JPS62245961A - Measuring method for enzyme activity - Google Patents

Abstract

PURPOSE:To exactly measure the activity of enzyme to be measured by bringing a phenyl group or napthyl group-contg. deriv. into contact with the enzyme to liberate a phenol or alpha-naphthol and bringing the same into contact with the diamine compd. expressed by the specific formula in the presence of an oxidizing agent. CONSTITUTION:The phenyl group or napthyl group-contg. deriv. as a substrate is brought into contact with the enzyme to be measured such as alkali phosphatase or acidic phosphatase to liberate the phenol or alpha-naphthol. The phenol or alpha-naphthol is brought into reaction with the diamine compd. expressed by the formula in the presence of the oxidizing agent such as potassium ferricyanide or chloramine T. The degree of coloration of the formed chromatic quinone dye is then measured. Since the quinone dye has a large molecular absorption coefft., the measurement sensitivity is high and therefore, the time for the reaction process is shortened; in addition, the measurement is hardly affected by abnormal serum having hemolysis and jaundice and therefore, the accuracy and preciseness of the measurement are improved.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for measuring enzyme activity, and in particular, to a method for measuring enzyme activity, in particular, phenol or α-naphthol is liberated by the action of an enzyme, and this liberated 7-enol or α-naphthol is The present invention relates to a method of producing a colored pigment from a substance and measuring enzyme activity from the degree of coloration of the pigment.

(Prior Art) Measurement of the activity of enzymes, such as alkaline phosphatase, acid phosphatase, kallikrein, and antithrone♂♂, is important in the clinical diagnosis of various diseases, and has already been widely used.

Klnd-Kin is currently widely used as a method for measuring the activity of alkaline phosphatase and acid phosphatase.
According to this conventional method, the above-mentioned enzyme is reacted with a phenyl group- or naphthyl group-containing derivative as a substrate, and the liberated phenol or α-naphthol and 4-7 onoantepyrine are combined with potassium ferricyanide as an oxidizing agent. The activity of the above-mentioned enzyme is determined by reacting in the presence of 9M to produce a yellow-red antipyrine dye and measuring the degree of coloration of this dye.

(Problems to be Solved by the Invention) The above Klnd-Klng method has a problem in that the measurement sensitivity is low. That is, the antipyrine dye produced when carrying out this conventional method has a low molecular extinction coefficient of 500 to 10,000 degrees, and therefore, when the concentration of free phenol or α-su7thole is low, the enzymatic reaction is difficult. Sensitivity is improved by setting a longer time to increase the amount of phenol or α-naphthol released.

The second problem is that the maximum absorption of antipyrine dye is 500n.
There is a reed near 500 nm, so the measurement wavelength is around 500 nm. That is, when enzyme activity is measured in the field of clinical testing, the specimen is serum, and sometimes it is an abnormal specimen such as hemolysis, chyle, yellow mites, etc. To further discuss this, hemoglobin rei 36 n
m, 530 to 570 nm, and K each have maximum absorption,
Chyle has absorbance over all wavelengths, but has a particularly strong absorbance at short wavelengths, while bilirubin has maximum absorption at 465 nm.
Therefore, since these are close to or overlap the wavelength of approximately 500 mm, which is the wavelength for measuring the absorbance of antipyrine dye, they act as interfering substances and cause measurement errors.

(Means and effects for solving the problems) The present invention focuses on the above-mentioned problems in the conventional methods, and has been made for the purpose of solving these problems. The degree of coloration of a colored quinone dye produced by contacting the enzyme to be measured with a substrate to liberate phenol or α-naphthol, and reacting this phenol or α-naphthol with a diamine compound in the presence of an oxidizing agent. The method is characterized in that the activity of the enzyme used is measured based on this measurement.

In the method of the present invention, the phenyl group- or naphthyl group-containing derivative as a substrate is converted into diphenol or α-
Those commonly used to liberate naphthol, such as disodium phenylphosphate and L-prolyl-L-phenylalanyl-L-arginine-1-naphthyl ester dihydrochloride, can be used.

Examples of the enzyme whose activity is to be measured include alkaline phosphatase, acid phosphatase, kallikrein, andesthrombin (2), and the like.

As an oxidizing agent, 7-potassium erythyanide, 9-m chloramine T
Chemical oxidizing agents can be used, such as sodium metaperiodate, and oxidation methods that utilize enzymatic reactions, such as a combination of hydrogen peroxide and peroxidase, can also be used.

The diamine compound has the formula (81 and R2 each mean alkyl, sulfone or acyl, or can be taken together and together with adjacent nitrogen atoms to form a heterocycle, and R3 and R4 are hydrogen, halogen, p-aminodiethylaniline is particularly preferred.

As an illustration of the reactions that occur when carrying out the method of the invention,
The case where alkaline phosphatase (ALP) is used as the activity measuring enzyme, phenyl phosphoric acid is used as the substrate, and p-aminodiethylaniline is used as the diamine compound is as follows.

H H As is clear from Test Example 1 below, the maximum absorption of the quinone dye produced in this reaction is around 670 nm. Even if it is an abnormal serum sample such as Dan,
Since the maximum absorption wavelength is different from that of hemoglobin and bilirubin, measurement errors caused by these can be significantly reduced.
Also, since the measurement wavelength is so high, the influence of chyle can be minimized as much as possible.

The molecular extinction coefficient of the above-mentioned quinone dye is approximately 20,000, which is 50, which is the molecular extinction coefficient of the yellow-red antipyrine dye that is the object of measurement by the conventional Kind-King method.
The sensitivity is about 2 to 4 times that of 00 to 10,000, so the sensitivity is high, and the time required for enzymatic reactions and oxidation reactions is only 5 minutes each, so it is 1/2 times as high as the conventional method. It will be shortened to about 3.

(Examples, etc.) Next, the present invention will be specifically described with reference to test examples and examples.

Test Example 1 A blue quinone dye-containing liquid obtained by reacting phenol using p-aminodiethylaniline as a diamine compound and potassium ferricyanide as an oxidizing agent was used as a test sample, and the above diamine compound The absorbance of each of these samples was measured at various wavelengths using a blind sample containing oxidizing agent and oxidizing agent but no phenol. The results are shown in Figure 1. It was found that the maximum absorption of the quinone dye in the sample was around 67 Onm. It has also been found that this molecular extinction coefficient is approximately 20,000, which is 2 to 4 times higher than that of the antipyrine dye that is the object of measurement in the conventional Kind-King method, and that the sensitivity is high.

Actual example 1m IC alkaline phosphatase activity measurement) (1
) Reagent (1) First reagent prioxyethylene octylphenyl ether at 0.1
Carbonate buffer (pl') of som yr7i containing %
il O,0) In 100 yd, 380 η of phenylphosphate disodium, 100 mF of potassium ferricyanide and io of magnehum chloride hexahydrate.

Prepared by adding and dissolving the cape. This test solution should be stored refrigerated.
It can be used for several months.

(2) P-aminodiethylaniline hydrochloride I in 1 ml of 1N hydrochloric acid as the second test solution.
Prepared by adding and dissolving Jun 36 Misaki, then adding purified water to make a total volume of 2 oul.

CI+) Measurement method (1) Measurement operation &) Collect the first test solution into a test tube addressed to 2.0111, and heat it at 37°C.
Prewarm.

b) Sample serum (T), purified water as a blind test, and standard serum (at) at 40μ! Collect the sample and put it in the test tube mentioned above.
Heat at 37°C for 5 minutes to react.

C) For both reaction solutions, the absorbance at a wavelength of 660 nm go(
g and Eo (fil) are measured using a blind control.

d) Immediately add 0.4 d of the second test solution, mix, and leave for 5 minutes.

e) For both reaction solutions, the absorbance Ef at a wavelength of 660 nm (
t) and Ef('H) are measured using a blind control.

ψ) Quantitative calculation The alkaline phosphatase activity value C in the sample serum is calculated according to the following formula.

Example 2 (Urine kallikrein activity measurement) (1) Reagents (1) Test t 0.2M phosphate buffer (pg, s) 7 oyd p-aminozoethylaniline hydrochloride 10 Ml, Pro-
P paper is immersed in a solution in which Phe-Arg-0Nip.2HCL (L-prolyl-L-) dinylalanyl-L-fulginine-1-naphthyl ester nohydrochloride) 611IF is dissolved and freeze-dried in vacuum.

This test paper was cut into 5×5 squares to produce 5×10 pieces.

(2) Coloring liquid 6ml chloramine T aqueous solution.

Alternatively, place one drop of the sample on the test paper and leave it for 2 minutes.

d) The test paper part exhibits blue color, so the color tone is
Prepared standard colorimetric coat (e.g. 0,20.40
The kallikrein activity in the sample is measured in comparison with 8ONU/1 (NU: 7 chill units), which exhibits a color tone corresponding to urine kallikrein.

Test Example 2 Regarding the activity measurement of alkaline phosphatase, we investigated whether hematoglobin, bilirubin, chyle, and ascorbic acid, which are causes of measurement errors in conventional methods, also affect the method of the present invention for measuring quinone pigments. The results of the investigation are as shown in Figures 2 to 5.
It was found that these substances had almost no effect.

Test Example 3 The results of investigating the simultaneous reproducibility of the method of the present invention were as shown in the table below, and it was found that the reproducibility was extremely good.

Test Example 4 Alkaline phosphatase activity was measured using the method of the present invention according to Example 1K and the conventional Klnd-King method, and the correlation between the two was investigated. The results were consistent as shown in FIG. It has been found that the two show a good correlation, in other words, the method of the present invention has sufficient practicality9.

(Effects of the Invention) In the method of the present invention, the degree of coloration of a colored quinone dye is measured for the purpose of measuring enzyme activity, and this quinone dye has a large molecular extinction coefficient, so the measurement sensitivity is high, and therefore the reaction treatment The time can be shortened, and since the above-mentioned quinone dye exhibits maximum absorption at a wavelength of around 67 Onm, it is possible to avoid hemolysis and hemolysis of the specimen.
Even if the serum is abnormal, such as chyle or yellow mites, it will hardly be affected, and therefore the measurement precision and accuracy will be high.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between wavelength and absorbance in a quinone dye-containing solution formed by reacting a phenol-containing test sample with p-7 minodiethylaniline and a phenol-free blind solution. Graph showing the results of adding hemoglobin to normal sample serum and investigating the effect of hemoglobin on the measured value in connection with the activity measurement of Figure 4 is the same as Figure 2, but the graph showing the results of examining the influence of chyle.
The figure is the same as Figure 2, except that it is a graph showing the results of investigating the influence of ascorbic acid.
It is a graph showing the correlation between the d-Klng method and the present invention. Patent applicant: Sanwa Kagaku Kenkyusho Co., Ltd.: +1: Figure 1: Baby (nm) Figure 2: Baby (mg/dl) Figure 3: Figure 4: Figure 5 (rw) ) 7
]) Sibi>e (mg/di) (Kind-King
y=0.968X 1.04r-0.98
41'? L=40

Claims (4)

[Claims] (1) Using a phenyl group- or naphthyl group-containing derivative as a substrate, contact the enzyme to be measured to liberate phenol or α-naphthol, and react this phenol or α-naphthol with a diamine compound in the presence of an oxidizing agent to produce it. A method for measuring enzyme activity, characterized by measuring the degree of coloration of colored quinone pigments. (2) The measuring method according to claim 1, wherein the diamine compound is an aniline. (3) The diamine compound has the formula ▲ Numerical formula, chemical formula, table, etc. can be formed, R^3 and R^
4 means hydrogen, halogen, alkyl or acyl) The measuring method according to claim 1 or 2, wherein (4) Claim 1, 2 or 3, characterized in that the diamine compound is p-aminodiethylaniline.
Measurement method described in Section.