JPS602199A - Measurement of alpha-amylase activity - Google Patents

Abstract

PURPOSE:To measure amylase activity in specimen, by using a malto oligosacharide having a reducing termninal group linked to a specific substituted aromatic group as a substrate, adding alpha- or beta-glucosidase and a known amount of a specimen to it, measuring absorption of liberated aglycone in a visible light range. CONSTITUTION:A substrate solution shown by the formula I is prepared. In the formula, n is 0-8; R is substituted aromatic group selected from a group shown by the formula II(X and Y are H, or halogen, and not H simultaneously), linked to reducing terminal group through alpha- or beta-bond, showing absorption spectrum different from that of cleaved aglycone. alpha-Glucosidase or alpha-glucosidase and beta- glucosidase and a known amount of a specimen are added to the substrate solution. Absorption of liberated aglycone is measured in a visible light range, and amylase activity in the specimen is obtained.

Description

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

Measurement of α-amylase activity in body fluids such as serum, urine, and saliva has important significance in clinical diagnosis, especially in acute or chronic pancreatitis, pancreatic cancer, and mumps. It is an essential measurement item in the differential diagnosis of adenitis, etc.

Conventionally provided reagents for measuring α-amylase activity are ~
It can be classified as using the following measurement principles.

(1) Amyloclastic method using iodostarch reaction (8) Chromogenic method measuring free pigment from dye-bound starch (4) Turbidometric method measuring turbidity due to starch Substrates used in these methods These are modified forms of starch 1 or amylose, amylopectin, etc. derived from starch, and in either case they rely on natural starch. However, in the case of natural starch, its quality and properties are inconsistent, and the reliability of α-amylase activity measurement values is inevitably low.
- There were also problems in that the relationship between strand scission by amylase and the measured characteristic value was unclear, and furthermore, the measurement operation was complicated.

Therefore, the coupled enzyme method has attracted attention as a method that does not have these drawbacks, and the following measurement methods are being considered.

(A) Starch N-dextrin or oligosaccharide is used as a substrate, and after cleavage with α-amylase, glucose is released from the 7 fragments by the action of α-glucosidase as a follower enzyme system. How to measure by means.

(B) Maltose produced by chain cleavage by 1α-amylase using starch N-dextrin or oligosaccharide as a substrate is decomposed by the action of a following enzyme (maltose phosphorylase), and the produced glucose-1% acid is further processed by phosphoglucosidase. A method for measuring the amount of glucose-6-phosphate produced by decomposition by the action of glucose-6-phosphate dehydrogenase and a coenzyme by ultraviolet absorbance measurement.

(0) Using limit dextrin prepared using phosphorylase and β-amylase as a substrate, maltodextrin phosphorylase is applied to the fragment generated by the action of α-amylase to release glucose-1-phosphate, and this is used in the above (B). ) method of measurement.

(D) A method in which glucoamylase is applied to a fragment produced by the action of α-amylase using starch modified by carboxymethylation or the like as a substrate, and the glucose produced therein is measured by known means.

(Using a maltooligosaccharide in which a P-nitrophenyl group is glucosidically bonded to the reducing end as a substrate, glucosidase is allowed to act as a follower enzyme after chain cleavage by α-amylase, and the P-nitrophenol produced here is compared. Method for quantifying color (Japanese Unexamined Patent Publication No. 57-53079).

(F) A maltooligosaccharide in which a substituted or unsubstituted phenyl group is glucosidically bonded to the reducing end is used as a substrate, and after chain cleavage by 1α-amylase, glucosidase is reacted as a follower enzyme, and the 4-amino A method of oxidative condensation of a chromogen such as antipyrine and colorimetrically quantifying the resulting pigment.

In the coupled enzyme methods (A) to (F) above, when natural starch is used (including cases where dextrin 1 limit dextrin or modified starch is used as a substrate), N (1) to (4) above is used. It has the same flaws as mentioned in . However, when using oligosaccharides themselves or oligosaccharides with a color-forming group (aglycone) glucoside-linked to their terminal groups as substrates, the structural formula must be clearly understood and highly purified substances must be used, so natural starch cannot be used. There is no fluctuation as mentioned in the case of 1α-amylase, and the stoichiometric relationship between the number of chain cleavages by 1α-amylase and the measured characteristic value becomes clear, and highly accurate and reliable results can be obtained.

From this point of view, methods (A), (B), (B), and (E) are better, but since glucose and maltose are present in body fluids, especially serum and urine, In the methods ((A), (E), and CD) that go through the data as a whole, the measured characteristic values are high, and even if special elimination methods such as the rate method (RateAssay) are used, their effects cannot be completely eliminated. It is difficult to do so.

From the above points of view, the substrate used for α-amylase measurement is one in which a chromogenic group (a substituted aromatic group that exhibits a different spectral absorption than that of the substrate upon cleavage) is glucosidically bonded to the reducing end of an oligosaccharide. good. However, when a maltooligosaccharide in which a 4-nitrophenyl group is glucosidically bonded to the reducing end is used as a substrate, various problems have become clear. One of them is that the optimum range for α-amylase in blood or urine is 6.6 to 7.0, whereas the maximum range is 4-2). In addition, an increase in temperature causes an increase in ε and chloride).An increase in IJ content causes an increase in ε, and an increase in the amount of albumin causes an increase in ε.Additionally, there is a problem that the amylase activity measuring reagent using 4-nitrophenol lacks sensitivity. Problems can be raised.

Taking these circumstances into consideration, the present inventors have conducted various studies (X and Y are each selected from H and halogen, but are not simultaneously H), and have determined that a substituted aromatic group selected from the group consisting of If the bound maltooligosaccharide is used as a substrate -
Knowing that these drawbacks can be overcome, the present invention has been completed.

That is, the present invention measures the amylase activity in one sample by adding a-glucosidase or α-glucosidase and β-glucosidase and a sample to a substrate represented by the following formula, and measuring the visible absorption of the released aglycone. This is a method for measuring amylase activity characterized by:

As a measuring method, either the rate method (Rate As5ay) in which the reaction of α-amylase is continuously monitored, or the endo method (in which the reaction is stopped and measured after reacting for a certain period of time) may be used.

The malto-oligosaccharide used in the present invention may be any malto-oligosaccharide as long as it is alpha- or beta-bonded to maltopentaose, maltohexaose, maltoheptaomalto-oligosaccharide and its reducing end, is easily released by glucosidase, and is easily quantified. . Examples of the liberated compounds include 2-chloro-4-ditrophenol-2,6-cyclo-4-ditrophenol, J6-dipromo-4-ditrophenol, 12-bromo-4-nitrophenol, and the like.

Those with α bond at the reducing end have poor solubility, whereas β
A bonded one is particularly preferable because it has good solubility.

It is preferable to add calcium chloride or the like to stabilize the reagent used in the present invention. Furthermore, the presence of EDTA, which is often used as an anticoagulant in serum, makes amylase unstable, but the presence of oa ions stabilizes amylase.

In the present invention, amylase activity in a sample such as serum or urine is measured by the following reaction.

(2-chloro-4-nitrophenyl)maltopentaoside↓α-amylasezek)recose + 2-chloro-4-nitrophenol↓ 0H- 2-chloro-4-nitrophenol anion χmaX4
00nm As in all enzymatic reactions, the reaction solution is usually kept at a constant concentration.

The maltooligosaccharide bonded to the photoactive end is synthesized from the above substituted aromatic group and the maltooligosaccharide according to a conventional method. Chemically, it can be synthesized by acetylating a maltooligosaccharide, bonding the acetylated maltooligosaccharide with a substituted aromatic group, and then deacetylating it (see Jikken Kagaku Kakuza Vol. 24, p. 304, 1958). Biochemically, it can be synthesized by reacting cyclodextrin lycosyltransferase, a substituted aromatic, and soluble starch (or α-cyclodextrin or white dextrin). The α-glycosidase used in the present invention may be of any origin, such as animal, plant, or microbial origin, but those obtained from yeast are particularly preferred in terms of its substrate specificity. In other words, the yeast-derived Nobiichi glucosidase has a broad aglycone specificity and is particularly suitable for the purpose of the present invention in that it acts well on glycosides with maltotriosides or less but does not act on glycosides with maltotetraosides or more. There is.

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

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

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example L 2-rioo-4-nitrophenylmaltopentaoside (
α body/β body = 50150) was dissolved in 50 mM ME8 buffer (Ill-17) to prepare a 4 mM substrate solution, and 0.5- was taken. A 0.5-unit enzyme solution was prepared by dissolving 30 units/- of α-glucosidase and 0.005 units/- of β-glucosidase in 50 mM MES buffer (pl-17). Serum diluted to various concentrations (0 to 500 Somogyi 1 unit/61) of 0.02- was added to the above measurement reagent, and the increase in absorbance was measured for 4 minutes from 3 minutes after S at 37°C, and the change in absorbance for 1 minute was determined. . The results are shown in FIG.

Example 2 Almost the same as Example 1, except that 4mM 2-chloro-4-nitrophenylmaltopentaoside (α form/
β body = 50150) and various concentrations (0 to 5
The difference was that a bond solution diluted to 0.00 Somogi 1 unit/61) was used. The results of measurements made in the same manner as in Example 1 are shown in FIG.

Comparative Example L Almost the same as Example 1, but instead of 4mM 2-chloro-4-nitrophenylmaltopentaoside as the substrate, 14mM 4-nitrophenylmaltopentaoside (α
The difference was that the body/β body = 50150) was used. FIG. 3 shows the results measured in the same manner as in Example 1.

Comparative Example 2 Almost the same as Example 2, but instead of 4mM 2-chloro-4-nitrophenylmaltopentaoside as the substrate, 4mM 4-nitrophenylmaltopentaoside (α
The difference was that the body/β body = 50750) was used. The results of measurements made in the same manner as in Example 1 are shown in FIG.

Examples & 2-chloro-4-nitrophenylmaltopentaoside (
α-form/β-form; 50150) was dissolved in 50 mM MKS buffer (pH 7). Calcium chloride (0-5 oomf/l) was added to this substrate solution.

Enzyme solution used in Example 1, 1500 Somogi 1 unit/d
KM saliva was added to / at 37°C and the change in absorbance from 3 minutes to 4 minutes was corrected to 1 somogi unit. The results are shown in FIG.

Example Almost the same as Example 1, but with 4mM2.6-
-) chloro-4-nitrophenylmaltopentaoside (
The difference was that α-form/β-form = 50150) was used.

The results of measurement in the same manner as in Example 1 are shown in FIG.

Example 5゜Almost the same as Example 1, except that 4mM 2-chloro-4-nitrophenylmaltopentaoxide (β form) was used as the substrate and β-glucosidase was set at 0.01 unit/-. . FIG. 7 shows the results measured in the same manner as in Example 1.

[Brief explanation of the drawing]

Item 1 II shows the relationship between serum at various concentrations and absorbance change over 1 minute when the substrate is 2-chloro-4-nitro-7enylmaltopentaoside (α form/β form = 5o/ao). show. The relationship between various concentrations of phlegm and the change in absorbance over 1 minute is shown. The third [N] shows the relationship between various concentrations of serum and absorbance change over 1 minute when the substrate is 4-nitrophenylmaltopentaoside. Shows the relationship with light intensity change. FIG. 5 shows the relationship between calcium chloride concentration in the reaction solution and amylase activity. Figure 6 shows the relationship between serum at various concentrations and the absorbance change over 1 minute when the substrate is 2,6-dichloro-4-:)0 phenylmaltoside (α form/β form = 50150) (D). Figure 7 shows the relationship between the serum in various outer sacs and the change in absorbance over 1 minute when the substrate is 2-chloro-4-nitrophenylmaltopentaoside (β form).$1 Figure Figure 21! 1 3rd page 4th white memo key tsubosuta 10J!-Soumi A゛2 petition/dl thread 51
! '1 Cac12 咒吻0 (No. 6 Figure 7 Procedures Amendment (spontaneous) L Indication of the case 1981 Patent Application No. 111.1! Section 96 Title of the invention α-Amylase Activity Measuring Method 8 Amendment Relationship with the case of a person who does (2) On page 7, line 9, next to ``There are problems such as shortage.''
"In particular, the two major problems with 4-nitrophenol are the large ε change due to temperature changes and the lack of sensitivity."
Insert. (3) The following Comparative Example 3 and Example 6 are inserted between the 17th line and the 18th line on page 14. Comparative Example 3 4-Nitrophenol was dissolved in 50 mM MIIES buffer (p+(7) to give 1 x 1 O-2 cape/-. 2-chloro-4-nitrophenol was dissolved in 50 mM MIIES buffer
5x l〇-Misaki/d in 0mM MKS buffer (pH 7)
It was dissolved so that Changes in absorbance were examined by varying the temperature of both solutions. The results are shown in FIG. Example 6 Almost the same as Example 1, but with 4111'M in the substrate.
The difference was that g-ri1ffO-4-nitrophenyl maltohebutaoside (α form) was used. The results measured in the same manner as in Example 1 are shown in Figure 9.
4-A) Shows the relationship between temperature and absorbance of lophenol and 2-chloro-4-=)ophenol. In the figure, ■ is 4-
Nitrophenol, ■ indicates 2-chloro-4-ditrophenol 0 Figure 9 shows serum at various concentrations and absorbance for 1 minute when the substrate is 2-chloro-4-nitrophenyl maltohebutaoside (α form). 0” (5) indicating a relationship with change.
Insert figure 8 and factor 9. 8I2 Song 9

Claims (1)

[Scope of Claims] 1α-glucosidase or α-glucosidase represented by the following formula,
A method for measuring amylase activity, which comprises adding fucosidase and β-glucosidase and a known amount of the sample, and measuring the visible absorption of the liberated aglycone, thereby measuring the amylase activity in the sample.