトロフエ
ニルα−マルトペンタオシド10gとN,N′−ジ
シクロヘキシルカルボジイミド15gを溶解し、こ
れにジクロロ酢酸2.0mlとジメチルスルホキシド
20mlの混合物を加え、よく混合し、20〜25℃で50
分間反応させた。これに、メタノール30mlにシユ
ウ酸(2水塩)7gを溶解した液を加え、反応を
停止し、この反応混合液に2−アミノピリジン溶
液(2−アミノピリジン45g、水60ml、酢酸15ml
及びシアノ水素化ホウ素ナトリウム16gを混合し
た液)を加え、30℃で12時間反応した。水素化反
応後、水1.5を加え、生じた沈澱を別し、こ
の液を6規定塩酸でPH1.0とした。過剰のシア
ノ水素化ホウ素ナトリウムを分解後、1規定水酸
化ナトリウムでPH4〜5とした。これにリゾプス
デレマー由来のグルコアミラーゼ30mgを加え40℃
で10時間インキユベートした後減圧濃縮した。こ
の濃縮物を水に溶解し、ゲル過した。カラムは
10mM重炭酸アンモニウムで平衡化したBiogel
P−2(Bio Rad社製)を充填した直径3.0cm、高
さ180cmのものを使用し、高分子画分を集め凍結
乾燥した。精製は高速液体クロマトグラフイーを
用い、Cosmosil5C18(半井化学、C18逆相)を充
填したカラム(10×250mm)を使用し、溶出液に
0.8%1−ブタノールを含む0.1M酢酸を使用し、
3.5ml/minの流速で行ない、目的物83mgを得た。
〔構造の確認〕
得られたピリジルアミノ4−ニトロフエニルα
−マルトペンタオシド中のp−ニトロフエニル基
に対するグルコース残基の数を次のようにして測
定した。ピリジルアミノ4−ニトロフエニルα−
マルトペンタオシドを1.4規定塩酸−メタノール
で90℃、2時間メタノリシスした。濃縮後、トリ
メチルシリル化し、2%OV−17(0.4×200cm)の
カラムを用い、110℃から250℃まで4℃/minの
昇温プログラムを用いて、グルコース量を定量し
た。また、p−ニトロフエニル基は0.1M酢酸中
での305nmの吸光度から定量した。その結果、
ピリジルアミノ4−ニトロフエニルα−マルトペ
ンタオシドのグルコース/p−ニトロフエノール
の比は3.6であつた。
上で得た結果と、ピリジルアミノ4−ニトロフ
エニルα−マルトペンタオシドのグルコアミラー
ゼの作用を受けないことから、この構造は次のも
のであると考えられる。第1図にピリジルアミノ
4−ニトロフエニルα−マルトペンタオシドの
D2O中の1H−NMRスペクトルを示す。
実施例 2
下記の試薬を用い、下記方法によりブランク値
を測定した。
試薬:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
β−グルコシダーゼ 10U/ml
第1表に示される基質 2mM
Example 1 4-nitrophenyl O-6-deoxy-6-
[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O Synthesis of -α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (pyridylamino 4-nitrophenyl α-maltopentaoside) 10 g of 4-nitrophenyl α-maltopentaoside and N,N in 200 ml of dimethyl sulfoxide Dissolve 15 g of '-dicyclohexylcarbodiimide, add 2.0 ml of dichloroacetic acid and dimethyl sulfoxide to this.
Add 20ml of the mixture, mix well and heat at 20-25℃ for 50
Allowed to react for minutes. To this, a solution of 7 g of oxalic acid (dihydrate) dissolved in 30 ml of methanol was added to stop the reaction, and to this reaction mixture was added a 2-aminopyridine solution (45 g of 2-aminopyridine, 60 ml of water, 15 ml of acetic acid).
and 16 g of sodium cyanoborohydride) was added, and the mixture was reacted at 30°C for 12 hours. After the hydrogenation reaction, 1.5 liters of water was added, the resulting precipitate was separated, and this liquid was adjusted to pH 1.0 with 6N hydrochloric acid. After decomposing excess sodium cyanoborohydride, the pH was adjusted to 4 to 5 with 1N sodium hydroxide. Add 30mg of glucoamylase derived from Rhizopus deremer to this and hold at 40°C.
After incubating for 10 hours, the mixture was concentrated under reduced pressure. This concentrate was dissolved in water and subjected to gel filtration. The column is
Biogel equilibrated with 10mM ammonium bicarbonate
Using a container with a diameter of 3.0 cm and a height of 180 cm filled with P-2 (manufactured by Bio Rad), the polymer fraction was collected and freeze-dried. Purification was carried out using high-performance liquid chromatography using a column (10 x 250 mm) packed with Cosmosil5C 18 (Hani Chemical, C 18 reverse phase), and the eluate was
Using 0.1M acetic acid containing 0.8% 1-butanol,
It was carried out at a flow rate of 3.5 ml/min, and 83 mg of the target product was obtained. [Structure confirmation] Obtained pyridylamino 4-nitrophenyl α
- The number of glucose residues relative to the p-nitrophenyl group in maltopentaoside was measured as follows. Pyridylamino 4-nitrophenyl α-
Maltopentaoside was subjected to methanolysis with 1.4N hydrochloric acid and methanol at 90°C for 2 hours. After concentration, trimethylsilylation was performed, and the amount of glucose was quantified using a 2% OV-17 (0.4 x 200 cm) column and a temperature increase program of 4°C/min from 110°C to 250°C. Moreover, the p-nitrophenyl group was quantified from the absorbance at 305 nm in 0.1M acetic acid. the result,
The glucose/p-nitrophenol ratio of pyridylamino 4-nitrophenyl α-maltopentaoside was 3.6. Based on the results obtained above and the fact that pyridylamino 4-nitrophenyl α-maltopentaoside is not affected by glucoamylase, this structure is considered to be as follows. Figure 1 shows pyridylamino 4-nitrophenyl α-maltopentaoside.
1 H-NMR spectrum in D 2 O is shown. Example 2 A blank value was measured using the following reagents and the following method. Reagents: 50mM glucosidase (PH7.0) α-glucosidase 80U/ml β-glucosidase 10U/ml Substrate shown in Table 1 2mM
【表】
測定法:
上記試薬3mlを取り、試薬ブランクの経時変化
を見た。(測定波長400nm、温度37℃)
その結果を第2表に示す。[Table] Measurement method: 3 ml of the above reagent was taken and the change over time of the reagent blank was observed. (Measurement wavelength: 400 nm, temperature: 37°C) The results are shown in Table 2.
【表】
本発明の試薬A、B、Cは試薬D、E、Fと比
較して、試薬ブランクの上昇が小さい。
実施例 3
試料中のα−アミラーゼ活性を、下記の試薬を
用い、下記方法により測定した。
試薬A:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
ピリジルアミノ2−クロロ−4−ニトロフエニ
ルα−マルトペンタオシド 2mM
試薬B:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
2−クロロ−4−ニトロフエニルα−マルトペ
ンタオシド 2mM
測定法:
試薬A又はB各3mlを取り、37℃での試薬ブラ
ンク経時変化を400nmで測定した。
試料(血清)50μに、試薬A又はB各3mlを
添加し、添加後5〜6分後の吸光度変化(α−ア
ミラーゼ活性)を測定した。(測定波長400nm、
反応温度37℃)
試薬ブランクの経時変化を第3表に、血清の吸
光度測定を第4表に示す。[Table] Compared with reagents D, E, and F, reagents A, B, and C of the present invention have a smaller increase in reagent blank. Example 3 α-amylase activity in a sample was measured using the following reagents and the following method. Reagent A: 50mM gum buffer (PH7.0) α-glucosidase 80U/ml Pyridylamino 2-chloro-4-nitrophenyl α-maltopentaoside 2mM Reagent B: 50mM gum buffer (PH7.0) α-glucosidase 80U/ml 2-chloro -4-Nitrophenyl α-maltopentaoside 2mM Measurement method: 3ml each of Reagent A or B was taken and the change over time of a reagent blank at 37°C was measured at 400nm. 3 ml each of reagent A or B was added to 50 µ of sample (serum), and the change in absorbance (α-amylase activity) was measured 5 to 6 minutes after the addition. (Measurement wavelength 400nm,
Reaction temperature: 37°C) Table 3 shows the change over time of the reagent blank, and Table 4 shows the absorbance measurement of the serum.
【表】【table】
【表】
本発明の試薬Aは試薬Bに比較して、試薬ブラ
ンクの上昇は小さく、α−アミラーゼ活性値は試
薬A、Bともに差はない。
実施例 4
試料中のα−アミラーゼ活性を、下記の試薬を
用い、下記方法により測定した。
試薬A:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
グルコシダーゼ 10U/ml
トシルオキシ4−ニトロフエニルα−マルトヘ
プタオシド 2mM
試薬B:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
トシルオキシ4−ニトロフエニルα−マルトヘ
プタオシド 2mM
試薬C:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
グルコシダーゼ 10U/ml
4−ニトロフエニルα−マルトヘプタオシド
2mM
測定法:
試薬A、B又はC各3mlを取り、37℃での試薬
ブランク経時変化を400nmで測定した。試料血
清50μに試薬A、B又はC各3mlを添加し、添
加後5〜6分後の吸光度変化(アミラーゼ活性)
を測定した。(測定波長400nm、反応温度37℃)
試薬ブランクの経時変化を第5表に血清の吸光
度変化を第6表に示す。[Table] Reagent A of the present invention shows a smaller increase in reagent blank than Reagent B, and there is no difference in α-amylase activity value between Reagents A and B. Example 4 α-amylase activity in a sample was measured using the following reagent and the following method. Reagent A: 50mM Glucosidase (PH7.0) α-glucosidase 80U/ml Glucosidase 10U/ml Tosyloxy 4-nitrophenyl α-maltoheptaoside 2mM Reagent B: 50mM Glucosidase (PH7.0) α-Glucosidase 80U/ml Tosyloxy4- Nitrophenyl α-maltoheptaoside 2mM Reagent C: 50mM Gut buffer (PH7.0) α-glucosidase 80U/ml Glucosidase 10U/ml 4-Nitrophenyl α-maltoheptaoside
2mM measurement method: 3ml each of reagents A, B, or C was taken, and the change over time in a reagent blank at 37°C was measured at 400nm. Add 3 ml each of reagents A, B, or C to 50 μ of sample serum, and observe the change in absorbance (amylase activity) 5 to 6 minutes after addition.
was measured. (Measurement wavelength: 400 nm, reaction temperature: 37°C) Table 5 shows the change in reagent blank over time, and Table 6 shows the change in absorbance of serum.
【表】【table】
2−(N−モルホリノ)エタンスルホン酸を50
mM、塩化ナトリウムを20mM含むように調製し
た水溶液に、水酸化ナトリウムを加えてPH6.9に
した。この溶液に実施例1で合成したピリジルア
ミノ4−ニトロフエニルα−マルトペンタオシド
を1mM、α−グルコシダーゼを10U/ml、グル
コアミラーゼを20U/mlになるように添加し、溶
解した。
〔測定操作〕
試液2mlに検体血清50μを加えて混合し、37
℃に加温して405nmにおける1分後から2分間
の吸光度変化を測定した。
別に、α−アミラーゼ活性既知の標準検体を用
い、上記と同様に操作し、検量関係を求め、この
検量線から検体のα−アミラーゼ活性を求めた。
このときの標準検体の各希釈段階に於けるα−ア
ミラーゼ活性(Somogyi単位/dl)と波長405n
mに於ける吸光度増加量(OD/min)との関係
を第2図に示す。
実施例 6
4−ニトロフエニルO−6−アニリノ−6−デ
オキシ−α−D−グルコピラノシル−(1→4)
−O−α−D−グルコピラノシル−(1→4)−
O−α−D−グルコピラノシル−(1→4)−O
−α−D−グルコピラノシル−(1→4)−O−
α−D−グルコピラノシド(アニリノ4−ニト
ロフエニルα−マルトペンタオシド)の合成
実施例1において、2−アミノピリジン45gの
代りにアニリン45gを用いた以外は、実施例1と
同じ試薬を用い、同様の操作を行つて、目的物
170mgを得た。
[構造の確認]
得られたアニリノ4−ニトロフエニルα−マル
トペンタオシド中のp−ニトロフエニル基に対す
るグルコース残基の数を、実施例1と同様の操作
法により測定した。その結果、アニリノ4−ニト
ロフエニルα−マルトペンタオシドのグルコー
ス/4−ニトロフエノールの比は3.8であつた。
実施例 7
4−ニトロフエニルO−6−デオキシ−6−n
−プロピルアミノ−α−D−グルコピラノシル
−(1→4)−O−α−D−グルコピラノシル−
(1→4)−O−α−D−グルコピラノシル−
(1→4)−O−α−D−グルコピラノシル−
(1→4)−O−α−D−グルコピラノシド(n
−プロピルアミノ4−ニトロフエニルα−マル
トペンタオシド)の合成
実施例1において、2−アミノピリジン45gの
代りにn−プロピルアミン29gを用いた以外は、
実施例1と同じ試薬を用い、同様の操作を行つ
て、目的物75mgを得た。
[構造の確認]
得られたn−プロピルアミノ4−ニトロフエニ
ルα−マルトペンタオシド中のp−ニトロフエニ
ル基に対するグルコース残基の数を、実施例1と
同様の操作法により測定した。その結果、n−プ
ロピルアミノ4−ニトロフエニルα−マルトペン
タオシドのグルコース/4−ニトロフエノールの
比は3.8であつた。
実施例 8
試薬中のα−アミラーゼ活性を、下記の試薬を
用い、下記の方法により測定した。試薬A:50m
Mグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
グルコアミラーゼ 10U/ml
アニリノ4−ニトロフエニルα−マルトヘプタ
オシド 2mM
試薬B:50mMグツドバツフアー (PH7.0)
α−グルコシダーゼ 80U/ml
グルコアミラーゼ 10U/ml
n−プロピルアミノ4−ニトロフエニルα−マ
ルトペンタオシド 2mM
測定法:
試薬A又はB各3mlを取り、37℃での試薬ブラ
ンク経時変化を400nmで測定した。
試料(血清)50μに、試薬A又はB各3mlを
添加し、添加後5〜6分後の吸光度変化(α−ア
ミラーゼ活性)を測定した。(測定波長400nm、
反応温度37℃)
試薬ブランクの経時変化を第7表に、血清の吸
光度測定を第8表に示す。
50% of 2-(N-morpholino)ethanesulfonic acid
Sodium hydroxide was added to an aqueous solution prepared to contain 20 mM sodium chloride to adjust the pH to 6.9. To this solution were added and dissolved pyridylamino 4-nitrophenyl α-maltopentaoside synthesized in Example 1 at 1 mM, α-glucosidase at 10 U/ml, and glucoamylase at 20 U/ml. [Measurement procedure] Add 50μ of sample serum to 2ml of test solution and mix.
The absorbance change was measured at 405 nm from 1 minute to 2 minutes after heating to ℃. Separately, using a standard specimen with known α-amylase activity, the same procedure as above was performed to determine the calibration relationship, and the α-amylase activity of the specimen was determined from this calibration curve.
α-Amylase activity (Somogyi units/dl) and wavelength of 405n at each dilution stage of the standard sample at this time
Figure 2 shows the relationship between the absorbance increase amount (OD/min) in m and the absorbance increase amount (OD/min). Example 6 4-Nitrophenyl O-6-anilino-6-deoxy-α-D-glucopyranosyl-(1→4)
-O-α-D-glucopyranosyl-(1→4)-
O-α-D-glucopyranosyl-(1→4)-O
-α-D-glucopyranosyl-(1→4)-O-
Synthesis of α-D-glucopyranoside (anilino-4-nitrophenyl α-maltopentaoside) The same reagents as in Example 1 were used, except that 45 g of aniline was used instead of 45 g of 2-aminopyridine. to the target object.
Obtained 170 mg. [Structure Confirmation] The number of glucose residues relative to the p-nitrophenyl group in the obtained anilino-4-nitrophenyl α-maltopentaoside was measured by the same procedure as in Example 1. As a result, the glucose/4-nitrophenol ratio of anilino 4-nitrophenyl α-maltopentaoside was 3.8. Example 7 4-nitrophenyl O-6-deoxy-6-n
-Propylamino-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-
(1→4)-O-α-D-glucopyranosyl-
(1→4)-O-α-D-glucopyranosyl-
(1→4)-O-α-D-glucopyranoside (n
Synthesis of -propylamino 4-nitrophenyl α-maltopentaoside) In Example 1, except that 29 g of n-propylamine was used instead of 45 g of 2-aminopyridine.
Using the same reagents and performing the same operations as in Example 1, 75 mg of the target product was obtained. [Structure Confirmation] The number of glucose residues relative to p-nitrophenyl groups in the obtained n-propylamino 4-nitrophenyl α-maltopentaoside was measured by the same procedure as in Example 1. As a result, the glucose/4-nitrophenol ratio of n-propylamino 4-nitrophenyl α-maltopentaoside was 3.8. Example 8 The α-amylase activity in the reagent was measured using the following reagent and the following method. Reagent A: 50m
M glucosidase (PH7.0) α-glucosidase 80U/ml Glucoamylase 10U/ml Anilino 4-nitrophenyl α-maltoheptaoside 2mM Reagent B: 50mM glucosidase (PH7.0) α-glucosidase 80U/ml Glucoamylase 10U/ml n-Propylamino 4-nitrophenyl α-maltopentaoside 2mM Measurement method: 3ml each of Reagent A or B was taken and the change over time in a reagent blank at 37°C was measured at 400nm. 3 ml each of reagent A or B was added to 50 µ of sample (serum), and the change in absorbance (α-amylase activity) was measured 5 to 6 minutes after the addition. (Measurement wavelength 400nm,
(Reaction temperature: 37°C) Table 7 shows the change over time of the reagent blank, and Table 8 shows the absorbance measurement of the serum.
【表】【table】
以上述べた如く、本発明は、新規な基質を用い
たα−アミラーゼ活性の新規な測定法を提供する
ものであり、本発明の方法によれば、基質がα−
グルコシダーゼ及びグルコアミラーゼの作用を全
く受けないので、試薬ブランクの上昇が著しく抑
制され、共役酵素液、基質液の一液化が可能とな
る点及びグルコアミラーゼの併用が可能な為、測
定感度が上昇する点等に顕著な効果を奏する発明
である。
As described above, the present invention provides a novel method for measuring α-amylase activity using a new substrate. According to the method of the present invention, the substrate is α-amylase.
Since it is completely unaffected by glucosidase and glucoamylase, the increase in reagent blank is significantly suppressed, and measurement sensitivity is increased because the conjugated enzyme solution and substrate solution can be combined into one solution, and glucoamylase can be used in combination. This invention has a remarkable effect on points, etc.
第1図は、4−ニトロフエニルO−6−デオキ
シ−6−〔(2−ピリジル)アミノ〕−α−D−グ
ルコピラノシル−(1→4)−O−α−D−グルコ
ピラノシル−(1→4)−O−α−D−グルコピラ
ノシル−(1→4)−O−α−D−グルコピラノシ
ル−(1→4)−α−D−グルコピラノシド(ピリ
ジルアミノ4−ニトロフエニルα−マルトペンタ
オシド)のD2O中の1H−NMRスペクトルを示し
たものである。第2図は、実施例5に於て得られ
た検量線を示し、横軸の各α−アミラーゼ活性
(Somogyi単位/dl)について得られた吸光度増
加量(OD/min)を縦軸に沿つてプロツトした
点を結んだものである。
Figure 1 shows 4-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4). -O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (pyridylamino 4-nitrophenyl α-maltopentaoside) D 2 O This shows the 1 H-NMR spectrum of the sample. Figure 2 shows the calibration curve obtained in Example 5, and the increase in absorbance (OD/min) obtained for each α-amylase activity (Somogyi units/dl) on the horizontal axis is plotted along the vertical axis. The points plotted are connected.
Claims (1)
NH−R4(但し、R4は炭素数1〜6のアルキル
基、フエニル基又はピリジル基を示す。)を示し、
R2は、ハロゲン原子又は/及びニトロ基を置換
基として有していてもよいフエニル基を示し、n
は1〜6の整数を示す。]で表わされる非還元末
端を修飾したオリゴサツカライド誘導体を基質と
して、α−グリコシダーゼ及び/又はグルコアミ
ラーゼ及び必要によりβ−グルコシダーゼの共存
下に、試料を接触させ、遊離するフエノール系化
合物を測定することにより、試料中のα−アミラ
ーゼ活性を測定することを特徴とするα−アミラ
ーゼ活性測定法。 2 式 (式中、nは2〜4の整数を示す。) で表わされる非還元末端を修飾したオリゴサツカ
ライド誘導体。[Claims] 1 General formula (): [In the formula, R 1 is a halogen atom, [Formula] -O-R 3 (However, R 3 represents an alkyl group having 1 to 6 carbon atoms) or -
NH-R 4 (wherein R 4 represents an alkyl group having 1 to 6 carbon atoms, a phenyl group or a pyridyl group),
R 2 represents a phenyl group which may have a halogen atom or/and a nitro group as a substituent, and n
represents an integer from 1 to 6. ] A sample is brought into contact with an oligosaccharide derivative modified with a non-reducing end represented by the following as a substrate in the presence of α-glycosidase and/or glucoamylase, and optionally β-glucosidase, and the liberated phenolic compound is measured. 1. A method for measuring α-amylase activity, which comprises measuring α-amylase activity in a sample. 2 formulas (In the formula, n represents an integer of 2 to 4.) An oligosaccharide derivative modified with a non-reducing end represented by:
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24289083A JPS60237998A (en) | 1983-12-22 | 1983-12-22 | Method of measurement of alpha-amylase activity |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP24289083A JPS60237998A (en) | 1983-12-22 | 1983-12-22 | Method of measurement of alpha-amylase activity |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60237998A JPS60237998A (en) | 1985-11-26 |
JPH0424999B2 true JPH0424999B2 (en) | 1992-04-28 |
Family
ID=17095746
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP24289083A Granted JPS60237998A (en) | 1983-12-22 | 1983-12-22 | Method of measurement of alpha-amylase activity |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60237998A (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4649108A (en) * | 1984-07-26 | 1987-03-10 | Genzyme Corporation | Alpha amylase assay |
JPS637797A (en) * | 1986-06-27 | 1988-01-13 | Toyobo Co Ltd | Activity measurement of alpha-amylase |
JPH0630602B2 (en) * | 1986-07-11 | 1994-04-27 | 和光純薬工業株式会社 | A novel method for producing non-reducing end-modified oligosaccharide derivatives |
US5192666A (en) * | 1986-07-11 | 1993-03-09 | Wako Pure Chemical Industries, Ltd. | α-amylase assay using modified oligosaccharide and process for producing said modified oligosaccharide |
US5229270A (en) * | 1986-11-17 | 1993-07-20 | Kanto Kagaku Kabushiki Kaisha | Reagent for the determination of chlorine ion |
DE3743908A1 (en) * | 1987-12-23 | 1989-07-06 | Boehringer Mannheim Gmbh | NEW OLIGOGLUCOSIDE |
JPH0687797B2 (en) * | 1988-12-29 | 1994-11-09 | 三光純薬株式会社 | Method for measuring α-amylase |
DE3929355A1 (en) * | 1989-09-04 | 1991-03-07 | Boehringer Mannheim Gmbh | PROCESS FOR SPECIFIC DETERMINATION OF PANCREAS (ALPHA) AMYLASE |
JP3087891B2 (en) | 1998-03-31 | 2000-09-11 | 東洋紡績株式会社 | Electrolyte measurement reagent composition |
JP2002199898A (en) * | 2000-12-28 | 2002-07-16 | Japan Science & Technology Corp | Method for assaying enzyme activity of glycohyrolase |
-
1983
- 1983-12-22 JP JP24289083A patent/JPS60237998A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS60237998A (en) | 1985-11-26 |
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