JPS60237998A - Method of measurement of alpha-amylase activity - Google Patents

Abstract

PURPOSE:In the titled method of measurement using a novel-amylase substrate, to provide method of measurement with a small error and improved reproducibility, by using an alpha- and/or beta-phenylmatooligoside having a modified nonreducing end as a substrate. CONSTITUTION:An alpha- and/or beta-phenylmaltooligoside (formula) obtained by modifying a nonreducing end of maltooligosaccharide such as maltotetraose, maltopentaose, etc. with a halogen atom, -X-R<3> or glucopyranose residue is used as a substrate. A test specimen is brought into contact with it in the presence of alpha-flucosidase and/or glucoamylase, and, if necessary, beta-glucosidase, and a free phenolic compound is measured, to measure alpha-amylase activity in the test specimen.

Description

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

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---1 unit amylasemaltose + 1waretotriose+21
Maltose+maltotriose 1]7 Glucose produced here is measured by a known method, such as a commutase/glucose-6-phosphz-)fhydrogenase/NADH system.

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-Nitrophenylmaltopentaosite (Special Publication 1984)
-53079 Publication) p-Nitrophenylmaltohexaoside (Special Publication No. 1983)
-53079 Publication) p-Nitrophenylmaltohebutaoside (Special 1:11
i1 Publication No. 54-51892) 2,4-Dichlorophenylmaltopentaosite (Japanese Patent Application Laid-open No. 35998-1987) Examples of the measurement format for amylase activity using these compounds as substrates are as follows.

In the case of p-nitrophenyl-α-maltopentasite, fil p-nitrophenyl-α-maltopentaoside-LLmuniie → p-nitrophenyl-α-maltoside + maltotriostrophenol + glucose Also in this system, α-glucosidase acts on the substrate p-nitrophenyl-a-maltopentaoside, increasing the blank value.

(112,4-dichlorophenyl-β-maltohentaoside yst22→2,4-dicyclophenyl-β
-Maltoside + maltotriose (2-a) 2.
4-cyclophenyl-β-α-glucosymaltoside + maltotriose □ Dase 111 → 2.4-dichlorophenyl-β-glucoside + glucose (2-b) 2.4-dichlorophenyl-β- β-glucosidase glucoside 2.4-dichlorophenol+glucose In this system as well, the blank increases due to the action of the substrate 2,4-dichlorophenyl-β-maltopentaocytoid α-glucosidase.

In the end, in the measurement system that uses conventionally provided substrates,
Since α-glucosidase acts on the substrate, there is a problem in that the reagent blank value increases significantly. Further α
- Consolidation of glucosidase solution and substrate solution into one solution has a common problem in that the stability of the reagent is significantly impaired due to substrate decomposition of α-glucosidase.

5- Therefore, as a result of various studies, the present inventors found that these problems could be solved by introducing a substituent at the terminal of the non-selecting group, and thus completed the present invention.

That is, the present invention has the following characteristics: (In the formula, R- represents a halogen atom, -X-R'', or a glucobylanose residue.

X represents 0, ooc, oso, or NHe;
2 represents a substituted or unsubstituted phenol residue. n is 0
Indicates an integer of ~8. ) using α- and/or β-phenylmalto-oligoside modified with a non-reducing end as a substrate in the presence of α-glucosidase and/or glucoamylase and β-glucosidase if necessary. By measuring the released phenolic compounds, the α-amylase activity in the sample can be determined. I1. This is a T method for measuring α-amylase activity, which is characterized by determining II.

In the substrate of the present invention, the non-reducing end of the maltooligosalt is modified with a rogen atom, -X-R' or a glucobylanose residue.

The maltooligo salt used in the present invention has 2 to 10 sugars, and specific examples include maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose. Particularly preferred are maltotetraose, maltopentaose, maltohexaose, and maltoheptaose.

Examples of the nitrogen atom to be modified in the substrate of the present invention include nitrogen, bromine, fluorine, and iodine.

1X-R” modified to the substrate of the present invention, X is 0,
OOC%O8O, or NH, R' represents an alkyl group having 1 to 6 carbon atoms, a substituted alkyl group, a phenyl group represents an alkyl-substituted phenyl group, a substituted phenyl group such as an alkoxy-substituted phenyl group, or a pyridyl group; Specifically, alkoxy groups such as methoxy and ethoxy, phenoxy groups, alkyl-substituted phenoxy groups, alkoxy-substituted phenoxy groups, pyridyloxy groups, alkylcarbonyloxy groups, pentyloxy groups, alkyl-substituted benzoyloxy groups, and alkoxy-substituted hendiyloxy groups. group, pyridylcarbonyloxy group, alkylsulfonyloxy group, phenylsulfonyloxy group, alkyl-substituted phenylsulfonyloxy group such as tosyloxy, alkoxy-substituted phenylsulfonyloxy group, hyridylsulfonyloxy group,
Examples include monoalkylamino groups, dialkylamino groups, anilino groups, alkyl-nucleus-substituted anilino groups such as toluidino, alkoxy-substituted anilino groups such as anisidine, and pyridylamine groups.

Examples of the glucobylanose residues to be modified in the substrate of the present invention include α and β (l→6) glucobylanose residues.

The substrates of the present invention are also α- and/or β-phenyl malto-oligosides having a substituted or unsubstituted phenyl group at the reducing end.

Substituted or unsubstituted phenols bonded to the substrate of the present invention through α- and/or β-bonds and phenols having a koxy group, an alkoxycarbonyl group, or a nitro group, such as chlorophenol, dichlorophenol, hydroxyphenol 1 Alkylphenols, alkoxyphenols, benzoic acids or nitrophenols,
Examples include halogenated nitrophenol, alkylated nitrophenol, alkoxylated nitrophenol, nitrated benzoic acid, and dinitrophenol.

In particular, phenols having at least one nitro group,
Example LI' 4-nitrophenol, 2-chloro-4-
ditrophenol, 2,6-dichloro-4-nitrophenol, 2,6-dipromo-4-nitrophenol, 2
-promo 4-ditrophenol, 2-nitrophenol, 2-hydroxy-9-4-nitrophenol, 3-hydroxy-4-nitrophenol and the like are preferred.

Next, the substrate fi+ of the present invention will be further explained with specific examples 4-
Nitrophenol 0-6-Tai 40-6-deoxy-α
-D-glucopyranosyl-((i→4)-〇-σ-D-
Glucopyranosyl-1-o-methoxy-6-dioxy-
α-0-glucopyranosyl-((i→4)-〇-α-D
-glucopyrano β-maltopentaoside] 4-nitrophenol α-D-glucopyranoyl-(
(1→4)-〇-σ-D-glucopyrano l〇-Silu ni'-”-β-D-glucopyranoside [4-nitrophenyl-β-maltopentaosiglucopyranosyl-
((l→4)-〇-α-D-glucopyranosyl)s
-o-β-D-glucopyradeoxy-6-((2-pyridyl)amino]-α-D-glucopyranosyl-((l→
4) -〇-4hi0 -D-glucoelectrocyanosyl-, -〇-α-D-gluenyl-α-maltopentaoside] 4-nitrophenol 0-6-p-)luenesulfonyloxy〇-α-D -glucopyranosyl-((1→4
)-〇-α-D-glucopyranosyl), -〇-α-D-
Glucopyranoside [tosyloxy 4-nitrophenyl-α-maltoheptaoside] The substrate fIl of the present invention is a novel compound, for example fa
The chloro-substituted substrate at the non-reducing end of t is produced by reacting a compound represented by the formula flTl with p-)luenesulfonyl chloride, and reacting the resulting compound represented by formula (1) with tetramethylammonium chloride. Odori et al.'s research report L? 4-POMICHXrJ, old oe
hem 6.84, 835-841 (197B)), the bl methoxy group-substituted substrate is produced by reacting a compound represented by the formula (ml) with methanol.
N.

BOLLKNBACK REPORT CMETHODS IN
CARBOHYDRATBCHEMISTRY vow
, [326].

(c) The tosyloxy group-substituted substrate means a compound represented by the formula tit.

The fdl pyridylamino group-substituted substrate is produced by reacting a compound represented by the formula (kawa) with pyridylamine. Research report by Odori et al. (K, OMICHI IJ, Biochem, 93
．． 1055-1060 (1983)].

A substrate substituted with t, l anilino group, toluidino group or anisidino group is produced by reacting a compound represented by the formula +fi+ with aniline, toluidine or anisidi. t
Refer to the method of dl.

(fl The phenoxy group-substituted substrate is produced by reacting a compound represented by the formula (TII) with phenol. (bl
The method was referred to.

Igl benzoyloxy group-substituted substrate is obtained by reacting a compound represented by the formula tut with benzyl alcohol. 13
- Manufacture. (Reference was made to the method of bl.

In addition, instead of the compound represented by the formula + ITI, α, β(
,) tetramethylammonium chloride tb + methanol lcl p -) luenesulfonyl chlorite (d) pyridylamine + -) aniline, toluidine or anisidine Tfl phenol (gl) is reacted with benzyl alcohol, respectively, to give a free fcl non-reducing end (C-6 position) Substrate with tosyloxy group l substitution Anif, l non-reducing end (C-6 position), Y+ Substrate with lino group, toluidino group or anisidino group substitution 14- ffl non-reducing end (C-6 position) phenoxy group substitution There is a method of producing a substrate (gl substituted with a benzyloxy group at the non-reducing end) and then reacting with cyclodextrin transferase to produce the desired maltooligosaccharide.

The α-glucosidase used in the present invention may be of any origin, such as animal, parasitic, or microbial origin, but it is particularly preferable to use one obtained from yeast in view of its substrate specificity. That is, α-glucosidase derived from yeast has a broad aglycone specificity, and furthermore, it acts well on glycosides smaller than maltotrioside, but does not act on glycosides larger than maltotetraoside, so it is particularly suitable for the purpose of the present invention. ing.

β-glucosidase of any origin may be used; for example, one obtained from almonds can be used.

Glucoamylase of any origin may be used; for example, glucoamylase obtained from Rhizopus deremer can be used.

Specifically, the usage of α-glucosidase, β-glucosidase, and glucoamylase includes (1) α-glucosidase (1) α-glucosidase and β-glucosidase (+111 glucoamylase (1v) glucoamylase and α- glucosidase f
Vl glucoamylase and β-glucosidase (vl
l Glucoamylase, α-glucosidase and β-
Any method of using glucosidase may be used, especially methods using α-glucosidase and glucoamylase (1v) and (vl)
is preferred.

In the method of the present invention, other additives may be added as necessary.

In the present invention, the α-amylase activity in the sample is measured by contacting the sample with the above-mentioned substrate in the presence of a follower enzyme and measuring the liberated phenolic compound.

As a method for measuring phenolic compounds, when the phenolic compound dissociated from the substrate exhibits a spectral absorption different from that of the substrate, the spectral absorption of the reaction product is directly measured.

In addition, when the phenolic compound dissociated from the substrate exhibits almost the same spectral absorption as the substrate, it is oxidized and condensed with a coloring reagent, such as a compound such as 4-aminoantipyrine,
Measure the color intensity.

As a method for measuring the phenolic compound, either a rate method in which the reaction of α-amylase is continuously monitored or a method in which the reaction is stopped after a certain period of time and the measurement is performed may be used.

In the present invention, by using the above-mentioned substrate, the action of α-glucosidase on the substrate is prevented, and the rise of the reagent blank is significantly suppressed, making it possible to integrate the follower enzyme solution and the substrate solution into one solution.

In addition, it has become possible to use glucoamylase (maltooligosaccharides with unsubstituted reducing ends serve as substrates for glucoamylase), which was previously not applicable to maltooligosaccharide substrates with unsubstituted non-reducing ends.

17-α-glucosidase acts well on low-molecular-weight glycosides below maltotrioside, but does not act on glycosides above maltotetraoside.

When a high molecular weight glycoside of maltoheptaoside or higher is used as an amylase substrate, a glycoside of maltoheptaoside or higher is produced. When d-glucosidase is used as a follower enzyme, J, which acts only on low-molecular-weight glycosides below maltotriocytate, which are the ice-melting products produced by amylase,
When glucoamylase is used, the advantage of using glucosidase is that it acts on glycosides of multitracytate or higher, increasing the sensitivity of the measurement system.

The measurement method of the present invention is an excellent method that can be easily applied to automatic analyzers.

The present invention will be explained below using examples.

Example 1 A blank value was measured using the following reagents and the following method.

Reagent: 50mM Gud buffer pH 7,0 α-glucosidase 80 U/ml 18-β-glucosidase IOU/resistance 2mM substrate shown in Table 1 Table 1 Foot measurement method: Take the above reagent 3 wg and observe the change over time in the reagent blank. saw. (Measurement wavelength 400nrn, temperature 37℃) Check out the results! @2 Shown in table.

Reagents A and B of the present invention have a smaller rise in reagent blanks than reagents C and D.

Example 2 Nobiichi amylase activity in a sample was measured using the following reagent and the following method.

Reagent A: 50mM Gudbuffer-pI (7,0
2mM Reagent B: 50mM Gut buffer - pH 7,0-
Maltopentaoside 2mM Measurement method: Three samples each of reagent A or B were taken, and the change over time in a reagent blank at 37°C was measured at 400 nm.

Reagent A or B was added to 50/'j of the sample (serum), and the change in absorbance (α-amylase activity) was measured 5 to 6 minutes after the addition. (Measurement wavelength: 400 nm, 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.

Table 4 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 Reagent ASB and Reagent ASB.

Example 3 α-amylase activity in a sample was measured using the following reagents and the following method.

Reagent A: 50mM Gud buffer (pH 7,02
1-α-glucosidase 80U/d Glucoamylase 10 # Tosyloxy 4-nitrophenyl-α-maltoheptaoside 2mM Reagent B: 50mM Gutdbuffy-(pH 7,0
) α-glucosidase 80 U/l/tosyloxy 4-nitrophenyl-α-maltoheptaoside 2mM Reagent C: 50mM Gutud buffer (pH 7, α-
Glucosidase 80 U/ag Gel coavalase 10 U/d 4-nitrophenyl-α-maltoheptaoside 2 mM Measurement method: Take 3 ml each of reagents A, B, or C and measure the change over time in a reagent blank at 37°C at 400 nm. sample serum 5
3 d each of reagents A, B, or C were added to 01Lt, and the change in absorbance (amylase activity) was measured 5 to 6 minutes after the addition. (Measurement wavelength: 400 nm, reaction temperature: 37° C.) Changes in reagent blank over time are shown in Table 5, and changes in serum absorbance are shown in Table 6.

22-Table 5 Table 6 Reagent A or B of the present invention has a smaller increase in reagent blank than Reagent C. α-amylase activity values are for reagents B and C.
There is no difference. The sensitivity of reagent A increased with the addition of glucoamylase.

Patent Applicant Toyobo Co., Ltd. Procedural Amendment (Method) % Formula % 1 Indication of Case 1982 Patent Application No. 242890a Title of Invention a - Amylase Activity Measuring Method & Person Who Makes Amendment Relationship with Case Patent Applicant 2-2-8 Dojimahama, Kita-ku, Osaka City, May 8, 1985 (Delivery date: May 28, 1985) a Contents of amendment 1 Specification, page 1, line 3, RA-lamylase activity measurement method.” amended to "alpha-amylase activity measurement method".

Claims (1)

[Scope of Claims] In the formula C, or represents a norogen atom, --R1, or a glucobylanose residue. X is 0. OOC, 080, or NU, and a S represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted phenyl group, or a pyridyl group. Further, R' represents a substituted or unsubstituted 71nol residue. n represents an integer of 0 to 8. ) in the coexistence of α-glucosidase and/or glucoamylase and optionally β-glucosidase, using α- and/or β-phenyl malto-oligoside modified with a non-reducing end as a substrate,
1. A method for measuring σ-amylase activity, which comprises measuring α-amylase activity in a sample by contacting the sample and measuring the liberated phenolic compound.