JPH0542275B2 - - Google Patents
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- Publication number
- JPH0542275B2 JPH0542275B2 JP2843884A JP2843884A JPH0542275B2 JP H0542275 B2 JPH0542275 B2 JP H0542275B2 JP 2843884 A JP2843884 A JP 2843884A JP 2843884 A JP2843884 A JP 2843884A JP H0542275 B2 JPH0542275 B2 JP H0542275B2
- Authority
- JP
- Japan
- Prior art keywords
- amylase
- glucosidase
- measuring
- nad
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明はG6P結合オリゴ糖または６−PG結合
オリゴ糖を基質として用いるα−アミラーゼの測
定法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring α-amylase using a G6P-linked oligosaccharide or a 6-PG-linked oligosaccharide as a substrate.
α−アミラーゼは人体内では主として膵臓又は
唾液腺で生成され、ある種の疾患では血清や尿等
の体液中ヘアミラーゼが逸脱してくる。血清や尿
中α−アミラーゼを測定することによつて臨床上
有用な診断が可能である。例えば血清中のα−ア
ミラーゼ活性は健康人ではほぼ一定の値を示す
が、急性膵炎の患者の場合には血清中のα−アミ
ラーゼ含量は上昇し、膵機能等の重要な臨床上の
パラメーターになる。 In the human body, α-amylase is mainly produced in the pancreas or salivary glands, and hair amylase in body fluids such as serum and urine becomes deviant in certain diseases. Clinically useful diagnosis is possible by measuring α-amylase in serum or urine. For example, α-amylase activity in serum shows a nearly constant value in healthy people, but in patients with acute pancreatitis, the content of α-amylase in serum increases and affects important clinical parameters such as pancreatic function. Become.
従来、α−アミラーゼの測定法はヨウ素−デン
プン反応法、比濁法、ブルースターチ法や各種の
酵素法が報告されまたは実施されているが非常に
問題が多い。例えばα−アミラーゼの基質として
デンプンを使用するヨウ素−デンプン反応法や比
濁法においては一定品質のデンプンを常時得るこ
とが著しく困難である。またブルースターチ法で
は定量操作中遠心分離を必須とするため、自動分
析機器への応用が困難となる他、レイトアツセイ
法で反応速度を測定することができない。酵素法
においても、血清や尿サンプルからの持ち込みの
グルコースやマルトースの消去が難しい。オリゴ
糖の還元性末端水酸基に芳香族置換体（パラニト
ロフエノール（pNP）等）を基質として用いる
発色法も提案されている。これらの基質は何れも
α−アミラーゼにより該分子中のα−1.4−グル
コシド結合が切断され、生じた生成物はさらに外
部より添加したα−グルコシダーゼ、β−グルコ
シダーゼ等の共役酵素により分解され、最終的に
遊離のpNPが生じる。この生じたpNPを400nｍ
付近にて測定することにより、α−アミラーゼ活
性を測定しようという方法である。 Conventionally, methods for measuring α-amylase have been reported or practiced, such as the iodine-starch reaction method, the turbidimetric method, the blue starch method, and various enzyme methods, but these methods have many problems. For example, in the iodine-starch reaction method and nephelometry, which use starch as a substrate for α-amylase, it is extremely difficult to consistently obtain starch of constant quality. In addition, the blue starch method requires centrifugation during the quantitative operation, making it difficult to apply to automatic analysis equipment and making it impossible to measure reaction rates using the late assay method. Even with enzymatic methods, it is difficult to eliminate glucose and maltose carried over from serum and urine samples. A coloring method using an aromatic substituted substance (such as paranitrophenol (pNP)) for the reducing terminal hydroxyl group of an oligosaccharide has also been proposed. In each of these substrates, the α-1,4-glucosidic bond in the molecule is cleaved by α-amylase, and the resulting product is further degraded by externally added coupled enzymes such as α-glucosidase and β-glucosidase, resulting in the final product. free pNP is generated. This generated pNP was measured at 400nm.
This method attempts to measure α-amylase activity by measuring in the vicinity.
しかしながらこの方法において、最終測定対象
となるpNPの吸収極大が400nｍ付近にあるため、
ビリルビンやヘモグロビン等の干渉物質の影響を
受けやすく、正確なアミラーゼ値が得られないと
いう欠点を有している。 However, in this method, the absorption maximum of pNP, which is the final measurement target, is around 400 nm, so
It has the disadvantage that it is easily affected by interfering substances such as bilirubin and hemoglobin, and accurate amylase values cannot be obtained.
そこで本発明者等は鋭意研究を重ねた結果、前
記欠点を解決し、本発明を完成するに至つた。 As a result of extensive research, the inventors of the present invention have solved the above-mentioned drawbacks and completed the present invention.
α−アミラーゼ活性を精度よく、しかも迅速に
測定できる方法としてはα−アミラーゼの基質と
してG6P結合オリゴ糖または6PG結合オリゴ糖を
使用することによつて目的が達せられる。 As a method for accurately and rapidly measuring α-amylase activity, the objective can be achieved by using a G6P-linked oligosaccharide or a 6PG-linked oligosaccharide as a substrate for α-amylase.
すなわち、本発明はα−アミラーゼ活性を測定
する際に、G6P結合オリゴ糖または6PG結合オリ
ゴ糖を基質としてこれにα−アミラーゼを含有す
る試料を添加すると同時に、または添加後にα−
グルコシダーゼまたはβ−グルコシダーゼを作用
させ、その生成物であるG6Pまたは6PGを酵素的
に測定することを特徴とするα−アミラーゼ測定
法である。 That is, when measuring α-amylase activity, the present invention uses a G6P-linked oligosaccharide or 6PG-linked oligosaccharide as a substrate and adds an α-amylase-containing sample simultaneously or after the addition of α-amylase.
This is an α-amylase measurement method characterized by allowing glucosidase or β-glucosidase to act and enzymatically measuring the product G6P or 6PG.
G6Pまたは6PGを酵素的に測定する方法として
は、共役酵素としてG6PDHまたは6PGDHを補
酵素としてNADまたはNADPを用い、G6Pまた
は6PGを最終的にNADHまたはNADPHの340n
ｍにおける吸光度の変化量としてとらえようとす
るものである。 A method for enzymatically measuring G6P or 6PG is to use G6PDH or 6PGDH as a conjugate enzyme and NAD or NADP as a coenzyme, and finally convert G6P or 6PG into 340n of NADH or NADPH.
It is intended to be understood as the amount of change in absorbance at m.
本発明によれば、最終的に生じたNADHまた
はNADPHを340nｍの紫外線にて測定するため、
上記ビリルビンやヘモグロビンの干渉物質を受け
ず、さらに単一化合物であるため一定した品質を
有し、自動分析機器への応用も可能である。 According to the present invention, in order to measure the finally produced NADH or NADPH using 340 nm ultraviolet light,
It is not affected by the above-mentioned interfering substances such as bilirubin and hemoglobin, and since it is a single compound, it has a constant quality and can be applied to automatic analysis equipment.
また、エンドポイントだけでなくレイトアツセ
イ法にも使用でき、アミラーゼ活性をグルコース
量として測定する系でないため、グルコースやマ
ルトースの除去をも必要としない特徴を有する。 In addition, it can be used not only for endpoints but also for late assays, and because it is not a system that measures amylase activity as the amount of glucose, it has the characteristic that it does not require the removal of glucose or maltose.
つぎに本発明における反応を式により説明する
と次のとおりである。 Next, the reaction in the present invention will be explained using the following formula.
(A) 基質として例えばG6P結合マルトペンタオー
ス（G5−G6P）を用いる場合
G5−G6P＋H2Oα−アミラーゼ
−−−−−−−→
マルトトリオース＋G2−G6P
G5−G6P＋2H2Oα−グルコシダーゼ
−−−−−−−−−→
２グルコース＋G6P
G6P＋NAD(P)+G6P脱水素酵素（G6PDH）
−−−−−−−−−−−−−→
６−PG＋NAD(P)Ｈ
(B) 基質として例えば６−PG結合マルトペンタ
オース（G5−６−PG）を用いる場合
G5−６−PG＋H2Oα−アミラーゼ
−−−−−−−→
マルトトリオース＋G2−６−PG
G2−６−PG＋2H2Oα−グルコシダーゼ
−−−−−−−−−→
２グリコース＋６−PG
６−PG＋NAD(P)+６−PG脱水素酵素（６−PGDH）
−−−−−−−−−−−−−−→
リブロース−５−リン酸＋NAD(P)Ｈ
上記反応により生成したNAD(P)Ｈを340nｍの
吸光度の増加で測定してもよいし、その後酸化還
元色素系を連結して発色で測定することも可能で
ある。さらに(A)の場合６−PGDHを加えること
によつて感度を２倍に上げることもできる。(A) For example, when G6P-bound maltopentaose (G 5 -G6P) is used as a substrate, G 5 -G6P+H 2 Oα-amylase−−−−−−→ maltotriose+G 2 −G6P G 5 −G6P+2H 2 Oα− Glucosidase −−−−−−−−→ 2 glucose + G6P G6P + NAD(P) + G6P dehydrogenase (G6PDH) −−−−−−−−−−−−−→ 6−PG + NAD(P)H (B) For example, when using 6-PG-bonded maltopentaose (G 5 -6-PG) as a substrate, G 5 -6-PG + H 2 Oα-amylase −−−−−−→ maltotriose + G 2 −6-PG G 2 −6−PG+2H 2 Oα-glucosidase−−−−−−−−−→ 2glyose+6−PG 6−PG+NAD(P) + 6−PG dehydrogenase (6−PGDH) −−−−−−−−− −−−−−→ Ribulose-5-phosphate + NAD(P)H The NAD(P)H produced by the above reaction may be measured by the increase in absorbance at 340 nm, or after that, the redox dye system may be connected. It is also possible to measure by color development. Furthermore, in the case of (A), the sensitivity can be doubled by adding 6-PGDH.
本発明によればα−アミラーゼを含有する試料
中のα−アミラーゼ活性を従来法に比し短時間で
精度よく、しかもブランク値の上昇もなく、また
内因性のグルコースやマルトースのえいきようを
全くうけないで測定することが可能となるので本
発明は産業上極めて有意義な方法である。 According to the present invention, α-amylase activity in a sample containing α-amylase can be measured in a shorter time and more accurately than conventional methods, and there is no increase in blank values, and the effects of endogenous glucose and maltose can be measured. The present invention is an industrially extremely useful method because it enables measurement without any exposure.
実施例 １
50ｍＭ FIPES（PH6.8）、５ｍＭ MgCl2、１
ｍＭ NAD+、２ｍＭ G5−G6P、10u／ml α
−グルコシダーゼおよび2u／mlG6PDHを含む反
応液２mlにヒトアミラーゼ溶液を０〜50μ入れ
25℃で340nｍの吸光度の増加を５分間測定し、
１分間あたりの反応速度を読みとつた。
Example 1 50mM FIPES (PH6.8), 5mM MgCl 2 , 1
mM NAD + , 2mM G5 -G6P, 10u/ml α
- Add 0-50μ of human amylase solution to 2ml of reaction solution containing glucosidase and 2u/ml G6PDH.
Measure the increase in absorbance at 340 nm at 25°C for 5 minutes,
The reaction rate per minute was read.
この結果、サンプル量に対して酵素活性が比例
しており、充分α−アミラーゼ活性の測定が可能
であることが認められた。 As a result, it was confirmed that the enzyme activity was proportional to the sample amount, and that α-amylase activity could be sufficiently measured.
実施例 ２
50ｍＭ PIPES、５ｍＭ MgCl2、１ｍＭ
NADP+、２ｍＭ G5−６−PG、10u／ml α−
グリコシダーゼおよび3u／ml6PGDHを含む反応
液２mlにヒトアミラーゼ溶液を０〜50μ入れ25
℃で340nｍの吸光度の増加を５分間測定し、反
応速度を読みとつた。
Example 2 50mM PIPES, 5mM MgCl2 , 1mM
NADP + , 2mM G5-6 -PG, 10u/ml α-
Add 0 to 50μ of human amylase solution to 2ml of reaction solution containing glycosidase and 3u/ml6PGDH25
The increase in absorbance at 340 nm was measured at ℃ for 5 minutes to read the reaction rate.
この結果、サンプル量に対して酵素活性が比例
しており、充分α−アミラーゼ活性の測定が可能
であることが認められた。 As a result, it was confirmed that the enzyme activity was proportional to the sample amount, and that α-amylase activity could be sufficiently measured.
Claims (1)
料を添加すると同時に又は添加した後に、α−グ
ルコシダーゼまたは／およびβ−グルコシダーゼ
を作用させ、その生成物であるグルコース−６−
リン酸（G6P）または６−ホスホグルコン酸（６
−PG）を測定することを特徴とするα−アミラ
ーゼの測定方法。[Claims] 1. When measuring α-amylase, (wherein n is an integer in the range of 3 to 6) or (wherein n is an integer in the range of 3 to 6) is used as a substrate, and at the same time or after the addition of a sample containing α-amylase, α-glucosidase or/and β-glucosidase is allowed to act on it. , its product glucose-6-
Phosphoric acid (G6P) or 6-phosphogluconic acid (6
-PG).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2843884A JPS60172299A (en) | 1984-02-20 | 1984-02-20 | Method of measurement of amylase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2843884A JPS60172299A (en) | 1984-02-20 | 1984-02-20 | Method of measurement of amylase activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60172299A JPS60172299A (en) | 1985-09-05 |
| JPH0542275B2 true JPH0542275B2 (en) | 1993-06-28 |
Family
ID=12248671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2843884A Granted JPS60172299A (en) | 1984-02-20 | 1984-02-20 | Method of measurement of amylase activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60172299A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5043436A (en) * | 1986-11-20 | 1991-08-27 | Kurita Water Ind., Ltd. | Substrate for measurement of alpha-amylase activity |
-
1984
- 1984-02-20 JP JP2843884A patent/JPS60172299A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60172299A (en) | 1985-09-05 |
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