JPH075616B2 - Novel phosphoric acid derivative and method for measuring acid phosphatase activity using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION 3.1 Field of Industrial Application The present invention has the general formula (I) [Wherein X is a halogen atom, R is-(CH ₂ ) _n CH ₃ (n = 0 to 0
3). ] The novel phosphoric acid derivative or its salt represented by these, and the measuring method of the acid phosphatase activity characterized by using it as a substrate.

The present invention is a novel phosphoric acid derivative and a measurement method using the same, and according to the present invention, acid phosphatase activity can be measured accurately and simply, and a medical test method for measuring acid phosphatase It is extremely useful in the fields of physical treatment and clinical examination.

3.2 Conventional Technology Acid phosphatase (hereinafter referred to as Acp) is an enzyme that hydrolyzes phosphate monoesters under acidic conditions (pH 4 to 6). Prostate cancer, breast cancer or bone disease with bone metastasis, liver and kidney. Acp activity in serum or urine is increased in patients with disease, and it is markedly increased in prostate cancer in particular, and the measurement of Acp activity as a tumor marker has attracted attention.

Conventionally, as methods for measuring acid phosphatase activity, methods using various synthetic substrates have been reported as shown below, and some have been put to practical use in daily clinical tests.

(A) Method using β-glycerophosphate as a substrate [Bodansky, A .: J. Biol. Chem., 101 , 93, (1933)]: β-glycerophosphate is hydrolyzed by Acp to give glycerin. Inorganic phosphorus is produced, and this inorganic phosphorus is developed and measured.

(B) Method using p-nitrophenyl phosphate as a substrate [Hudson, PB: J. Urol., 58 , 89, (1947)]: p-nitrophenol produced by hydrolysis with Acp was treated with an alkali. Color and measure.

(C) Method using phenyl phosphate as a substrate: The phenol produced by hydrolysis with Acp was converted to Folin-Ciocalt.
Method to develop color with en reagent [King, EJ, Armstrong, AR: C
anad, Med.Assoc.J., 31 , 376, (1934)], and a method for measuring the red quinone produced by oxidative condensation of the resulting phenol with 4-aminoantipyrine [Kind, PRNKin
g, EJ: J.Clin.Path., 7 , 322, (1954)].

(D) Method using naphthyl phosphate as a substrate [Hillman, G .:
Z.Klin.Chem., Klin.U.Biochem., 9 , 237, (1971)]: Fast red TR is reacted with naphthol produced by hydrolysis with Acp to obtain an azo dye, and colorimetric measurement is performed.

(E) Method using 2,6-dichloro-4-nitrophenyl phosphate as a substrate [Teshima, S. Hayashi, Y. Ando, M .: Clin. Chi
m.Acta., 168 , 231, (1987)]: A method for colorimetrically measuring the yellow color tone of 2,6-dichloro-4-nitrophenol produced by hydrolysis with Acp at 400 nm.

3.3 Problems to be Solved by the Invention These measuring methods have various problems, which cause inaccuracy of measured values. For example, in (a), since normal serum contains inorganic phosphorus, the inorganic phosphorus in the test serum must be measured in advance, and complicated operation is required, which is a practical problem. (B) is the optimum pH of Acp
After enzymatic reaction in the acidic region, p-nitrophenol, which is a chromogen, does not develop color unless it is made alkaline with caustic soda aqueous solution or the like, and therefore rate assay cannot be performed.
Also, at the measurement wavelength of 405 nm, it is the slope of the UV spectrum of the chromogen, and is strongly influenced by bilirubin in serum, which causes an error in the measurement value. Similar to (b), (c) requires a reaction for coloring phenol generated by hydrolysis with Acp, and cannot perform rate assay. Further, the coloration between phenol and 4-aminoantipyrine is also unstable, which causes an error in measured values. (D)
Fast Red TR, which reacts with free naphthol, is unstable. In addition, this method can also perform rate attention.
Although it can be applied to automatic analyzers, there is a large lag time in the reaction, which causes measurement error.

(E) can be rate-assayed without requiring color reaction and can be applied to automatic analyzers, but the measurement wavelength is 40
Since it is near 0 nm, it is easily affected by bilirubin and hemoglobin in serum, and the substrate itself is unstable in an aqueous solution, and spontaneous hydrolysis occurs.

As described above, the conventional methods for measuring Acp activity have various drawbacks and have practical problems such as causing an error in the measured values.

3.4 Means for Solving the Problems We have earnestly studied to solve the drawbacks of the conventional method and arrived at the present invention. That is, a novel compound represented by the general formula (I) is synthesized, and Acp by the UV method using such a compound as a substrate.
When the activity measurement was examined, this method can use a wavelength of about 320 to 370 nm as a measurement wavelength, it is extremely stable against non-enzymatic hydrolysis, and Acp in serum is measured.
We obtained knowledge that it specifically reacts with.

Therefore, by using this substrate, the Acp activity can be measured extremely accurately and reproducibly, and the measurement having various other advantages becomes possible.

That is, the invention has the general formula (I) [Wherein X is halogen and R is-(CH ₂ ) _n CH ₃ (n = 0
~ 3). ] The novel phosphoric acid derivative or its salt represented by these, and this novel phosphoric acid derivative or its salt are used as a substrate, The acidic phosphatase activity measuring method characterized by the above-mentioned.

R in the above formula (I) is methyl, ethyl, propyl or butyl. X is a halogen atom such as chlorine, bromine or fluorine. Examples of the salt of the novel phosphoric acid derivative of the above formula (I) include alkali metal salts such as sodium salt and potassium salt; amine salts such as tris (hydroxymethyl) aminomethane salt, cyclohexylamine salt and dicyclohexylamine salt. Can be mentioned.

The novel phosphoric acid derivative can be synthesized, for example, by the reaction scheme shown below.

That is, 2,6-dihalophenol (II) is reacted with an acid anhydride to be esterified to give 2,6-dihalophenol ester (III), and then Fries in the presence of a catalyst such as aluminum chloride or zinc chloride. Rearranges to obtain 2,6-dihalo-4-acylphenol (IV), and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide [MetOH (VI)]
After reacting with the corresponding salt (V) to react with phosphorus oxychloride (POCl ₃ ) and hydrolyzing, the compound of formula (I)
The novel phosphoric acid derivative of is obtained. All of these reactions are known per se and the reaction conditions are the same as known reactions.

The salt of the novel phosphoric acid derivative is obtained by a known method using a compound of formula (I) and an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide; or an amine such as tris (hydroxymethyl) aminomethane or cyclohexylamine. It is obtained by treating with.

Next, the Acp activity measuring method of the present invention using the novel phosphoric acid derivative will be described by taking 2,6-dichloro-4-acetylphenylphosphoric acid (hereinafter referred to as DCAP-P) as an example.

The UV spectra of DCAP-P (a) and 2,6-dichloro-4-acetylphenol (b) are shown in FIG. DCAP-P is A
When hydrolyzed by the action of cp, phosphoric acid and 2,6-dichloro-4
-Produces acetylphenol. 30 for phosphoric acid and DCAP-P
There is almost no UV absorption above 0 nm. 2,6-dichloro-
4-Acetylphenol absorbs UV below 370 nm. Therefore, in the method of measuring Acp activity by the UV method,
Using DCAP-P as a substrate, the reaction can be followed at a measurement wavelength of 300 to 370 nm, in which case it is less likely to be interfered with by other serum components. Therefore, the increase in 2,6-dichloro-4-acetylphenol can be accurately followed, and Ac
It is possible to measure p activity accurately. Also, as described below, DCAP-P has many excellent advantages.

Therefore, Acp using the novel phosphoric acid derivative of the general formula (I)
Specific examples of the activity measuring method include the following measuring methods. That is, by mixing a sample containing Acp with the novel phosphoric acid derivative represented by the general formula (I) or a salt thereof, and then measuring the absorbance, particularly at 300 to 370 nm,
This is a method for measuring Acp activity.

In the method using 2,6-dichloro-4-nitrophenyl phosphate as a substrate described above, it is possible to rate-assay in the acidic region where the optimum pH of Acp is, but since the measurement wavelength is 400 nm, serum components such as bilirubin and hemoglobin are used. Receive strong interference. On the other hand, in the measurement wavelength of the present invention of about 300 to 370 nm, the interference is not so much received, so that the optimum measurement condition is easy. Further, the novel phosphoric acid derivative of the present invention, for example, 2,6-dichloro-4-acetylphenol produced by hydrolysis of DCAP-P has a maximum absorption in the vicinity of 300 nm, and thus the measurement wavelength can be set to a peak. . This means that the difference in molecular extinction coefficient caused by the problem of wavelength accuracy of the analyzer becomes very small, and the difference between measured values of analyzers becomes very small.

Furthermore, the novel substrates of the invention, eg DCAP-P, are very stable to non-enzymatic hydrolysis. For example, pH 5.4 is 100
Almost no hydrolysis occurred in 10 minutes at 37 ° C in mM citrate buffer (Fig. 5). This result shows that non-enzymatic hydrolysis can be neglected during the measurement, and the Acp activity can be accurately measured.

Further, the novel substrate phosphoric acid derivative of the present invention has a high affinity for Acp and is suitable for measuring Acp activity.

Citric acid, acetic acid, succinic acid, phthalic acid, etc. can be used as a buffering agent for keeping the pH constant when measuring the activity of Acp. Buffering agents other than the above can be used as long as they can maintain the buffering capacity at pH 4.0 to 6.0.

For example, when DCAP-P was used as a substrate, the optimum pH of Acp was around 5.4 in 100 mM citrate buffer (see Fig. 4). As described above, since DCAP-P has non-enzymatic hydrolysis stability at pH 5.4, the assay method of the present invention can perform the reaction at the optimum pH of Acp.

3, 5 Effects of the Invention The Acp activity measuring method of the present invention solves the problems of the conventional method in various respects. The advantages of the present invention are as follows.

(1) The reaction mechanism of the measurement system is simple and clear, and there are very few causes of measurement error.

(2) It can be measured at the peak wavelength (330 nm).

(3) The novel phosphoric acid derivative of the present invention used as a substrate, for example,
Since DCAP-P is very stable against non-enzymatic hydrolysis, reproducibility of measured values is good.

(4) Since it is not necessary to make a sample blank for each sample, simple and quick measurement can be performed, and a large number of samples can be processed.

(5) Since the novel phosphoric acid derivative of the present invention, such as DCAP-P, is stable, it can be reacted at the optimum pH (5.4).

(6) The novel phosphoric acid derivative of the present invention, for example, 2,6-dichloro-4-acetylphenol produced by hydrolysis of DCAP-P has a sufficient molecular extinction coefficient at the measurement wavelength at the optimum pH (5.4) of Acp. Because it is big, like p-nitrophenol,
The reaction can be continuously tracked without any operation such as once stopping the reaction, making it alkaline, and measuring the colorimetrically.

(7) It can be easily applied to an automatic analyzer.

(8) The novel phosphoric acid derivative of the present invention has a high affinity for Acp and is suitable for measuring Acp activity.

As described above, the method for measuring Acp activity of the present invention solves the problems of the conventional method, has many advantages and characteristics, can accurately and simply measure Acp activity, and can measure Acp activity in daily clinical tests. It can contribute enough.

3, 6 Examples Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1. Synthesis of 2,6-dichloro-4-acetylphenylphosphoric acid (1) Dissolve 50.0 g of 2,6-dichlorophenol in 28 ml of acetic anhydride, add 1 or 2 drops of concentrated sulfuric acid, and add 130 ° C. After reacting for 10 minutes and acetylating, the reaction solution was poured into 300 ml of ice water after the reaction, extracted with ethyl acetate, the ethyl acetate phase was dried over anhydrous magnesium sulfate overnight, and ethyl acetate was concentrated to give a colorless oily substance 58.2 g. Got Next, the oily substance was added to nitrobenzene 150.
56.8 g of powdered aluminum chloride was gradually added thereto, and the mixture was reacted at 60 to 70 ° C. for 27 hours to carry out Fries rearrangement. After the reaction was completed, the reaction solution was poured into cold dilute hydrochloric acid 1,
The organic phase and aqueous phase were separated, and the organic phase was extracted with 2N aqueous sodium hydroxide solution. After extraction, the solution was made acidic by adding 5N hydrochloric acid, further extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated to obtain 38.5 g of crude 2,6-dichloro-4-acetylphenol crystals. The crystals were recrystallized from hot ethyl acetate to obtain 19.9 g of pure white crystals of 2,6-dichloro-4-acetylphenol.

2,6-Dichloro-4-acetylphenol (3,5-dichloro-4-hydroxyacetophenone) C ₈ H ₆ Cl ₂ O ₂ mp 160-162 ° C Elemental analysis value [%] Found. Calcd. C; 47.05 46.86 H; 2.78 2.95 (2) Dissolve 8.9 g of 2,6-dichloro-4-acetylphenol in acetone, and under ice cooling, 2N aqueous sodium hydroxide solution.
Add 10.8 ml.

Acetone and ether were added to this solution again to precipitate crystals.
Let The crystals collected by filtration are dried under reduced pressure. Pale yellow 2,6-
Sodium salt of dichloro-4-acetylphenol 8.04
got g. Next, 2.61 g of this sodium salt is cooled to about 10 ° C.
Slowly add to 10.4 ml of phosphorus oxychloride prepared. After addition
Incubate at 12-15 ℃ for 30 minutes. Precipitated sodium chloride
After filtering off, the filtrate was concentrated and a colored oil was obtained. This oil
About 100 ml of cold water was added to the mixture, and the mixture was kept at 0 ℃ to room temperature for 45 minutes.
A hydrolysis reaction was performed. After separating the insoluble matter by filtration,
The liquid is extracted with ethyl acetate and dried over anhydrous magnesium sulfate.
After that, it was concentrated with ethyl acetate to give 2,6-dichloro-4-a
1.22 g of crude crystals of cetylphenylphosphoric acid are obtained. This coarse
The crystals were recrystallized from hot ethyl acetate / n-hexane to give 682 mg of crystals.
I got crystals. Furthermore, this crystal was subjected to column chromatography.
(Stationary phase; Sephadex Purified with LH-20, solvent: methanol)
And white 2,6-dichloro-4-acetylphenylphosphate
521 mg of colored crystals were obtained.

2,6-Dichloro-4-acetylphenylphosphate C ₈ H ₇ Cl ₂ O ₅ P mp 165 to 170 ° C (dec) Elemental analysis value [%] Found. Calcd. C 33.38 33.71 H 2.53 2.48 U, V spectrum 1 and IR spectrum respectively,
It is shown in FIG.

Example 2 Method for measuring serum Acp activity (1) 100 mM citrate buffer pH 5.4 (25 ° C.) (2) Specimen (3) 7.8 mM substrate (DCAP-P) solution (1) Sample 0.1 in 2.0 ml of buffer Add ml for 2-10 minutes
Preheat at 37 ℃, add 0.5 ml of the substrate solution of (3) to it,
At the same time, start the stopwatch exactly 1 minute, 2
The absorbance at 330 nm is measured to determine the change in absorbance per minute. Figure 3 shows the time course.

Human prostate-derived Acp (manufactured by Sigma) was used as a sample. The Acp activity value is calculated by the following formula.

1) ΔOD / min is the amount of change in absorbance per minute at a measurement wavelength of 330 nm.

2) The molecular extinction coefficient of 2,6-dichloro-4-acetylphenol at a wavelength of 330 nm is 18500.

From the above equation, the Acp activity of the sample used was 205 (IU / l). As shown in FIG. 3, linearity was shown over 10 minutes. This indicates that the automatic analyzer can be used.

Example 3 The pH of the buffer solution of (1) of Example 2 was changed from 4.8 to 6.2, and the optimum pH of Acp in this method was determined. Buffer p
All but Example H were according to Example 2. The results are shown in FIG. The optimum pH was 5.4 under these conditions.

Example 4 0.5 ml of the substrate solution of (3) was added to 2.0 ml of the buffer solution of (1) of Example 2.
Was added, and the mixture was placed in a 37 ° C heat-retaining cell, and the change in absorbance at a wavelength of 330 nm was followed over time to examine the non-enzymatic hydrolysis stability of the substrate. The result is 10 as shown in Fig. 5.
It was almost stable until the minute. Substrate DCAP-P has an optimum pH of 5.
Since it is stable at 4, it is not necessary to measure a reagent blank for each sample.

Example 5 In accordance with Example 2, the relationship between the dilution ratio of hiro prostate-derived Acp and the enzyme activity was examined (FIG. 6). The sample dilution was performed using (1) of Example 2. As shown in Fig. 6, the sample dilution and enzyme activity are in a linear proportional relationship passing through the origin.
It was revealed that Acp activity can be measured widely from low to high units.

Example 6 The line solution of (3) of Example 2 was used after being appropriately diluted.
The Km value for this substrate was determined from the aver-Burk plot and found to be 0.14 mM / l (Fig. 7). From this, it was revealed that the substrate has a high affinity for Acp and has sufficient adaptability to this reaction system.

Example 7 Synthesis of 2,6-dibromo-4-acetylphenylphosphoric acid (1) 5.0 g of 2,6-dibromophenol was dissolved in 5 ml of acetic anhydride, and 1 drop of concentrated sulfuric acid was added. After shaking by hand occasionally and the heat generation subsided, the solution was poured into 50 ml of ice water, and the precipitated crystals of acetic acid-2,6-dibromophenyl were collected by filtration, washed with cold water, and dried under reduced pressure. . Next, this acetic acid-2,6-dibromophenyl was dissolved in 20 ml of nitrobenzene, to which 4.0 g of powdered anhydrous aluminum chloride was gradually added to 60-70.
Fries rearrangement was performed by reacting at 50 ° C for 50 hours. After completion of the reaction, the reaction solution was poured into 100 ml of cold dilute hydrochloric acid and left at 5 ° C overnight,
The precipitated crystals were collected by filtration, washed with n-hexane and dried under reduced pressure to obtain 3.87 g of crude crystals of 2,6-dibromo-4-acetylphenol. The crystals were recrystallized from hot ethyl acetate to give 2.42 g of pure, light and colored crystals of 2,6-dibromo-4-acetylphenol.

2,6-Dibromo-4-acetylphenol (3,5-dibromo-4-hydroxyacetophenone) C ₈ H ₆ Cl ₂ O ₂ mp 180-183 ° C Elemental analysis value [%] Found. Calcd. C; 32.71 32.69 H; 1.90 2.06 (2) 2.21 g of 2,6-dibromo-4-acetylphenol was dissolved in acetone and 2N sodium hydroxide aqueous solution 3.75 under ice cooling.
Add ml. Acetone and ether are added to this solution again, and the precipitated crystals are collected by filtration and dried under reduced pressure. Light yellow 2,6
1.88 g of the sodium salt of dibromoacetylphenol was obtained. Next, 0.90 g of this sodium salt is suspended in 10 ml of n-hexane, and when cooled to -10 ° C, 2 ml of phosphorus oxychloride is added dropwise. After the dropping, the temperature of the reaction solution is returned to room temperature and the reaction is performed for 20 hours. The precipitated sodium chloride was filtered off, the filtrate was concentrated, and a colored oily matter was obtained. 50 ml of ice water is added to this oily substance to carry out hydrolysis reaction at 0 to 15 ° C for 1 hour and at 30 to 45 ° C for 1 hour. The insoluble matter was filtered off, sodium chloride was added to the filtrate to make it saturated, and the mixture was extracted with ethyl acetate.
After drying over anhydrous magnesium sulfate, ethyl acetate was distilled off to give crude crystals of 2,6-dibromo-4-acetylphenol.
23 mg was obtained. The crude crystals were recrystallized with dimethyl sulfoxide (DMF) and ether to obtain 234 mg of pure light and colored crystals of 2,6-dichloro-4-acetylphenylphosphoric acid.1 / 2DMF.

C ₈ H ₇ Br ₂ O ₅ P / 1 / 2C ₃ H ₇ NO mp149-154 ° C Elemental analysis value [%] Found. Calcd. C; 28.03 27.80 H; 2.82 2.56 N; 1.75 1.71 Example 8 2,6- Synthesis of dichloro-4- (n-butyryl) phenylphosphoric acid (1) 2,6-dichlorophenol (10.0 g) was added to n-butyric anhydride 10
Dissolve in ml and add 1 drop of concentrated sulfuric acid. Shake the container from time to time to stir, and when the fever subsides, pour the solution into 100 ml of ice water. This solution was extracted with n-hexane, washed with saturated brine, dried over anhydrous magnesium sulfate overnight, and then n-hexane was distilled off to give colorless oily n-butyric acid-2,6-dichlorophenyl 13.22. got g. Next, this butyric acid-2,6-dichlorophenyl was dissolved in 25 ml of nitrobenzene, and 13.8 g of powdered anhydrous aluminum chloride was gradually added to it, and the temperature was adjusted to 55 to 65 ° C.
Fries rearrangement was performed by reacting for 40 hours. After the reaction,
The reaction mixture was poured into cold dilute hydrochloric acid (500 ml), left overnight at 5 ° C., the aqueous phase was separated, and the organic phase was extracted with 2N aqueous sodium hydroxide solution. After extraction, the solution was acidified with 5N hydrochloric acid, the precipitated crystals were extracted with ethyl acetate, washed with saturated brine, dried over anhydrous magnesium sulfate overnight, and the ethyl acetate was distilled off.
2.09 g of crude crystals of 6-dichloro-4- (n-butyryl) phenol were obtained. The crystals are recrystallized from ether and n-hexane to give pale and pure 2,6-dichloro-
1.95 g of crystals of 4- (n-butyryl) phenol were obtained.

2,6-Dichloro-4- (n-butyryl) phenol C ₁₀ H ₁₀ O ₂ Cl ₂ mp 93-96 ° C Elemental analysis value [%] Found. Calcd. C; 51.58 51.53 H; 4.35 4.32 (2) 2,6 2.33 g of dichloro-4- (n-butyryl) phenol is dissolved in acetone, and 2.5 ml of 4N sodium hydroxide aqueous solution is added under ice cooling. Acetone and ether were again added to this solution, and the precipitated crystals were collected by filtration and dried under reduced pressure to obtain 2.14 g of pale and colored sodium salt of 2,6-dichloro-4- (n-butyryl) phenol. Then this sodium salt
2.14 g are suspended in 20 ml of n-hexane, cooled to -10 ° C, and 7.25 ml of phosphorus oxychloride is added dropwise. After the dropping, the temperature of the reaction solution is returned to room temperature and reacted for 24 hours. After filtering off the precipitated sodium chloride, the filtrate was concentrated and a colored oily matter was obtained. Add 75 ml of ice water to this, 0 ℃ -15 ℃ for 1 hour, 40-50 ℃
The hydrolysis reaction was carried out for 1 hour. After the reaction, the insoluble matter was filtered off, sodium chloride was added to the filtrate to make it saturated, and the mixture was extracted with ethyl acetate. After drying over anhydrous magnesium sulfate, ethyl acetate was distilled off to obtain 2.06 g of crude crystals of 2,6-dichloro- (n-butyryl) phenylphosphoric acid. The crude crystals were recrystallized from DMF ether to obtain 1.79 g of pure white crystals of 2,6-dichloro-4- (n-butyryl) phenylphosphoric acid.DMF.

2,6-dichloro-4-(n-butyryl) Fuenirurin acid _{mp78~80 ℃ C 10 H 11 Cl 2} O 5 P · C 3 H 9 NO elemental analysis [%] Found Calcd C;. . 40.39 40.43 H; 5.18 4.70 N; 3.61 3.63 Example 9 2,6-dibromo-4-acetylphenyl phosphate and 2,6-
Acp activity measurement method using dichloro-4- (n-butyryl) phenyl phosphate as a substrate As an alternative to the substrate (DCAP-P) of (3) of Example 2, 2,
6-dibromoacetylphenyl phosphate (DBAP-P) and 2,6
-Dichloro (n-butyryl) phenyl phosphate (DCBP-P)
Was used to measure the Acp activity. Reagents and operations were in accordance with Example 2. The time course at that time is shown in FIGS. 8 and 9. In addition, the Acp activity of the sample was 103 for DBAP-P.
IU / l and DCBP-P were 109 IU / l. The molecular extinction coefficients are 18900 and 11200, respectively. As shown in FIGS. 8 and 9, linearity was shown over time for 10 minutes. This indicates that an automated analyzer can be used with these substrates as well.

[Brief description of drawings]

Figure 1 shows (a) DCAP-P (concentration 50 nM) and b) 2,6-
100m of dichloro-4-acetylphenol (concentration 50nM)
3 shows a UV spectrum in M sodium citrate buffer pH 5.4 (25 ° C.). Figure 2 shows the IR spectrum of DCAP-P. Figure 3 shows the reaction time course when DCAP-P was used as the substrate. Fig. 4 shows the optimum pH of Acp. FIG. 5 shows the non-enzymatic hydrolysis stability of DCAP-P. FIG. 6 shows the relationship between Acp dilution and enzyme activity. FIG. 7 shows the SV curve and the Lineweaver-Burk plot. FIG. 8 shows a reaction time course when DBAP-P is used as a substrate. FIG. 9 shows a reaction time course when DCBP-P is used as a substrate.

Claims (2)

[Claims] 1. A general formula (I) [In the formula, X is a halogen atom, and R is-(CH ₂ ) _n CH ₃ (n = 0 to
3). ] The novel phosphoric acid derivative or its salt represented by these. 2. A sample containing acid phosphatase, and a compound of general formula (I) Wherein, X is a halogen atom, R is _{- (CH 2) n CH 3} (n = 0
~ 3). ] The novel phosphoric acid derivative represented by
A method for measuring acid phosphatase activity in a sample by measuring the absorbance at 0 to 370 nm.