JPH0424040B2 - - Google Patents

Description

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

α-Amylase activity in test samples such as biological samples, particularly in human saliva, pancreatic juice, blood, and urine, is important in medical diagnosis. For example, pancreatitis,
In pancreatic cancer and parotitis, α-
Amylase activity shows a significant 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, chromogenic method, and satcalogenic method.

Among the amyloclastic methods, the callaway method has been the most widely used, but because coexisting proteins inhibit the coloration of starch and iodine,
There are problems such as poor reproducibility due to the short reaction time.

The chromogenic method is generally referred to as the blue starch method, and is a method for measuring soluble pigments produced in an enzymatic reaction using an insoluble substrate in which a pigment is bound to starch or amylose. 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 Somogyi method is a typical Satskalogenik method, but it shows high values due to glucose in the sample.
There are problems such as 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-glycoside bond chains, so α-1,4-
Polysaccharides with glycosidic bonds or substrates with modified groups introduced into these polysaccharides (among these,
α- with a modification group introduced at least at the non-reducing end
A substrate that is a polysaccharide having a 1,4-glycosidic bond is hereinafter abbreviated as a modified substrate. ), but α-amylase is treated with a modified substrate among these substrates, glucoamylase is used as a conjugate enzyme, and the glucose produced is measured to determine α-amylase.
There is a method to determine amylase activity. Conventionally, this method has mainly been carried out using the so-called rate assay method, which spectroscopically captures and measures the reaction rate under certain conditions.

The present inventors believe that if a termination agent is found that can effectively stop reactions using modified substrates that provide such a late assay method, one-point assay method (i.e., measuring the rate of reaction) will be possible. (a method in which the reaction is assumed to be progressing at a constant rate, the reaction is stopped after a certain period of time, the concentration of the substance produced in the reaction system is measured, and the concentration is converted into the reaction rate). Focusing on the fact that a method for measuring amylase activity can be easily obtained, as a result of intensive research into such a reaction terminator, 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-propanediol, 2-amino-1,4-butanediol, monoethanolamine, diethanolamine, triethanolamine or salts thereof are highly effective as such reaction terminators. We have discovered that this is the case, and have completed the present invention.

That is, in measuring the enzymatic activity of α-amylase using glucoamylase as a conjugated enzyme and a modified substrate, the present invention uses 2-amino-2-hydroxymethyl-1,3 as a reaction terminator during the progress of the reaction. -Propanediol [tris(hydroxymethyl)aminomethane], 2-amino-1,3
-Propanediol, 2-amino-2-methyl-
1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, 2-amino-
The enzymatic reaction is stopped using 1,4-butanediol, monoethanolamine, diethanolamine, triethanolamine or salts thereof, and α
- A method for measuring α-amylase activity characterized by quantitatively measuring the enzymatic activity of amylase.

According to the present invention, since glucoamylase activity can be effectively stopped, α-amylase activity can be quantitatively measured with accurate and reproducible values.

Compounds that can be used as reaction terminators in the present invention are listed below along with their structural formulas. (1) to (8).

(1) 2-amino-2-hydroxymethyl-1,3
-Propanediol [tris(hydroxymethyl)aminomethane] (2) 2-amino-1,3-propanediol (3) 2-amino-2-methyl-1,3-propanediol (4) 2-Amino-2-ethyl-1,3-propanediol (5) 2-amino-1,4-butanediol (6) Monoethanolamine (7) Diethanolamine NH (C ₂ H ₄ OH) ₂ (8) Triethalamine N (C ₂ H ₄ OH) ₃ The compounds represented by (1) to (8) are preferably salts, preferably minerals such as hydrochloric acid or sulfuric acid. It can also be used as a salt of an acid or an organic acid such as maleic acid or oxalic acid.

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

For example, the literature Agricultural Biological Chemistry 41 , 2149-2161 (1977)
According to [Agric.Biol.Chem., 41 2149-2161 (1977)], tris(hydroxymethyl)aminomethane in particular has an activity-inhibiting effect on glucoamylase, and its activity-inhibiting effect can be observed in Aspergillus awamori. After heating 0.39 units of glucoamylase derived from tris(hydroxymethyl)aminomethane 50mM and pH 5.3 at 37℃ for 5 minutes, add soluble starch (0.2%) and measure the residual activity from the amount of glucose produced. I'm looking for it. The residual activity at that time was 49.9%.

Also, other references Agricultural Biological Chemistry 41 , 2139-2148 (1977)
According to [Agric.Biol.Chem., 41 , 2139-2148 (1977)] Mucor rouxianns
The derived glucoamylase is inhibited by tris(hydroxymethyl)aminomethane at 5mM and pH5.3, but the residual activity is 26.7% or 28.8%.

As described above, these documents introduce that tris(hydroxymethyl)aminomethane has an inhibitory effect on glucoamylase, but the inhibitory effect is not complete and considerable residual activity is observed. On the other hand, in the one-point assay method that stops the oxygen reaction in progress, the performance of the reaction terminating agent required to obtain accurate and reproducible enzyme activity measurement results is as follows: Moreover, it is necessary to almost completely inhibit enzyme activity. Therefore, it was naturally determined that tris(hydroxymethyl)aminomethane, 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, the enzyme action of α-amylase can be used. As a result, the substrate for glucoamylase is produced little by little, so the concentration ratio of the inhibitor to the substrate increases, leading to an idea that the inhibition efficiency improves.

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

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

(In the above formula, G represents a glucose unit, X represents a modifying group such as a carboxymethyl group, and n and m represent an integer of 2 or more.) There are many known methods for quantifying glucose, and none of these methods Needless to say, it 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 quantitatively oxidizes the chromogen through the peroxidase present, and the amount of glucose in the reaction solution can be measured by comparing the color of the generated oxidized chromogen. . The reaction formula is shown below.

Glucose + O ₂ + H ₂ O glucose oxidase----------→ H ₂ O ₂ + gluconic acid H ₂ O ₂ + chromogen peroxidase----------→ Oxidation type chromogen + H ₂ O 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, and on the other hand, NAD is reduced to NADH, so the By measuring the increase in absorbance, the amount of glucose in the reaction solution can be measured.
The reaction formula is shown below.

Glucose + ATP hexokinase----------- → Mg ²⁺ Glucose-6-phosphate + ADP Glucose-6-phosphate + NAD Glucose-6-phosphate dehydrogenase----------- −−――――――→ 6-phosphogluconolactone + NADH ATP; Adenosine-5′-triphosphate ADP; Adenosine-5′-diphosphate NAD; Nicotinamide adenine dinucleotide NADH; Reduced form - Nicotinamide When measuring glucose produced by the action of glucoamylase, a conjugated enzyme, simultaneously with adenine dinucleotide α-amylase or subsequently, it may be affected by glucose coexisting in the specimen, especially biological samples such as serum and urine. , eliminating existing glucose prior to the α-amylase reaction is an effective means for carrying out this measurement method.

Glucose oxidase is a method of elimination.
There are various methods such as a method using catalase, a method using hexonase (JP-A-57-47495), and a 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 with modified groups at their non-reducing ends to the extent that they can serve as substrates for α-amylase, such as sodium starch glycolate (carboxymethylated starch), “Clinica Himica Acta 76
(1977) 277–283 [Clinica Chimica Acta, 76
(1977) 277-283] or carboxymethylated amylose (Japanese Patent Application No. 57-148756), those containing a carboxyvinyl group as a modifying group are particularly preferred from the viewpoint of solubility, but in addition to carboxymethyl groups, Those containing a 2-pyridylamino group, 3-pyridylamino group, anilino group, 2-hydroxyanilino group, methylanilino group, carboxyphenylamino group, etc. can be used without problems, and other groups can also be used, especially as modifying groups. It is not subject to any restrictions. α- such as starch or amylose
When a polysaccharide having a 1,4-glycosidic bond is used as a substrate, in the method of measuring glucose produced by conjugating glucoamylase to α-amylase, glucoamylase is capable of producing amylose independently of α-amylase. α from non-reducing end
Since it is an exotype enzyme that hydrolyzes -1,4-glycosidic bonds, it has the disadvantage of degrading the substrate regardless of the α-amylase reaction.
(1) Inability to contain both substrate and glucoamylase in the measurement reagent solution, (2) Inability to use sufficient glucoamylase for measurement, (3) Increase in reagent blinding.
This is not preferable because problems such as the following may occur.

Furthermore, as a measurement condition for carrying out the present invention, a particularly effective concentration of the reaction terminator according to the present invention 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,
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 preferable example is a pH of about 6 to 8. Buffers that maintain the optimum pH can be freely used, such as phosphate and gas buffers. In addition, the final liquid property varies slightly depending on the glucose measurement system, but preferably has a pH of 6.5 to 8.5.

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 measure a large number of samples. As a result, special automatic analysis equipment was required as a means to avoid these problems. However, according to the present invention, since the reaction is stopped and measured using a reaction terminator, special automatic analysis equipment Does not require analytical equipment,
The practical range of the measurement method has been expanded by allowing manual measurement, and it has become possible to measure multiple samples simultaneously using the 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 the industry in these respects as well.

Examples will be shown below and explained in more detail,
The present invention is not limited to these.

Example 1 Reagent (1) Test solution 1 Gutud buffer (PIPES) 10 mmol, glucoamylase 20,000 units, glucose oxidase 100,000 units, mutarotase 200 units, catalase 500,000 units,
Take 1 mmol of N,N-diethylxylidine, 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 1.

(2) Test solution 2 Gutud buffer (PIPES) 20 mmol, peroxidase 5000 units, sodium chloride 15 mmol, carboxymethylated amylose 6 g, sodium nitride
Take 15 mmol and 0.5 mmol of 4-aminoantipyrine, dissolve in purified water, and adjust the pH to 6.9 with sodium hydroxide to bring the total amount to 1.

(3) Reaction stop solution Tris(hydroxymethyl)aminomethane
Take 0.6mol and dissolve it in purified water, add hydrochloric acid to make the pH 7.5.
Let the total amount be 1.

Measurement method: Add 10 μl of sample serum to 1 ml of test solution 1 and incubate at 37℃ for 5 minutes.
Warm for a minute. Add 1 ml of test solution 2 to this and heat at 37°C.
Perform the reaction for 10 minutes. Add 2 ml of reaction stop solution to this solution.
Add to stop the reaction, and measure the absorbance at 620 nm.

Separately, a standard specimen with known α-amylase activity is used to prepare a calibration curve in the same manner as above, and the α-amylase activity of the specimen is determined 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 20 mmol, glucoamylase 3
10,000 units, hexokinase 10,000 units, glucose-
6-phosphate dehydrogenase 10,000 units, magnesium chloride 5 mmol, NAD (oxidized nicotinamide adenine dinucleotide) 2 mmol, ATP (adenosine≡
phosphoric acid) 2 mmol, carboxymethyl starch 3
Take g, dissolve it in purified water and add potassium hydroxide to pH 7.0.
The total amount is 1.

(2) Reaction stop solution Tris(hydroxymethyl)aminomethane
Take 0.4 mol and dissolve it in purified water, add hydrochloric acid to make the pH 7.0.
Let the total amount be 1.

Measurement method: Add 20 μl of sample serum to 2 ml of test solution 1 and incubate at 37℃ for 10 minutes.
Warm for a minute. Add 1 ml of reaction stop solution to this solution to stop the reaction, and measure the absorbance at 340 nm.

As a sample-blind test, the 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 Test Solution 1 and carrying out the same procedure.

Separately, a standard sample with known α-amylase activity was used in the same manner as above, and a calibration curve was created using the values obtained by subtracting the sample blindness. From this calibration curve, the α-
Determine amylase activity.

Example 3 Reagent (1) Test solution 1 Gutud buffer (PIPES) 10 mmol, glucoamylase 20,000 units, glucose oxidase 100,000 units, mutarotase 100 units, catalase 500,000 units,
Take 1 mmol of N,N-diethylxylidine, 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 1.

(2) Test solution 2 Gutud buffer (PIPES) 20 mmol, peroxidase 5000 units, sodium chloride 15 mmol, carboxymethylated amylose 6 g, sodium nitride
Take 15 mmol and 0.5 mmol of 4-aminoantipyrine, dissolve in purified water, and adjust the pH to 6.9 with sodium hydroxide to bring the total amount to 1.

(3) Reaction stop solution Take 0.6 mol (36 g) of monoethanolamine, dissolve it in purified water, and add hydrochloric acid to adjust the pH to 7.5.
shall be.

Measurement method: Add 10 μl of sample serum to 1 ml of test solution 1 and incubate at 37℃ for 5 minutes.
Warm for a minute. Add 1 ml of reagent 2 to this and store at 37°C.
Perform the reaction for 10 minutes. Add 2 ml of reaction stop solution to this solution.
was added to stop the reaction, and the absorbance at 620 nm was measured.

Separately, a standard specimen with known α-amylase activity is used to prepare a calibration curve in the same manner as above, and the α-amylase activity of the specimen is determined from this calibration curve.

[Brief explanation of drawings]

Figure 1 shows that in Example 1, 80 Somogyi units/dl
Absorbance change after adding reagent 2 when using serum of
FIG. 3 is a diagram showing the change in absorbance after adding .

Claims (1)

[Scope of Claims] 1. Glucoamylase as a conjugate enzyme and α-1, into which a modification group has been introduced at least at the non-reducing end,
When measuring the enzymatic activity of α-amylase using a substrate that is a polysaccharide having 4-glycosidic bonds (hereinafter abbreviated as modified substrate), 2-amino-2-hydroxy is used as a reaction terminator during the reaction. Methyl-1,3-propanediol [tris(hydroxymethyl)aminomethane], 2-amino-1,3-propanediol, 2-amino-2-
Methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, 2-
The α-method is characterized in that the enzymatic reaction is stopped using amino-1,4-butanediol, monoethanolamine, diethanolamine, triethanolamine, or a salt thereof, and the enzymatic activity of α-amylase is quantitatively measured. Method for measuring amylase activity. 2 The modified substrate is carboxymethylated starch,
or carboxymethylated amylose, the method for measuring α-amylase activity according to claim 1. 3 The concentration of the reaction terminator at the time of reaction termination is 50 or more.
The method for measuring α-amylase activity according to claim 1 or 2, wherein the α-amylase activity is 100 mM or more.