JPS6030697A - Novel method and reagent for determination of alpha-amylase activity - Google Patents

Abstract

PURPOSE:To carry out the quantitative determination of alpha-amylase activity, in high accuracy and reproducibility, by using a specific aminoalcohol as a termination agent of enzymatic reaction. CONSTITUTION:The enzymatic activity of alpha-amylase is determined by using glucoamylase as conjugated enzyme and carboxymethylated starch, carboxymethylated amylose, etc. as modified substrate. In the above process, the enzymatic reaction is stopped by adding a reaction terminater comprising the compound of formula I (R<1>-R<4> are H or 1-4C lower alkyl which may have OH group) or its salt in an amount to attain a concentration of >=50-100mM at the termination of the reaction, and thereafter, the alpha-amylase activity is determined by conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and reagent for measuring α-amylase activity.

Test samples such as biological samples, especially saliva, pancreatic juice,
α-Amylase activity in blood and urine is important in medical diagnosis. For example, in pancreatitis, pancreatic cancer, and parotitis, α-amylase activity in blood and urine shows a marked increase compared to normal values.

Various methods for measuring α-amylase activity have been published so far, but they can be mainly classified into three groups: amyloclastic method, chromodanzik method, and sakkalodanzik method.

Among the amyloclastic methods, the caraway method has been the most widely used, but it has problems such as coexisting proteins inhibiting the color formation between starch and iodine, and poor reproducibility due to the short reaction time. Yes 0 The chromodanck method is generally called the blue starch method,
This method uses an insoluble substrate in which a dye is bound to starch or amylose to measure the soluble dye produced in an enzymatic reaction. Although this method has been widely used recently, it has weak activity as a substrate, the reaction system is heterogeneous due to insolubility, and requires complicated operations, making it difficult to apply to automatic analyzers. There are other problems.

The memoji method is a typical example of the Sanorikirogenik method, but
There are problems such as a high value due to glucose in the sample and complicated operations.

As described above, each of the conventional methods for measuring α-amylase activity has its own drawbacks.

In addition, α-amylase is an enzyme that hydrolyzes α-1,4-glycosidic bond chains, so it can be used in polysaccharides with α-1,4-glycosidic bonds such as starch, amylose, and glycogen, or on their substrates. It acts on a modified substrate into which a modifying group has been introduced. α-amylase is made to act on a modified substrate among these substrates, glucoamylase is used as a conjugate enzyme, and the glucose produced is measured.
-There is a way to increase amylase activity. Conventionally, this method uses a so-called rate assay, which spectroscopically captures and measures the reaction rate under certain conditions.
Law was the main practice.

The present inventors believe that if a stopper that can effectively stop the reaction using a modified substrate that provides such a late assay method is found, a method and reagent for measuring α-amylase activity using a one-point assay method will be developed. As a result of extensive research into such reaction terminators, we have found that the compound represented by the general formula O or its salt is extremely effective as such a reaction terminator. H ) i.e.
The present invention uses glycoamylase as a coupling enzyme to
- In measuring the enzymatic activity of amylase, the enzymatic activity of α-amylase can be quantitatively determined using a one-point assay method in which the enzymatic reaction is stopped using a compound represented by the general formula or its salt as a reaction terminator as the reaction progresses. A method and reagent for measuring α-amylase activity.

According to the present invention, since glucoamylase activity can be effectively stopped, α
- Amylase activity can be determined in a limited manner.

Examples of compounds represented by general formula [1] that can be used as reaction terminators in the present invention are (1) to (
8).

(1) 2-amino-2-hydroxymethyl-1,3-propanediol (tris(hydroxymethyl)aminomethane) CHOH 1' HOCH2-C-NH2 CHOH (2) 2-amino-1,3-propanediol- Le HOC
H2-C-NH2 CI-I 0H (3) 2-amino-2-methyl-1,3-propanediol CH3 HOCH2-C-NH2 CH,,0H (4) 2-amino-2-ethyl-1,3 -Bronokundiol 2H5 HOCH2-C-NH2 CH20H (5) 2-amino-1,4-butanediol ■ HOCH2-C-C2H4OH H2 (6) Monoethanolamine ■ HOCH2-C-NH2 (7) Jetanolamine NH (C2H40H)2 (8) Triethalamine N(C2H40H)3 The compounds represented by (1) to (8) are preferably salts of mineral acids such as hydrochloric acid or sulfuric acid, or organic acids such as maleic acid or oxalic acid. It can also be used as a salt.

Among these compounds, tris(hydroxymethyl)aminomethane has been confirmed to have an activity inhibitory effect on glucoamylase.

For example, the literature Agriculture Biological Chemistry Soil 1, 2149-2161 (1977) (A
gric, Biol, Chem, 4±, 2149
2161 (1977)), 1-lis(hydroxymethyl)aminomethane in particular has an activity-inhibiting effect on glucoamylase, and its activity-inhibiting effect is inhibited by Aspergillus awamor.
i) glucoamylase 039 units derived from tris(hydroxymethyl)aminomethane 50mM and pH 5.3,
After heating at 37℃ for 5 minutes, soluble starch (02%
) and measure the residual activity from the amount of glucose produced. The residual activity at that time was 49.9
%.

In addition, other literature Agricultural Biological Chemistry, 2139-2148 (1977) (Ag
ric, Biol, Chem,,'dan, 2139
2148 (1977)), gel core mirror-1g+d derived from Mucor rouxianns was inhibited in 5 mM of tris(hydroxymethyl)aminomethane at pH 5.3, but the residual activity was It is 26.7% or 288%.

In this way, in these documents, tris(hydroxymethyl)
Although aminomethane has been shown to have an inhibitory effect on glucoamylase, the inhibitory effect is not complete and considerable residual activity is observed. On the other hand, in a one-point assay method that stops an enzymatic reaction in progress, the performance of a reaction terminating agent is required in order to obtain accurate and reproducible enzyme activity measurement results. It is necessary to inhibit enzyme activity almost completely.

Therefore, it was naturally determined that tris(hydroxymethyl)aminometh/, which does not have a complete inhibitory effect on glucoamylase, cannot be used as a reaction terminator for glucoamylase in progress.

However, the present inventors came up with the idea that the inhibitory effect of tris(hydroxymethyl)aminomethane on glucoamylase might be due to competitive inhibition with the substrate, and used a modified substrate to react tris(hydroxymethyl)aminomethane. When used as a reaction stopper for glucoamylase in progress, glucoamylase activity can be particularly effectively stopped, and α-amylase activity can be measured with accurate and reproducible values. After obtaining this knowledge and conducting extensive research on other similar compounds, the present invention was completed.

That is, although the mechanism of the inhibitory effect of tris(hydroxymethyl)aminomethane on glucoamylase has not yet been clarified, in the measurement of α-amylase activity using a modified substrate as in the present invention, glucoamylase is inhibited by the enzymatic action of α-amylase. Since the amylase substrate is produced little by little, the concentration ratio of the inhibitor to the substrate increases, and the idea was that the inhibition efficiency would improve accordingly.

Hereinafter, the present invention will be described in detail by citing its typical embodiments.

In the present invention, α-amylase activity is determined by measuring the glucose produced by reacting glucoamylase, a conjugated enzyme, on a modified substrate simultaneously or subsequently with α-amylase. An example of this reaction formula is shown below. show.

Gn-G-G~ (In the above formula, G represents a glucose unit, X represents a modifying group such as a caleboxymethyl group, and n and m represent an integer of 2 or more.) Many methods for quantifying glucose are known. , it goes without saying that any of these methods can be used.

The main glucose quantification methods are shown below.

First, when glucose oxidase is applied to glucose, it is oxidized and at the same time hydrogen peroxide is produced.

The generated hydrogen peroxide electrically oxidizes the chromogen through coexisting peroxidase, and the amount of glucose in the reaction solution can be measured by comparing the color of the generated oxidized chromogen. can. The reaction formula is shown below.

Glucose oxidase glucose + 0□ + H2O H2O□ + gluconate peroxidase H2O2 + chromogen oxidized chromogen H20 In addition, glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. The produced glucose-6-phosphate becomes 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase in the presence of NAD, while NAD is reduced to NADH.
Measure the amount of glucose in the reaction solution by measuring the increase in absorbance of H at around 340 nm;
, iJ'come.

The reaction formula is shown below.

Glucose - Glucose - 6-phosphate + NAD□ 6-phosphate dehydrogenase -------→6-phosphog! Reconolactone-NADH ATP; Adenosine-5'-triphosphate ADP;
Adenosine-57-diphosphate NAD; nicotinamide adenine dinucleotide NADH; reduced W-nicotinamide adenine dinucleotide α-amylase and glucoamylase, which is a coupled enzyme, acts simultaneously or subsequently to measure glucose produced. may be affected by glucose coexisting in the specimen, especially biological samples such as serum and urine, and it is important to carry out this measurement method to eliminate existing glucose prior to the reaction of it-amylase. This is an effective method for the above.

Methods of elimination include glucose oxidase-catalase and hexokinase (Japanese Patent Application Laid-open No. 1983-1999).
There are various methods such as the method using glucose oxidase-peroxidase) and the method using glucose oxidase-peroxidase, and any of these methods can be freely combined.

The sample to be measured in the present invention may be any specimen containing α-amylase, such as blood, serum, urine, etc. as biological components.

Glucoamylase is not particularly limited, but glucoamylase derived from animals or microorganisms can be used, for example.

Preferred substrates for use in the present invention are polysaccharides containing modified groups at non-reducing ends to the extent that they can serve as substrates for α-amylase, such as sodium starch glycolate (carboxymethylated starch) described in the literature "Clinica Himi Force Acta n (1977) 277-283 (C1in
ica Chimica Acta.

LfL (1977) 277-283. ,! J or carboxymethylated amylose (patent application 14875/1987)
No. 6) etc. Modifying groups 7) Those containing carboxyvinyl groups are particularly preferred from the viewpoint of solubility, but in addition to carboxymethyl groups, 2-pyridylamino groups, 3-pyridylamino groups, anilino groups, 2-hydroxyanilino groups, etc. Those containing a group, methylanilino group, carboxyphenylamino group, etc. can be used without any problem, and there are no particular restrictions on the modifying groups other than these. When a polysaccharide having an α-1,4-glycosidic bond such as starch or amylose is used as a substrate, glucoamylase is - α-1,4- from the non-reducing end of amylose regardless of amylase
Since it is an echin-type enzyme that hydrolyzes glycosidic bonds, it has the disadvantage of degrading the substrate regardless of the α-amylase reaction, so (1) it cannot contain both the substrate and glucoamylase in the measurement reagent solution. This is not preferable because problems such as 2) not being able to use enough glucoamylase for measurement, and (3) an increase in the number of reagents tested are caused.

Further, as a measurement condition for carrying out the present invention, a particularly effective concentration of the compound represented by the general formula (0) at the time of reaction termination is 50 to 100 mM or more.

The reaction temperature of the amylase reaction and the conjugate enzyme is not particularly limited, but is preferably about 25 to 40°C, and the reaction time can be freely selected depending on the purpose.

The optimum pH for the reaction of amylase and the conjugate enzyme is not particularly limited, but a preferred example is a pH of about 6 to 8. Buffers that maintain the optimum pH can be freely used, and include, for example, phosphate and gas buffers. - Although the final liquid properties vary slightly depending on the glucose measurement system, p
H6,5-85 are preferred.

Further, as an activator for α-amylase, for example, sodium chloride, calcium chloride, potassium chloride, etc. are used.

When performing manual late assay methods that do not use conventional reaction terminators, it is difficult to obtain accurate and reproducible measurement values unless strict restrictions on measurement conditions are observed, and it is difficult to obtain accurate and reproducible measurement values. However, according to the present invention, the reaction can be stopped using a reaction stopping agent.
-Measurements can be made manually without the need for special automatic analysis equipment, expanding the practical range of measurement methods.
Furthermore, it has become possible to measure multiple samples simultaneously using a manual method. Furthermore, since the reaction stops extremely quickly, there are few measurement errors and accurate values with good reproducibility can be obtained. Furthermore, since a strong acid or a strong alkali is not required and a mild liquid property around neutrality can be selected, it is easy to maintain stable conditions for coloring. There are great things to contribute to this industry in these axes as well.

Examples will be shown below and explained in more detail, but the present invention is not limited thereto.

Below: Spicy Example 1 Reagent (1) Test solution 1 Gud buffer solution (PI PES) ], Ornmo
l, glucoamylase 20,000 units, glucose oxidase l.

10,000 units, mutarotase 200 units, catalase 500,000 units, N,N-diethylxylidine 1 mmol, togram chloride 15 mmol, calcium chloride 5 rnrno7
Dissolve it in purified water and hydroxylate]・pH 6 in grams,
9 and the total amount as 11.

(2) Test solution 2 Kuhnod buffer (PI PES) 20 mmol, 5,000 units of oxidase/l, sodium chloride 1
Mu1 for 5 5rnmol, 4-aminoantipyri 7
Take 0.5 mmol, dissolve it in purified water, adjust the pH to 6.9 with sodium hydroxide, and bring the total amount to ]l.

(3) Reaction stop layer tris(hydroxymethyl)aminomethane 06mail
Dissolve in purified water, add hydrochloric acid to adjust the pH to 7.5, and bring the total volume to 14.

Measurement method Add 10 μl of sample serum to 1 ml of test solution 1 and heat at 37°C.
Heat for 5 minutes. Add 1 ml of test solution 2 to this,
The reaction is carried out at 7°C for 10 minutes. Add reaction stop solution 2 to this solution.
ml to stop the reaction, and measure the absorbance at 620 nm.

Especially! - Using a standard specimen with known amylase activity, prepare a calibration curve in the same manner as above, and calculate the α-ε-lase activity of the specimen from this calibration curve.

As is clear from FIG. 1, the absorbance after adding the reaction stop solution is constant.

Example 2 Reagent (1) Test solution 1 Potassium phosphate 20mITlc+l Glucoamylase 300,000 units, Hexokinase 10,000 units, Glucose-6-phosphate dehydrogenase 10,000 units, Magnesium chloride 5mmc
+1 rNAD (oxidized form = cotinamide adenine dinucleonade) 2 mmol, ATP (adenosine diphosphoric acid) 2 min'c+l + Dissolve 3 g of carboxymethyl starch in purified water and add potassium hydroxide (I)
H7. O and the total amount is 11.

(2) Reaction stop solution tris(hydroxymethyl)aminomethane 0.4mc+
Dissolve 1 in purified water and add hydrochloric acid to pH 7. 0
and the total amount is 11.

Measurement method: Add 20 μl of sample serum to 2 ml of sample solution 1 and heat at 37° C. for 10 minutes. 1 mi of reaction stop solution is added to this solution to stop the reaction, and the absorbance of 340 nlT] is measured.

As a sample-blind test, absorbance at 340' nm is measured using a test solution obtained by removing only carboxymethyl starch from Test Solution 1 in place of the above-mentioned Test Solution 1 and carrying out the same procedure.

Separately, a standard specimen with known α-amylase activity is used in the same manner as above, a calibration curve is prepared by subtracting the blinded value from each specimen, and the amylase activity of each specimen is calculated from this calibration curve.

Example 3 Reagent (1) Test solution 1 Gutud buffer e, (PIPES) 10 mmol, glucoamylase 20,000 units, glucose oxidase], about 100,000 years, mutarotase 100 units, catalase 500,000 units, N,N-diethylxylidine 1 mm0l Take 15 mmol of sodium chloride and 5 mmol of calcium chloride, dissolve in purified water, and adjust the pH to 6.9 with sodium hydroxide to bring the total amount to 11.

(2) Test solution 2 Gudo buffer (PIPES) 2Qmmol11, A-oxidase 5,000 units, thorium chloride 1
5rnmol.

6g carboxymethylated amylose, 11g sodium nitride
Take 5 mmol, 70.5 mmo7 of 4-aminoantipyri, dissolve it in purified water, adjust the pH to 6.9 with sodium hydroxide, and bring the total amount to 11.

(3) Dissolve 6 mol (36 g) of monoethanolamine o, the reaction stop solution, in purified water, add hydrochloric acid to adjust the pH to 7.5, and bring the total amount to 11
Measurement method: Add 10 μl of sample serum to 1 ml of test solution 1 and heat at 37° C. for 5 minutes. Add 1 ml of test solution 2 to this and
The reaction is carried out for 10 minutes at °C. Add 2 m of reaction stop solution to this solution.
1 is added to stop the reaction, and the absorbance at 6201 m is measured.

Separately, prepare a standard curve in the same manner as above using a standard sample with known α~amylase activity, and from this calibration curve
- Measure amylase activity.

[Brief explanation of drawings]

Figure 1 shows the absorption after adding test solution 2 when using 80 Somogyi units/di serum in Example 1.
Wako Pure Chemical Industries Co., Ltd. Procedural Amendments Showa I; 2nd year/Rho month 15th 1, Indication of incident Roda? 4-# Oreishu Mitsutashi /3ri3//艮L2
Title of the invention 3 Relationship to the case of the person making the amendment Patent applicant postal code 541 Consecutive address 4? Department (Tokyo) TEt, 03-270-8
5714 Date of amendment order5, number of inventions reduced by amendment6, column for title of invention, column for scope of claims, and column for detailed description of invention of the specification to be amended. 7. Contents of amendment (1) "Novel method and reagent for measuring α-amylase activity" written in the title of the invention column is amended to read "Novel method for measuring α-amylase activity". (2. The claims are amended as shown in the attached sheet. (3) "Glycoamylase" described on page 7, line 9 of the specification
Correct it with ``glucoamylase''. (4) "Glucose 6-phosphate" described from line 4 on page 15 of the specification to line 6 on page 15 is corrected to "glucose-6-phosphate." (5) Corrected to "N,N-diethylquinlysinej'krN,N-diethylquinlysine" as described in page 20, line 7 of the specification. (6) Amended to “N,N-diethylquinlysine Ji “N,N-diethylxylysine 7” as stated in page 23, line 7 of the specification.” Attachment 2, Claims (1) Glucoamylase α-
In measuring the enzymatic activity of amylase, the enzymatic reaction is stopped using a compound represented by general formula [1] or a salt thereof as a reaction terminator as the reaction progresses, and the enzymatic activity of α-amylase is quantitatively measured. A method for measuring α-amylase activity, characterized by: R' (However, R1-R4 represent hydrogen or a lower alkyl group having 1 to 4 carbon atoms which may have an -OH group.) (2) Modified substrate for measuring the enzymatic activity of α-amylase A method for measuring α-amylase activity according to claim 1 using the method. (3) The modified substrate is carboxine methylated starch. or carboxymethylated amylose, the method for measuring α-amylase activity according to claim 2. (4) The concentration of the compound represented by the general formula [1] or its salt that completely stops the enzyme reaction at the time of stopping the reaction is 50 to 100.
The method for measuring α-amylase activity according to claim 1, 2, or 3, wherein the α-amylase activity is +uM or more. (5) The compound represented by the general formula CD is 2-amino-2
-Hydroxymethyl-1,3-propanediol (tris(hydroxymethyl)aminomethane), 2-amino-
1,3-propanediol, 2-amino-2-methyl-
1,3-propanediol, 2-amino-2-ethyl-
Claims 1 and 2 are 1,3-propanediol, 2-amino-1,4-propanediol, monoethanolamine, jetanolamine, or triethanolamine. The method for measuring α-amylase activity described in Section 3 or Section 4.

Claims (1)

[Claims] (1) Using glucoamylase as a coupling enzyme to
In measuring the enzymatic activity of amylase, the enzymatic reaction is stopped using a compound represented by general formula (1) or a salt thereof as a reaction terminator as the reaction progresses, and the enzymatic activity of α-amylase is quantitatively measured. A method for measuring α-amylase activity, characterized by: I R3 HOCH2-C-N-R' -, general formula []]2 (However, R1-R4 represents hydrogen or a lower alkyl group having 1 to 4 carbon atoms which may have a -〇H group. −j′0)
(2) The method for measuring α-amylase activity according to claim 1, which uses a modified substrate in measuring the enzymatic activity of α-amylase. (3) The method for measuring α-amylase activity according to claim 2, wherein the modified substrate is carboxymethylated starch or carboxymethylated amylose. (4) The concentration of the compound represented by the general formula for stopping the enzyme reaction or its salt at the time of stopping the reaction is 50 to 100 mM
Claims 1, 2, or 3, which are the above.
The method for measuring α-amylase activity described in Section 1. (5) The compound represented by the general formula 〇〇 is 2-amino-2
-Hydroxymethyl-1,3-propanediol (tris(hydroxymethyl)aminomethane), 2-amino-
1,3-propanediol, 2-amino-2-methyl-
1,3-propanediol, 2-amino-2-ethyl-
Claims 1, 2, 3, or 1,3-propanediol, 2-amino-1,4-butanediol, monoethanolamine, diethanolamine, or triethanolamine; 5. The method for measuring α-amylase activity according to item 4. (α-glucoamylase was used as a coupling enzyme to
In measuring the enzymatic activity of amylase, the enzymatic reaction is stopped using a compound represented by the general formula symbol or its salt as a reaction terminator as the reaction progresses, and the enzymatic activity of α-amylase is quantitatively measured. A reagent for measuring α-amylase activity. RI R3 2 (However, RI-R4 represents hydrogen or a lower alkyl group having 1 to 4 carbon atoms which may have a -0H group.) (7
) The reagent for measuring α-amylase activity according to claim 6, which uses a modified substrate in measuring the enzymatic activity of α-amylase. (8) (A reagent for measuring α-amylase activity according to claim 7, wherein the Ili substrate is carboxymethylated starch or carboxymethylated amylose. (9) General formula for stopping the enzyme reaction. ] The concentration of the compound or its salt at the time of reaction termination is 50 to 100mM
Claims 6, 7, or 8 which are the above.
A reagent for measuring α-amylase activity as described in Section 1. . (10) The compound represented by the general formula [l] is 2-amino-2-hydroxymethyl-1,3-propanediol (
tris(hydroxymethyl)aminomethane). 2-amino-1,3-propanediol, 2-amino-
2-methyl-1,3-propanediol, 2-amino-
2-ethyl-1,3-furopanediol, 2-amino-
Measurement of α-amylase activity according to claim 6, 7, 8, or 9, which is 1,4-butanediol, monoethanolamine, jetanolamine, or triethanolamine. reagent for use.