JPS60172299A - Method of measurement of amylase activity - Google Patents

Abstract

PURPOSE:To obtain alpha-amylase activity in a short time in high accuracy, by using a specific oligosaccharide as a substrate, adding a test specimen containing alpha-amylase to it, treating simultaneously the test specimen with alpha-glucosidase to give a reaction product, measuring enzymatically the reaction product. CONSTITUTION:An oligosaccharide shown by the formula I (n is 3-6) or formula IIis used as a substrate, and a test specimen containing alpha-amylase is added to it. Simultaneously or after the addition, the test specimen is treated with alpha- glucosidase and/or beta-glucosidase to give glucose-6-phosphate or 6-phosphogluconic acid as a reaction product, which is measured. EFFECT:Measurement is possible without causing rise in a blank value and without being influenced by endogenous glucose and maltose at all.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring α-amylase using an 06F-linked oligosaccharide or a 6-PG-linked oligosaccharide as a substrate.

In the human body, α-amylase is mainly produced in the pancreas or salivary glands, and in certain diseases, amylase escapes into body fluids such as serum and urine. Clinically useful diagnosis is possible by measuring α-amylase in serum or urine. For example, α-amylase activity in serum shows a nearly constant value in healthy people, but in patients with acute pancreatitis, α-amylase activity in serum
- Amylase content is increased and becomes an important clinical parameter such as pancreatic function.

Conventionally, methods for measuring α-amylase have been reported or practiced, such as an iodine-starch reaction method, a turbidimetric method, a blue starch method, and various enzymatic methods, but these methods have many problems. For example α
-Iodine using 1 starch as a substrate for amylase-
In starch reaction methods and turbidimetric methods, it is extremely difficult to constantly obtain starch of constant quality. In addition, the blue starch method requires centrifugation during the operation of the fixed tray, which makes it difficult to apply to automatic analysis equipment, and it is not possible to measure the reaction rate using the late assay method. Also in the enzymatic method,
It is difficult to remove glucose and maltose from serum and urine samples. A coloring method using an aromatic substituted substance for the reducing terminal hydroxyl group of an oligosaccharide has also been proposed, but none of these substrates can be used for α-amylase to induce α-1, 2 or more positions in the molecule. The 4-glucosidic bond is cleaved, meaning that once expanded, the product produced by the reaction of α-amylase with the substrate will again become a substrate for α-amylase. In addition, not only α-amylase but also α-glucosidase acts on the product generated by α-amylase decomposition, and the two enzymes compete for substrate, making it impossible to accurately measure α-amylase activity. The company is clear.

Therefore, as a result of intensive research, the present inventors solved the above-mentioned drawbacks and completed the present invention.

This objective can be achieved by using a G6P-linked oligosaccharide or a Hiro6-PG-linked oligosaccharide as a substrate for α-amylase as a method for accurately and rapidly measuring α-amylase activity.

That is, in order to measure the Hiro α-amylase activity of the present invention, a G6P-linked oligosaccharide or an a6-PG-linked oligosaccharide was used as a substrate, and at the same time as a sample containing α-aminase was added thereto. This is a method for measuring the activity of α-amylase, which is characterized in that after that, α-glucosidase and/or β-glucosidase are allowed to act, and G6Pt or 6-PG is enzymatically measured as a product.

Next, the reaction in the present invention will be explained using the following formula.

■ As a substrate, for example, 06F-bonded maltopentaose (
When using (1, -06F) α-amyl g G, -06P + H, O maltotriose + (J, -G6
For example, 6-PG-bound maltopentaose ((j
a-amylase G, -6-PG + 1-1.0 + ff lutotriose + (], -]6-PGα-glucosidase, -6-P (1+211,0, 2 glucose +6
-P(i+NAIXP)H N A D (P) H produced by the above reaction) is 34
It is possible to measure by increasing the absorbance of 01m, or it is also possible to connect a redox dye system and measure by color development. Furthermore, in the case of (4), the sensitivity can be doubled by adding 6-PGDH.

According to the present invention, α-amylase in a sample containing α-amylase is
Compared to conventional methods, it is possible to measure amylase activity in a shorter time and with higher precision, without increasing the blank value, and without accounting for the effects of endogenous glucose or maltose. This is an extremely meaningful method industrially.

Example 1 50mM FIPBS (pH 6,8), 5m
M MgO1,.

1mMNAD, 2mM G, -G6P, 10
u/ml α-glucosidase and 2u/+++
A human amylase solution of θ~50 μ was added to 2 ml of the reaction solution containing jG6PDH, and the increase in absorbance at 34′ Onm was measured at 25° C. for 5 minutes, and the reaction rate per minute was read.

Figure 1. Linearity of α-amylase activity measurement 0 10 2
0 30 40 50 Temple amount (μt) As a result, it was confirmed that the enzyme activity was proportional to the sample amount, and that α-amylase activity could be sufficiently measured.

Example 2 50mM PIPBS, 5mM Mg0J? ,,1mM
NADP, 2mM G66PG, 10u
Add 0 to 50 μL of human amylase solution to reaction solution 2- containing /mt α-glucosidase and 3 u/m6 PGDH, measure the increase in absorbance at 340 nm at 25°C for 5 minutes,
I read the reaction rate.

Figure 2. Linearity of α-amylase activity measurement 0 10 2
0 30 40 50 Sample amount (μt) As a result, it was confirmed that the enzyme activity was proportional to the sample amount, and that α-amylase activity could be sufficiently measured.

Claims (1)

[Claims] (in the formula, n fl is an integer in the range of 3 to 6) or (in the formula, n is an integer in the range of 3 to 6) as a substrate,
At the same time as or after adding a sample containing α-amylase, α-glucosidase and/or β-glucosidase are allowed to act on this, and the product thereof is glucose-6-phosphorus (06F) or 6-phosphoglucon. α characterized by measuring acid (6-PG)
-Method for measuring amylase.