JPS603836B2 - Enzyme activity measurement method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for measuring enzyme activity.

The method for measuring enzyme activity of the present invention is based on the general formula (1) (wherein, X
is HO- or (R,, R2 is hydrogen or C, ~C5 alkyl group or ○(CH2)n1, n is an integer from 1 to 4), and Y is Q-NH-1-amino acid, or General formula (0), which uses a compound having an alcohol residue) as a substrate and is released from the substrate by the action of an enzyme.
(wherein X is the same as above) is combined with 4-aminoantipyrine or its derivative in the presence of an oxidizing agent, and the concentration of the resulting quinonimine dye is determined colorimetrically. This is a characteristic feature.

In recent years, the measurement of enzyme activity in biological body fluids such as serum has become important in the field of clinical testing as a means of disease diagnosis.However, conventional methods for measuring the activity of enzymes, especially acylase and esterase, are The method is complex;
The accuracy was also low and unsatisfactory for practical purposes.

The present inventors have conducted various studies to develop a simple and highly accurate testing method for enzymes, especially acylase and esterase.
If a substrate with the general formula is used and liberated by enzymatic action, it can be converted to a quinoneimine dye by binding with 4-aminoantipyrine in the presence of an oxidizing agent, and the resulting dye can be quantified colorimetrically. The present invention was completed based on the knowledge that enzyme activity can be easily measured using the following method.

The substrate used in the present invention can be easily obtained by acylating or esterifying these and the amino acid or alcohol corresponding to Y using a conventional method.

Production examples of p-dimethylaminobenzoylcholine and p-dimethylamino/hippuric acid, which are used as examples of substrates in the present invention, are shown below. '1) Manufacture of p-dimethylaminobenzoylcholine: Mix 20 parts of p-dimethylaminobenzoic acid and 150 parts of thionyl chloride, and add 60 parts to 60 parts.
heating for an hour, removing unreacted thionyl chloride under reduced pressure,
Recrystallization from V-Blue petroleum ether gave p-dimethylaminobenzoyl chloride.

p-dimethylaminobenzoyl chloride 10 mol
and low mol of choline chloride were mixed and heated under reflux at 120°C for 6 hours. The reaction solution was cooled to room temperature, and the pale yellow precipitate was washed with ether and n-hexane, and separated and purified by silica gel chromatography to obtain p-dimethylaminobenzoylcholine. '21 Production of p-dimethylaminohippuric acid: p-dimethylamino/benzoic acid 10 wmol and glycine ethyl ester low mol
was suspended in 100% of chloroform, low mol of jetylaminopropylcarbodiimide was added thereto, stirred for 3 hours, and the reaction solution was dissolved in water and 0.1NH
After thorough washing with CI and drying using anhydrous sodium sulfate, chloroform was distilled off under reduced pressure, the residue was dissolved in 20' methanol, 0.1N NaOH20' was added thereto, and the solution was dissolved at room temperature for 1. After stirring for an hour, the pH was adjusted to 4.7 and water was removed under reduced pressure.

The residue was separated and purified by silica gel chromatography to obtain p-dimethylami/hippuric acid. '31 o-,
Production of m-, p-hydroxyhippuric acid: Instead of the p-dimethylaminobenzoic acid used in [2}' above, o-
, m-, p-hydroxybenzoic acid,
O-, mH, p-hydroxyhippuric acid can be produced by a procedure similar to that described above.

As the oxidizing agent used in the present invention, chloramine T, chloramine B, potassium ferricyanide, periodic acid, periodate salts, etc. are suitable, and periodic acid and its salts are particularly preferred from a practical standpoint.

Next, the measurement method of the present invention will be explained with reference to the following examples. A specific example of measuring the activity of cholinesterase in serum using p-dimethylaminobenzoylcholine as a substrate is shown below.

To 1 part of a substrate solution (solution A, pH 7.5) containing 5M of p-dimethylaminobenzoylcholine, 2.5mM of 4-aminoantipyrine, and 5mM of sodium phosphate, 0.02% of serum was added. It was heated to 370℃.

After 10 minutes, 1 part of the reaction stop solution (solution B, sodium metaperiodate 17mM, neostigmine 7M) was added to the above reaction solution.
A pillow was added and the mixture was flooded with 370°C for 2 minutes, and the absorbance was measured using skin light at a wavelength of 55°.

Let this absorbance be C. As a blank test, the above A
After adding B solution 1' to solution 1', serum 0.02'
was added, and the absorbance was measured using light with a wavelength of 55 nm.

Let this absorbance be D. The activity unit U of cholinesterase in serum was determined by the following formula.

U = Barley x Poison x Poison... Mustache

U; 2 Sat 0.01 (Amol/wZ/min) CV=0
．． A specific example of measuring hiplicase activity using 5% p-dimethylaminohippuric acid as a substrate is shown below.

A 0.1M phosphate buffer containing 15mM p-dimethylaminohippuric acid and 2.5mM 4-aminoantipyrine (p
Add 100 μg of a liquid containing hipricase to H8.0) 2' and allow the reaction to proceed at 370 for 10 minutes. ) was added, and after flooding with 370℃ for 2 minutes, the absorbance was measured using light at a wavelength of 55℃.

Let the absorbance be A. As a blank test, reaction stop solution 1' was added to phosphate buffer 2' above, 100 ml of the above hypercase-containing liquid was added thereto, and the mixture was left at 37°C for 2 minutes. The absorbance was measured using the light of

Let the absorbance be B. The activity unit U of hiplicase was determined by the following formula.

U = 2 References X...Chapter x 3.

The experiment was repeated 30 times using the same hiplicase-containing liquid as Xmoo, and the determined activity units U and coefficient of variation CV were as follows.

U2.5±0.06 (mountain m○1/min)CV
= 2.4% Furthermore, a specific example of measuring the activity of hiplicase using p-hydroxyhippuric acid as a skin conditioner is shown below.

A 0.1 M phosphate buffer (pH 8.
0) Liquid containing hiplicase for 2 [100
was added and allowed to react for 10 minutes at 370°C. To this reaction solution was added a reaction stop solution (sodium metaperiodate 2
0 M, 2-mercaptoethanol (5 mM) 1' was added and heated to 37° C. for 2 minutes, and then the absorbance was measured using light with a wavelength of 50 orbitals.

Let the absorbance be A. As a blank test, reaction stop solution 1 was added to phosphate buffer 2, and 100A of the hiplicase-containing liquid was added thereto at 37°C.
After leaving it for 2 minutes, the absorbance was measured using a 50-wavelength desk lamp.

Let the absorbance be B. The activity unit U of hiplicase is
It was calculated using the following formula.

U = Boiled voice x Umami... 3 x 3. XmooActivity unit U determined by repeating the experiment 30 times using the same hiplicase-containing liquid
and the coefficient of variation CV were as follows.

Claims (1)

[Claims] 1. A compound having the following general formula (I) is used as a substrate and is released from the substrate by the action of an enzyme. A method for measuring enzyme activity, which comprises combining a benzoic acid derivative of the following general formula (II) with 4-aminoantipyrine or a derivative thereof in the presence of an oxidizing agent, and colorimetrically quantifying the produced quinoneimine dye. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ In the above formulas (I) and (II), X is HO- or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (R_1, R_2
is hydrogen or an alkyl group of C_1 to C_5 or HO(CH_2)_n-,
n is an integer of 1 to 4), and Y represents an α-NH-1-amino acid or alcohol residue. 2 A compound having the general formula (I) above ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (X is HO-, NH_2-, (CH_3)_2N-, ( C_2
H_5)_2N- or ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and Y is -NH- CH_2-COOH, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (Z is -H, -CH_3 or -CH_2-CH_3 ), ▲There are mathematical formulas, chemical formulas, tables, etc.▼), and the benzoic acid derivative of the above general formula (II) is a compound having the general formula 2. The method for measuring enzyme activity according to claim 1, wherein the enzyme is a benzoic acid derivative having the same formula as the benzoic acid derivative. 3. The enzyme activity measuring method according to claim 1 or 2, wherein the enzyme is acylase or esterase. 4. Claim 1 or 2, wherein the oxidizing agent is one or more of periodic acid, periodate salts, and potassium ferricyanide.
Enzyme activity measurement method described in section.