JPH0218079B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring α-amylase activity, which is characterized by using a novel substrate, that is, an oligosaccharide derivative. α-Amylase activity in samples, especially in human saliva, pancreatic juice, 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 have been published so far for measuring α-amylase activity, but the main ones are:
It can be classified into three groups: amyloclastic method, chromogenic method, and satcalogenic method. Among the amyloclastic methods, the callaway method has been most widely used, but it has problems such as coexisting proteins inhibiting the color development of starch and iodine, and the reaction time is short, resulting in poor reproducibility. 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. The disadvantage is that the reaction cannot be measured as a truly stoichiometric reaction. Starch, as is widely known, is a mixture of a linear glucan with α-1,4 bonds called amylose and a branched glucan with α-1,6 bonds called amylopectin. The mixing ratio, molecular weight, degree of branching, and branching structure of amylose and amylopectin vary depending on the type of raw material plant, harvest time, production area, etc., and it is a heterogeneous mixture. If a heterogeneous starch is used as a substrate, the reaction will not be stoichiometric and the kinetics of α-amylase cannot be detected. Kinetic detection of α-amylase is achieved by the use of oligosaccharides with 4 to 7 glucose chains. Recently, maltotetraose, maltopentaose, maltohexaose,
A method using an oligosaccharide such as maltoheptaose as a substrate (JP-A-50-56998, JP-A-53-37096), or an oligo with a chromogen such as P-nitrophenol attached to the reducing end. Method using saccharide (Japanese Unexamined Patent Publication No. 54-51892), etc.
Methods using homogeneous and well-structured substrates have been published. These methods usually use α-glucosidase (EC3.2.1.20, α-D-glucoside glucohydrolase) or glucoamylase (EC3.2.1.3, 1,4-α-D-glucan glucohydrolase) as the coupled enzyme for measurement. ) is required. These conjugated enzymes convert α-1,4 from the non-reducing end of the sugar chain with α-1,4-glycosidic
- It is an exo-type enzyme that hydrolyzes glycosidic bonds, and has the disadvantage that it degrades the substrate regardless of the α-amylase reaction. As a result, the reagent solution for measurement was unstable, and the reagent blind value was extremely high, significantly impairing measurement accuracy. Furthermore, it was not possible to use sufficient glucoamylase or α-glucosidase for measurement, making it difficult to assemble an accurate measurement method. The present inventors solved the problem by replacing the primary alcohol _-CH2OH at the 6-position of the non-reducing terminal glucose of these oligosaccharides with _-CH2NHR for oligosaccharides with 4 to 7 glucose chains. The substrate was synthesized. By introducing this group,
It is possible to spectrophotometrically detect this substrate as well as a product having a -CH ₂ NHR group produced as a result of α-amylase action, and also this substrate (hereinafter referred to as
It is called a modified oligosaccharide. ) has an excellent affinity for α-amylase and is a good substrate, and it has been discovered that it does not serve as a substrate for glucoamylase or α-glucosidase, leading to the completion of the present invention. That is, in the present invention, when measuring α-amylase activity, the primary alcohol (-CH ₂ OH) at the 6-position of the non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose units is expressed by the general formula -
This is a method for measuring α-amylase activity, which is characterized in that an oligosaccharide derivative having the following structural formula () substituted with a group represented by CH ₂ NHR is used as a substrate. (In the formula, the rightmost glucose unit is a reducing group, n
is an integer of 2 to 5, and R is an integer of 4 to 5.
A primary alcohol (-
CH ₂ OH), which has the general formula −
When substituted with a group represented by CH ₂ NHR, the structural formula
The alcohol (-CH ₂ OH) that becomes the oligosaccharide derivative having (1) is oxidized to the corresponding aldehyde (-CHO), which reacts with the aldehyde (-CHO) of the oxidized polysaccharide to form a Schiff base. Represents an organic residue having an amino group. ) Hereinafter, the present invention will be explained in detail by giving examples. First, regarding the modified oligosaccharide used in the present invention, dextrin is used as a raw material, and dimethyl sulfoxide and N,N'-dicyclohexylcarbodiimide are used to modify the first glucose unit of dextrin.
Partially oxidizes alcohols. For example, about 1 in 30 glucose groups is oxidized. This reaction was carried out using the method of Jones, G.H. et al.
Carbohydrate Chemistry Vol. Vl pp. 315-322 (1972) Academic Press, New York]. The oxidation reaction is stopped by adding oxalic acid, then an organic amine according to the present invention such as 2-aminopyridine, sodium cyanoborohydride is added, heated to 95°C and reacted for about 20 minutes, water is added to the reaction mixture, The resulting precipitate was separated and removed, and hydrochloric acid was added to this solution to bring the pH to about 1.0. Sodium cyanoborohydride was decomposed, the pH was neutralized, and the solution was gelated using a column packed with Biogel p-2. Collect the high molecular weight (modified dextrin) fraction. After reacting this modified dextrin with liquefaction type α-amylase derived from Bacillus subtilis in a buffer solution of about PH 5.5 at 37°C for about 30 minutes, the reaction solution is heated at a temperature of 70°C or higher for 20 to 60 minutes, Deactivate α-amylase. After the heat treatment, the reaction solution was cooled to about 20℃, the pH of the solution was adjusted to a neutral pH of 6 to 8, and then α-glucosidase or glucoamylase was added and heated at 37℃.
After 20 to 50 hours of action, a modified oligosaccharide produced by the action of α-amylase is obtained. This reaction is generalized and the general formula is shown below. (In the formula, G represents a glucose unit, and X is

Represents a group represented by -CH ₂ NHR such as [Formula]. Further, l, m, and n represent 0 or a positive integer. ) Next, this mixture was concentrated, and column chromatography was performed using a column packed with Biogel P-4 to obtain a modified oligosaccharide, that is, maltotetraose with an -NHR group such as a pyridylamino group at the non-reducing end. , maltopentaose, maltohexaose, and maltoheptaose can be obtained. That is, to summarize the present production method, the primary alcohol (-CH ₂ OH group) of dextrin is oxidized to form an aldehyde (-CHO), and then an organic amine according to the present invention such as 2-aminopyridine is reacted with the primary alcohol (-CH 2 OH group). It is used as a Schiff base and reduced to obtain a modified dextrin. The desired modified oligosaccharide is then obtained by enzymatic hydrolysis. In this production method, the raw material polysaccharide is α-
Any long-chain sugar having a 1,4-glycosidic bond can be used, and in addition to dextrin, starch, amylose, etc. can also be used as raw materials. In addition, in the step of obtaining a modified polysaccharide from an oxidized polysaccharide, in addition to 2-aminopyridine, a compound having an amino group that forms a Schiff base with an aldehyde in the oxidized polysaccharide (organic amine according to the present invention) may be used. In addition to 2-aminopyridine, for example, 3-aminopyridine, aniline, 2-hydroxyaniline, aminobenzoic acid,
Examples include methylaniline. In this case, the finally obtained modified oligosaccharides are oligosaccharides modified with the respective corresponding groups. That is, examples of modified oligosaccharides according to the present invention include oligosaccharides modified with pyridylamino groups such as 2-pyridylamino groups or 3-pyridylamino groups, oligosaccharides modified with anilino groups, and hydroxyanilino groups such as 2-hydroxyanilino groups. Examples include modified oligosaccharides, carboxyphenylamino group-modified oligosaccharides, and alkylanilino group-modified oligosaccharides such as methylanilino groups. Although α-amylase is used for the purpose of hydrolyzing modified dextrin into oligosaccharides, the type is not particularly limited, and for example, amylase derived from animal pancreas or microorganisms can be used. The aforementioned Bacillus
The liquefied amylase derived from P. subtilis can be used effectively because the reaction stops or slows down in the state of oligosaccharides with about 10 glucose chains, making it easy to control the product. As examples of measuring α-amylase using this modified oligosaccharide, high performance liquid chromatography (HPLC) method and spectrophotometry are listed, and the measurement principles and characteristics thereof are shown, but the scope of use of this modified substrate is limited. isn't it. [] High performance liquid chromatography (HPLC)
Method Pyridylamino group has fluorescence (excitation wavelength
320nm, fluorescence wavelength 400nm), and using this, we performed an α-amylase reaction using this modified substrate, and then formed a short chain with a pyridylamino group in the non-reducing terminal glucose (glucose group is 2 Or, by subjecting a solution containing modified oligosaccharides (3) to HPLC and comparing the outflow peak with the outflow peak of a standard specimen whose activity value is known and which was operated in the same way, α
- Amylase activity can be determined. In HPLC, the column and eluent are not particularly limited, but the column is TSK-Gel LS 410, 5μm,
C18 (Toyosoda Co) and 0.1M acetic acid containing 0.1% n-butanol can be effectively used as the eluent. By using the modified oligosaccharide of the present invention as a substrate for measuring α-amylase activity by HPLC method, it becomes possible to construct a measuring method having the following advantages (a) to (v). (a) Modified oligosaccharides are easily soluble in water and highly reactive with α-amylase. (b) In the method of the present invention, since a single compound is used as a substrate, the stoichiometry of the reaction is established, and the dynamics of α-amylase can be detected. (c) Since this method does not use a coupled enzyme system following the α-amylase reaction, it is easy to detect the dynamics of α-amylase. (d) This method separates and measures oligosaccharides with 2 or 3 glucose units containing pyridylamino groups or other modifying groups, which are generated in the α-amylase reaction, so there is no influence of existing glucose in the blood. I don't receive it. (e) Since detection is performed using fluorescent photometry, highly sensitive measurements are possible, and measurements can be made with a trace amount of sample. [2] Spectrophotometry Since glucoamylase or α-glucosidase is an exotype enzyme, this modified substrate cannot be used as a substrate. On the other hand, α-amylase is an endo-type enzyme that hydrolyzes any α-1,4 glycosidic bond in oligosaccharides, and can use this modified substrate as a substrate. is hydrolyzed and a new acyclic end is generated. α-glycosidase or glucoamylase acts on this newly generated non-reducing end to produce glucose, and α-amylase activity can be determined by measuring the amount of glucose produced. There are many known methods for quantifying glucose, and 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 quantitatively oxidizes the chromogen through the coexisting peroxidase, 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 - ― ― ― ― ― ― ― ― → Oxidized color Original substance + H ₂ O In addition, glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. The generated 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. 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 hexonase - - - - - - - → Mg ²⁺ Glucose-6-phosphate + ADP Glucose-6-phosphate + NAD Glucose-6-phosphate dehydratase―――――――――――――― ---→ 6-phosphogluconolactone+
NADH ATP; adenosine-5'-triphosphate ADP; adenosine-5'-diphosphate NAD; β-nicotinamide adenine dinucleotide NADH; reduced-β-nicotinamide adenine dinucleotide Spectroscopy using this modified oligosaccharide α− by photometric method
In the amylase activity measurement method, glucose produced by the action of α-amylase or a coupled enzyme glucoamylase or α-glucosidase is measured simultaneously or subsequently. Accurate measurement values may not be obtained due to the influence of glucose present in the blood, and eliminating existing glucose prior to the α-amylase reaction is an effective means of implementing this measurement method. Become. There are various methods for elimination, such as a method using glucose oxidase catalase, a method using hexokinase (Japanese Unexamined Patent Publication No. 57-47495), and a method using glucose peroxidase, and any of these methods can be freely combined. be. In addition, α-amylase activity can be measured by using the modified oligosaccharide as a substrate, allowing α-amylase to act on it, and measuring the maltose produced. For example, maltose measurement is described in Japanese Patent Application Laid-open No. 1983-
According to the measurement system described in 119296. The modified oligosaccharide of the present invention was determined by spectrophotometry
By using it as a substrate for measuring amylase activity, it is possible to construct a measuring method having the following advantages. (a) This modified oligosaccharide is easily soluble in water and has high reactivity with α-amylase. (b) In the method of the present invention, since a single compound is used as a substrate, the stoichiometry of the reaction is established, and the dynamics of α-amylase can be detected. (c) Since the modified oligosaccharide used in the present invention is not a substrate for glucoamylase (or α-glucosidase) but is a specific substrate for α-amylase,
No side reactions occur and reagent blind values are extremely small. (d) Since a sufficient amount of glucoamylase and α-glucosidase can be used, reactions after the α-amylase reaction are fast and accurate rate assays can be performed. (e) The test solution for measurement is stable. (f) Good adaptability to automatic analyzers. Next, examples will be shown and explained in more detail. Example 1 Preparation of modified oligosaccharides 10 ml of dextrin in 250 ml of dimethyl sulfoxide
g and N,N'-dicyclohexylcarbodiimide
Dissolve 15 g of the solution, add a mixture of 1 ml of dichloroacetic acid and 12.5 ml of dimethyl sulfoxide, mix well, and react at 20-25°C for 40 minutes. methanol 25ml
Add a solution of 6.3g of oxalic acid (dihydrate) to the
Stop the reaction. Add a 2-aminopyridine solution (2-aminopyridine 60g, water 90ml,
24 ml of acetic acid and 24 ml of sodium cyanoborohydride
Add the mixture of g) and heat at 95℃ for 20 minutes.
After the heating reaction, 1 part of water is added to form a precipitate, and the mixture is separated. This solution is adjusted to pH 1.0 with 6N hydrochloric acid, and after decomposing excess sodium cyanoborohydride, the pH is adjusted to 7.0 with 6N aqueous ammonia, and concentrated in vacuo. Take 350 mg of this concentrate and dissolve it in water.
Gel. The column was Biogel P-2 (manufactured by Bio Rad) equilibrated with 10mM ammonium bicarbonate.
A column with a diameter of 2.6 cm and a height of 113 cm was used and was passed through this column twice. This column operation was performed three times (the charge amount of the concentrate was 350 mg each) and the high molecular fractions were collected and freeze-dried. The yield here is approx.
700 mg, and the modification was 3.1% in glucose units based on the absorbance at 310 nm in 0.1 M acetic acid. The degree of modification was measured using the method of Hase, S. et al. [J.Biochem. 85 .
217. (1979)]. Dissolve 470 mg of this modified dextrin in 15 ml of water,
33mM acetic acid-calcium acetate buffer (PH5.5) 90
ml, add 50 units of liquefied amylase derived from Bacillus Subtilis, and incubate at 37°C for 25 minutes. Then, after heating at 100℃ for 15 minutes to inactivate amylase, glucoamylase
Add 150 units and incubate at 37℃ for 24 hours.
Add 150 units of glucoamylase and incubate for 24 hours. This hydrolyzate is concentrated and chromatographed using a column packed with Biogel P-4 (manufactured by Bio Rad) equilibrated with 20mM acetic acid. Column diameter 1.9
I used one with a height of 212 cm. Each fraction of 4 to 7 glucose chains of the modified oligosaccharide was collected and lyophilized. Substrate purification was performed using HPLC using TSK-Gel LS410 5μm.
A large column (7 x 300 mm) packed with C18 is used, and the eluent contains 0.045% n-butanol.
A 0.1M ammonium acetate buffer (PH3.9) was used at a flow rate of 3.0ml/min. Confirmation of structure The modified oligosaccharide was reduced with sodium borohydride and then incubated at 90% in a methanol solution containing 1.5N hydrochloric acid.
After thermal decomposition at 0.degree. C. for 4 hours, it is then trimethylsilylated in pyridine. Then, it was measured by gas chromatography. Column is Chromosorb
Use 80 to 100 meshes (0.5 x 300 cm) coated with 2% OV-17 on W, and heat from 120℃ to 185℃.
It was measured by chromatography at an elevated temperature (4°C/min). Using similarly operated maltose as a standard, the number of glucose in each fraction (1, 2, 3, 4, and 5) was determined from the ratio of glucose and glycitol, and from this, the glucose chain length of the oligosaccharide was 3, 4, and 5.
I scored 5, 6 and 7. (FG3, FG4, respectively
They are abbreviated as FG5, FG6, and FG7. ) Furthermore, since each modified oligosaccharide is not hydrolyzed by glucoamylase, it is thought that a modifying group (pyridylamino group, etc.) is included at the non-reducing end. Therefore, each fraction of FG3 to 7 was subjected to complete methylation using the Hakomori method [Biochemistry Experiment Course 4, Chemistry of Carbohydrates (Volume 2) P.506, Tokyo Kagaku Dojin], and then hydrolyzed with acid. It was confirmed by gas chromatography that methyl-2,3,4,6-tetra-0-methylglucoside was not produced at all. Furthermore, FG3 and FG5 fractions were taken, dissolved in 0.1M sodium acetate-10mM calcium acetate buffer pH 5.5, added with Taka amylase, and heated at 37°C for 3 hours for hydrolysis. When this hydrolyzate was examined by HPLC and TLC, FG3 and maltose were produced from FG5, but FG3 was not hydrolyzed by Taka amylase. Also add FG3 to 0.3N hydrochloric acid
When hydrolyzed at 100°C for 30 minutes, the product obtained was maltose, glucose and two types of fluorescent compounds, but no maltotriose was produced. Example 2 Reagent (1) Test solution 1 Sodium acetate 100 mmol, calcium acetate
10 mmol, maltopentaose containing a pyridylamino group at the non-reducing end prepared in Example 1
Take 0.36 mmol, dissolve it in purified water, and adjust the pH to 5.5 with sodium hydroxide to bring the total amount to 1. Measurement procedure: Add 5 μl of sample blood to 500 μl of test solution 1 and incubate at 37℃ for 15 minutes.
Warm for a minute. This reaction solution is subjected to high performance liquid chromatography, and the production amount of a modified oligosaccharide containing three glucose molecules in which the non-reducing end glucose has a pyridylamino group is determined from the peak area. Separately, a standard specimen with known α-amylase activity is used and operated in the same manner as above to determine a calibration relationship, and the α-amylase activity of the specimen is determined from this calibration curve. α− at each dilution stage of the standard sample at this time
Amylase activity (Somogyi units/dl) and free
The relationship with the amount of FG3 (μM) is shown in Figure 1. The α-amylase activity of the standard sample is 200 Somogyi units. Liquid chromatography column is TSK-Gel
LS410, 5μm, C18 (4 x 300mm, Toyosoda
Co.), the effluent contained 0.1% n-butanol.
Uses 0.1M acetic acid aqueous solution, flow rate is 1.7ml/min
This was done at room temperature. Example 3 Reagent (1) Test solution 2 Gutud buffer (PIPES) 40 mmol, glucoamylase 50,000 units, glucose oxidase 100,000 units, mutarotase 200 units, catalase 500,000 units,
Take 0.7 mmol of 4-aminoantipyrine, 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 3 40 mmol of Gud buffer (PIPES), 15,000 units of peroxidase, 15 mmol of sodium chloride, 5 mmol of calcium chloride, 3 g of maltopentaose prepared in Example 1 with a pyridylamino group at the non-reducing end, 15 mmol of sodium nitride. Take 10 mmol of phenol, dissolve it in purified water, and adjust the pH to 6.9 with sodium hydroxide to bring the total amount to 1. Measurement method: Add 20 μl of sample serum to 2 ml of test solution 2 and incubate at 37℃ for 5 minutes.
Warm for a minute. Add 1 ml of test solution 3 to this and heat at 37°C.
Measure the change in absorbance (ΔE/min) at 505 nm for 3 minutes from 2 minutes to 5 minutes. 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. The calibration curve at this time is shown in FIG. α of standard sample
-Amylase activity is 500 Somogyi units. The change in absorbance per 3 minutes in a reagent blind test was compared between using maltohexaose containing a pyridylamino group as a substrate and maltohexaose, and is shown below as a comparative example. Comparative Example 1 (1) Test Solution 4 Same as Test Solution 2 of Example 3 (2) Test Solution 5 Instead of maltopentaose containing a pyridylamino group at the non-reducing end in Test Solution 3 of Example 3, pyridylamino group was added to the non-reducing end. Use maltohexaose containing a diylamino group, and use the same method as Test Solution 3 below. (3) Test solution 6 Maltohexaose was used instead of maltopentaose containing a pyridylamino group at the non-reducing end in test solution 3 of Example 3, and the same as test solution 3 hereinafter. Measurement method: Add 20 μl of purified water to 2 ml of test solution 4 and heat at 37°C for 5 minutes. Add 1 ml of test solution 5 to this, react at 37°C, and measure the change in absorbance (ΔE/min) at 505 nm for 3 minutes from 2 minutes to 5 minutes. Moreover, the measurement is performed in the same manner as above using test solution 6 instead of test solution 5. The results are shown in Table 1.

【table】 [Brief explanation of drawings]

FIG. 1 shows the relationship between α-amylase activity (Somogyi units/dl) and the amount of released FG3 (μM/min) in Example 2. Figure 2 shows α-amylase activity (Somogyi units/dl) and absorbance change at 505 nm (ΔE/dl) in Example 3.
min).

Claims (1)

[Claims] 1. When measuring α-amylase activity, the primary alcohol (-CH ₂ OH) at the 6-position of the non-reducing terminal glucose of a linear oligosaccharide consisting of 4 to 7 glucose units is expressed by the general formula 1. A method for measuring α-amylase activity, which comprises using as a substrate an oligosaccharide derivative substituted with a group represented by -CH ₂ NHR and having the following structural formula (1). (In the formula, the rightmost glucose unit is a reducing group, n
is an integer from 2 to 5, and R is glucose 4
A primary alcohol (-
CH ₂ OH), which has the general formula −
When substituted with a group represented by CH ₂ NHR, the structural formula
The alcohol (-CH ₂ OH) that becomes the oligosaccharide derivative having (1) is oxidized to the corresponding aldehyde (-CHO), which reacts with the aldehyde (-CHO) of the oxidized polysaccharide to form a Schiff base. Represents an organic residue having an amino group. 2. The measuring method according to claim 1, wherein R of the group represented by the general formula -CH ₂ NHR of the oligosaccharide derivative having the structural formula (1) is [Formula].