JPS609799B2 - A new method for measuring amylase activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for measuring amylase activity that can be conveniently used for measuring amylase activity in serum or urine in tests of general organ function.

Generally speaking, the following methods for measuring amylase activity are: 1) A method in which a detection amylase is added to a starch solution and the decrease in viscosity of the solution due to starch hydrolysis is measured;
A method of adding sample amylase to a starch solution and measuring the decrease in turbidity of the solution (3' method of adding sample amylase to a starch solution and measuring the absorbance by coloring the remaining starch amount by a starch reaction) , {4) A method of adding sample amylase to a starch solution and measuring the amount of reducing sugar in the solution, and [5- Method of adding sample amylase to a solution of starch cross-linked with a dye and colorimetrically measuring the released dye. There is a method to do this, but using a viscometer (1)
The method of 21 or the method of 21 which uses a turbidity meter has the disadvantage of poor accuracy.

In addition, the method {3} which utilizes the iodostarch reaction has the disadvantage that it is difficult to continuously monitor the starch decomposition process by amylase activity, and the activity value appears differently depending on the quality of the starch used. Method (4), which measures the amount of reducing sugar, is difficult to continuously monitor the starch decomposition process, and the activity value is likely to be distorted due to the influence of glucose in the sample. Furthermore, the method [5], which involves color comparison of the released pigment, is similarly difficult to continuously monitor the starch decomposition process, and the measurement operation is cumbersome as it requires centrifugation. Except for the difficulties and drawbacks of various conventional methods, it has high precision, good reproducibility, and easy operation. The purpose of the present invention is to provide a novel method for measuring amylase activity, which is not surprising due to interference from glucose, proteins, etc., and which can obtain measurement results in about 10 minutes.

In the present invention, when amylase as a sample and glucosidase and mutarotase as auxiliary enzymes are added to a solution of a Q-glucoside bond having a polymerization degree of 5 or more as a substrate, the Q-glucoside bond is hydrolyzed and the final to produce an equilibrium mixture of Q-glucose and 8-glucose, the specific optical rotation of the equilibrium mixture is as low as about 1/4 of that of the Q-glucoside conjugate of the substrate, and the equilibrium mixture is produced from the Q-glucoside conjugate. By determining that the degree of amylase changes accurately depending on the activity value of the sample amylase, tracking the change in the optical rotation of the substrate solution, and comparing the results with those obtained using samples with known activity. Not only does it make it possible to know the activity of the amylase sample, but it also makes it possible to determine the international unit of enzyme activity simply by accurately measuring the change in its optical rotation.

The present invention will be explained in detail below based on examples thereof.

Maltobenose is used as the Q-glucoside bond having a degree of polymerization of 5 or more. Maltobentaose is hydrolyzed by Q-amylase to produce equivalent amounts of maltose (degree of polymerization 2) and maltotrise (degree of polymerization 3), and maltotriose is converted into Q-glucose by the action of Q-glucosidase. Furthermore, the Q-glucose is rapidly converted into an equilibrium mixture of Q-glucose and 8-glucose by the action of mutarotase. For example, if the wavelength is 5, the specific rotation of an aqueous solution of those substances is
When measured at 37°C using an 8-track sodium circle, maltobentaose is 1178.30, maltotriose is 11600, tomaltose is 1112.50, Q-glucose is 110, and Q-glucose is 1112.50. Since the equilibrium mixture with 8-glucose is expressed as 147.90°, the specific optical rotation of the aqueous solution in each reaction stage under the above conditions is 1178.30° and 1136.3° (i.e., 112.0°), respectively. .5160XO.5) "11020 and 147.
90 respectively. In this way, the specific rotation changes only in the decreasing direction from the initial stage to the final stage, and at the final stage, a remarkable decrease of about 1/4 times the initial value appears.
The degree of decomposition reaction of maltobentaose varies depending on the difference in amylase activity, and the change in optical rotation of the reaction solution also varies accordingly. This makes it possible to measure accurately. Since this measurement method only requires observation and recording using a polarimeter, it is easy to continuously track and record the reaction progress of maltobentaose, and as mentioned above, the specific optical rotation decreases from the initial stage to the final stage of the reaction. Since the amount of change in optical rotation is linearly proportional to the passage of reaction time because it changes only in the direction, in addition to the method of looking at the amount of change in optical rotation after a certain period of time, it is also possible to measure the rate of change of optical rotation continuously. It is also possible to determine the amylase activity using the method described above, and the glucose in the sample amylase (for example, glucose in serum when amylase activity in serum can be tested; this type of glucose has already undergone metarotation). It is possible to eliminate fluctuations in measured values due to optical rotation by simply performing numerical correction during optical rotation measurement, eliminating the need for troublesome chemical operations such as sample pretreatment, and eliminating interference caused by proteins in the sample at wavelengths longer than 40 degrees. It can be resolved by measurement, and the measurement results can be obtained within a short time of 10 to 2 minutes. At the same time, maltobentaose, which is the substrate, is easy to obtain with consistent quality and the reproducibility of the measurement results is good. It has excellent effects such as: Additionally, Q-glucosidase and mutarotase used as auxiliary enzymes are also conveniently available as commercial products. Even if an appropriate Q-glucoside bond with a degree of polymerization of 6 or more or starch is used as a substrate, maltotriose and maltose will be produced by the action of amylase, and as in the case of using maltobenose as a substrate, there will be no auxiliary When an enzyme is used, an equilibrium mixture of Q-glucose and 8-glucose is formed, which causes a large change in optical rotation, so amylase activity can be easily measured. Based on actual test results, the sensitivity is compared with that of maltobentaose as follows.

As described above, although the sensitivity to changes in optical rotation is somewhat reduced, it is possible to sufficiently measure amylase activity even if a Q-glucoside bond with a degree of polymerization of 6 or more is used as a substrate if necessary. be. The measurement method of the present invention will be further explained below using test examples.・Test example 1% of maltobentaose using a buffer solution with pH 6.9
Make a solution.

Then, 450 units/0.1 of the Q-glucosidase solution and 2500 units/0.1 of the mutarotase solution were combined with 3.8' of the maltobentaose solution and mixed. ℃ and placed in a cell with a polarimeter optical path length of 50 mm, 50 mg of serum with an amylase activity of 24 international units (Roche R ratio),
Mix 100A, 150〃〆, 20A and 250A, respectively, and unify the total amount of each test liquid in the cell to 4.25', and then test under the conditions of temperature: 370, wavelength: Soki skin. As a result of continuously measuring and recording changes in optical rotation in An almost perfectly linear proportional relationship as shown in Figure 1 is established, and the relationship diagram between the amount of change in optical rotation of each test liquid and the amount of added serum after 10 minutes has passed from the start of measurement is shown in Figure 2. It can be seen that the activity value of the sample amylase can be easily and accurately determined by the optical rotation measurement method according to the present invention through the Kendoh diagram as shown in FIG.

[Brief explanation of the drawing]

FIGS. 1 and 2 are test result charts of one test example of the present invention. Leopard 1 Multi 2 drawings

Claims (1)

[Scope of Claims] 1. Adding amylase as a specimen and glucosidase and mutarotase as auxiliary enzymes to a solution containing an α-glucoside bond having a degree of polymerization of 5 or more as a substrate, and measuring the change in the optical rotation of the solution. A method for measuring amylase activity characterized by: 2. The method for measuring amylase activity according to claim 1, wherein the α-glucoside bond is maltopentaose (degree of polymerization 5).