Determination of carbonic anhydrase activity based on indoleacetic acid ester

Abstract

The invention discloses a method for determining the activity of carbonic anhydrase based on indoleacetic acid ester, comprising the following steps: preparing a catalytic substrate indoleacetic acid ester solution, a Tris-HCl buffer and a carbonic anhydrase solution; the invention calculates the amount of indole-based substances generated by carbonic anhydrase catalysis according to an indole-based concentration-absorbance curve, and then determines the catalytic activity of carbonic anhydrase. The invention catalyzes the decomposition of indoleacetic acid ester into indole-based substances by carbonic anhydrase, and uses a spectrophotometer to detect the absorbance to determine the amount of indole-based substances generated, so that the carbonic anhydrase activity can be quantitatively determined, with high sensitivity; moreover, the method is simple and easy to operate, and is green, environmentally friendly and pollution-free.

Description

Technical Field

[0001] The present invention relates to a method for determining enzyme activity, in particular to a method for determining esterase activity based on indoleacetic acid ester, and belongs to the technical field of biocatalysis. Background Art

[0002] Carbonic anhydrase (CA) is a zinc-based metalloenzyme whose core active site histidine, cysteine ​​and / or glutamine residues coordinate with metal ions, forming a tetrahedral coordination with the metal, catalyzing the reversible hydration of CO2 to form bicarbonate and protons. Currently, eight CA families have been identified, namely α, β, γ, δ, ζ, η, θ and the recently reported ι. Although the families differ in terms of structural folding, active site residues coordinated with metals, and metals used in catalysis, they can all catalyze the reversible hydration of CO2. The catalytic mechanism of CA can be summarized as a two-step ping-pong mechanism. The first step is the nucleophilic attack of CO2 to produce HCO3 - , the second step is to regenerate the zinc-bound hydroxyl group by proton transfer.

[0003] The existing methods for determining carbonic anhydrase activity include manometry, colorimetry, pH electrode method and esterase method.

[0004] The manometry method uses NaHCO3 as a substrate and phosphate as a buffer to determine the activity of carbonic anhydrase by measuring the pressure changes at different time periods after a certain enzyme solution. However, the manometry method is cumbersome and the experimental accuracy is extremely dependent on the pressure measuring instrument, and there are large errors in the experiment.

[0005] The colorimetric method refers to the process of quickly mixing a CO2 saturated aqueous solution, a phosphate buffer and an enzyme solution at room temperature, and then using phenol red as an indicator to record the absorbance change curve at a wavelength of 560nm over time. The carbonic anhydrase activity is calculated based on the change curve. However, in this method, both the indicator and the buffer will inhibit the activity of carbonic anhydrase, which is not conducive to true determination.

[0006] The pH electrode method refers to adding an enzyme solution to a barbiturate buffer at 0°C, then adding a saturated CO2 aqueous solution, and using a pH electrode to monitor the time required for the pH to drop from 8.0 to 6.3, and then calculating the enzyme activity. This method has harsh experimental conditions and high equipment requirements, and the linear response range of enzyme activity is small, so small changes in activity cannot be accurately measured.

[0007] The esterase method refers to indirectly indicating the activity of carbonic anhydrase by using carbonic anhydrase to catalyze the hydrolysis of esters. The existing esterase method is to use p-nitrophenyl acetate as a substrate, add an enzyme solution to a Tris-H2SO4 buffer, and measure the absorbance at 348nm, with no enzyme solution added as a control group. This method is simple to operate and has good sensitivity, but there is autolysis.

[0008] Carbonic anhydrase exists in almost all living organisms, including mammals, protozoa, diatoms, and even viruses. It is the fastest known enzyme. Currently, the CO2 hydration reaction catalyzed by carbonic anhydrase has been widely used in medicine, materials, environment and other fields. However, the existing methods for determining the activity of carbonic anhydrase have certain problems. Therefore, it is an inevitable trend to develop a fast, efficient and accurate method for determining the activity of the enzyme. Summary of the invention

[0009] Purpose of the invention: In view of the problems existing in the existing methods for determining the activity of carbonic anhydrase, the present invention provides a method for determining the activity of carbonic anhydrase esterase based on indoleacetic acid ester, indoleacetic acid ester is hydrolyzed by carbonic anhydrase to generate indole group, and the amount of indole group generated is used to quantify the catalytic activity of carbonic anhydrase.

[0010] Technical solution: The present invention provides a method for determining carbonic anhydrase activity based on indoleacetic acid ester, comprising the following steps:

[0011] (1) preparing a catalytic substrate indoleacetic acid ester solution, a Tris-HCl buffer solution and a carbonic anhydrase solution;

[0012] (2) Mix a certain amount of carbonic anhydrase solution and Tris-HCl buffer, and then add indoleacetic acid ester solution; after reacting at a certain temperature for a sufficient time, detect the absorbance at a wavelength of 375 nm using a multifunctional microplate reader; and the group without the addition of carbonic anhydrase solution is used as the control group;

[0013] (3) According to the indole concentration-absorbance equation, the amount of indole acetate catalyzed by CA to generate indole is calculated, and the CA enzyme activity is obtained according to the enzyme activity calculation equation.

[0014] Carbonic anhydrase has esterase activity and can decompose indoleacetic acid ester to form indole group and acetate. Indole group can absorb light with a wavelength of 375nm. The mass of indole group generated by carbonic anhydrase in a certain period of time is measured, and the control without adding enzyme is used to eliminate the influence of environment. Finally, the molar amount of indole group generated by enzyme catalysis in 1 minute is taken as its enzyme activity.

[0015] In step (1), the concentration of the indoleacetic acid ester solution is 10-15 mmol / L, the concentration of the Tris-HCl buffer is 30-40 mM, pH=7.5-8, and the concentration of the acid anhydrase solution is 1.0-1.5 mg / mL.

[0016] In step (2), the amount of indoleacetic acid ester solution is 200-240 μL, the amount of Tris-HCl buffer is 2010-2250 μL, and the amount of carbonic anhydrase solution is 250-300 μL. After the Tris-HCl buffer and the carbonic anhydrase solution are mixed at a temperature of 35-40° C., indoleacetic acid ester is added, and the absorbance is measured at a wavelength of 375 nm after reacting for 100-130 minutes.

[0017] In step (3), the indole concentration-absorbance equation is:

[0018] C=OD 375 / (ε*L)

[0019] The derivation process of the formula is as follows:

[0020] When monochromatic light radiates through the solution of the substance being measured, the amount of light absorbed by the substance being measured is proportional to the concentration of the substance being measured and the thickness of the liquid layer, that is:

[0021] OD 375 is the absorbance of indole solutions of different concentrations at 375 nm, C is the concentration of indole solution (mmol / L), I0 is the intensity of incident monochromatic light, I is the intensity of transmitted monochromatic light, T is the transmittance of indole, ε is the indole extinction coefficient of 5600 / mol, and L is the optical path length, i.e. the height of the wells of a 96-well plate.

[0022] In step (3), the enzyme activity A is calculated as:

[0023]

[0024] The calculation formula of N is as follows:

[0025] N=C*V R .

[0026] The unit of enzyme activity A is 1U, 1U means that carbonic anhydrase catalyzes the generation of 1 μmol of indole in 1 minute, N is the amount of substance (μmol) catalyzed by CA to generate indole acetate, t is the reaction time (min), V R is the volume of the reaction solution (L), V E is the volume of carbonic anhydrase solution (L).

[0027] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows: (1) the present invention uses indoleacetic acid ester as a substrate, and based on the esterase activity of carbonic anhydrase, decomposes indoleacetic acid ester into indole groups, and quantitatively characterizes the carbonic anhydrase activity by measuring the amount of indole groups generated; at the same time, the method is simple and easy to operate; (2) the present invention quantitatively characterizes the carbonic anhydrase activity by the amount of indole groups generated, which is simple to operate, highly sensitive and does not cause autolysis, and at the same time, the amount of carbonic anhydrase used is small, which can be suitable for large-scale determination; (3) the chemical raw materials used in the present invention are green, environmentally friendly, pollution-free, and harmless to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For (a) spectrophotometer and (b) 96-well plate.

[0029] Figure 2 The CA activity change curves at different pH;

[0030] Figure 3 For different Ca 2+ The CA activity change curve under different concentrations;

[0031] Figure 4 For different Mg 2+ The CA activity change curve under different concentrations;

[0032] Figure 5 For different CO3 2- CA activity change curve under different concentrations;. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0034] In order to better determine the activity of carbonic anhydrase, the present invention provides a method for determining the activity of carbonic anhydrase, which is based on the catalytic hydrolysis of indoleacetic acid ester by carbonic anhydrase, and can quantify the catalytic activity of carbonic anhydrase simply, efficiently and with high sensitivity.

[0035] Example 1

[0036] (1) preparing a 10 mmol / L indoleacetic acid ester solution, preparing a 30 mmol / L Tris-HCl buffer solution at pH 7, 7.5, and 8, respectively; preparing a 1.0 mg / ml carbonic anhydrase solution;

[0037] (2) At 35°C, 2010 μL of the above 30 mmol / L Tris-HCl buffer with different pH values ​​was mixed with 250 μL of 1.0 mg / ml carbonic anhydrase solution, and then 240 μL of 10 mmol / L indoleacetic acid ester solution was added to a 96-well plate. After reacting for 120 min, the absorbance was measured at a wavelength of 375 nm. The plate without enzyme was used as the control group;

[0038] (3) According to the indole concentration-absorbance equation, the amount of indole substance N formed by CA catalysis at pH 7, 7.5, and 8 was 841, 887, and 921 μmol, respectively. The enzyme activity A was then calculated according to the following formula:

[0039] C=OD 375 / 5.6

[0040] N=C*V R

[0041]

[0042] Among them, OD 375 is the absorbance of different concentrations of indole solution at 375nm, C is the concentration of indole solution (mmol / L), the extinction coefficient ε is 5600 / mol, and the optical path is 1mm. The unit of enzyme activity A is 1U, 1U means that carbonic anhydrase catalyzes the generation of 1μmol of indole in 1 minute, N is the amount of substance (μmol) catalyzed by CA to generate indole acetate, t is 120min, V R 2.5ml, V E is 0.25ml.

[0043] The spectrophotometer range is 0.01, according to C = OD 375 / 5.6, that is, C = 0.01 / 5.6 = 0.00017mmol / L≈ = 0.17μmol / L, that is, when the change in indole concentration is greater than 0.17μmol / L, it can be measured, and the sensitivity is 0.17μmol / L.

[0044] Finally, the carbonic anhydrase activity at different pH values ​​was calculated as Figure 2 As shown in (a), the carbonic anhydrase activities at pH = 7, 7.5, and 8 were 0.073 U, 0.077 U, and 0.08 U, respectively. The carbonic anhydrase activities measured by p-nitrophenyl acetate at different pH values ​​are shown in Figure 2 (b) The carbonic anhydrase activity measured by the two methods showed the same trend, that is, the carbonic anhydrase activity increased with the increase of pH.

[0045] Example 2 Different Ca 2+ Enzyme activity determination at different concentrations

[0046] (1) Prepare 10 mmol / L indoleacetic acid ester solution and 30 mmol / L Tris-HCl buffer at pH 8, and add CaCl2 to make the Ca content of Tris-HCl buffer 2+ The concentrations are 0, 20, 40, 60, 80, 100 mmol / L respectively; 1.0 mg / ml carbonic anhydrase solution is prepared;

[0047] (2) At 35°C, 2010 μL of different Ca 2+ After mixing 30mmol / L pH=8 Tris-HCl buffer with 250μL of 1.0mg / ml carbonic anhydrase solution, 240μL of 10mmol / L indoleacetic acid ester was added to the 96-well plate, and the absorbance was measured at a wavelength of 375nm after 120min of reaction. The control group was used without enzyme addition;

[0048] (3) According to the indole concentration-absorbance equation, Ca 2+ The mass N of the indole-based substance formed by CA catalysis at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L is 922, 1048, 887, 864, 783, and 530 μmol, respectively. The enzyme activity A is calculated according to the formula:

[0049] C=OD 375 / 5.6

[0050] N=C*V R

[0051]

[0052] Among them, OD 375 is the absorbance of different concentrations of indole solution at 375nm, C is the concentration of indole solution (mmol / L), the extinction coefficient ε is 5600 / mol, and the optical path is 1mm. U is the enzyme activity unit of enzyme activity A, 1U means that carbonic anhydrase catalyzes the generation of 1μmol of indole in 1 minute, N is the amount of substance (μmol) catalyzed by CA to generate indole acetate, t is 120min, V R 2.5ml, V E is 0.25ml.

[0053] The calculated different Ca 2+ The enzyme activity changes under different concentrations. Figure 3 As shown in (a), Ca 2+The enzyme activities at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L were 0.080, 0.091, 0.077, 0.075, 0.068, and 0.046 U, respectively. 2+ Carbonic anhydrase activity was measured by p-nitrophenyl acetate at Figure 3 (b) The carbonic anhydrase activity measured by the two methods showed the same trend. 2+ The effect on CA activity is as follows: C(Ca 2+ )=0-40mM (slightly decreased at 40mM), and reached the maximum at 20mM, compared with the case without adding Ca 2+ increased by about 23%; when C(Ca 2+ )>40mM, the activity of CA decreased slightly between 40-80mM, while further increasing Ca 2+ concentration, the activity will decrease rapidly, that is, high concentration of Ca 2+ It will inhibit CA from catalyzing the hydration reaction of CO2 and significantly reduce the enzyme activity.

[0054] Example 3 Different Mg 2+ Enzyme activity determination at different concentrations

[0055] (1) Prepare 10 mmol / L indoleacetic acid ester solution and 35 mmol / L Tris-HCl buffer at pH 8, and add MgCl2 to increase the Mg content of the Tris-HCl buffer. 2+ A 1.0 mg / ml carbonic anhydrase solution was prepared at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L;

[0056] (2) At 35°C, 2010 μL of different Mg 2+ After mixing 35mmol / L pH=8 Tris-HCl buffer with 250μL of 1.0mg / ml carbonic anhydrase solution, 240μL of 10mmol / L indoleacetic acid ester was added to the 96-well plate, and the absorbance was measured at a wavelength of 375nm after 120min of reaction. The control group was used without enzyme addition.

[0057] (3) According to the indole concentration-absorbance equation, Mg 2+ The mass N of the indole-based substance formed by CA catalysis at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L was 922, 1106, 1405, 1751, 1797, and 1555 μmol, respectively. The enzyme activity A was calculated according to the formula:

[0058] C=OD 375 / 5.6

[0059] N=C*V R

[0060]

[0061] Among them, OD 375 is the absorbance of different concentrations of indole solution at 375nm, C is the concentration of indole solution (mmol / L), the extinction coefficient ε is 5600 / mol, and the optical path is 1mm. U is the enzyme activity unit of enzyme activity A, 1U means that carbonic anhydrase catalyzes the generation of 1μmol of indole in 1 minute, N is the amount of substance (μmol) catalyzed by CA to generate indole acetate, t is 120min, V R 2.5ml, V E is 0.25ml.

[0062] Calculated different Mg 2+ The enzyme activity changes under different concentrations. Figure 4 As shown, Mg 2+ The enzyme activities at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L were 0.080, 0.096, 0.122, 0.152, 0.156, and 0.135 U, respectively. 2+ Carbonic anhydrase activity was measured by p-nitrophenyl acetate at Figure 4 (b) The carbonic anhydrase activity measured by the two methods showed the same trend. 2+ The concentration of Mg increased with the increase of concentration. In the concentration range of 0-80mM, Mg 2+ The enhancement effect on the activity of CA continued to increase, reaching the best at 80 mM, where the enzyme activity reached its maximum value; C(Mg 2+ )>80mM, Mg 2+ The activity is improved and the benefit is reduced, but compared with C(Mg 2+ ) = 0 mM, it still increased by 35.1%.

[0063] Example 4 Different CO3 2- Enzyme activity determination at different concentrations

[0064] (1) Prepare 15 mmol / L indoleacetic acid ester solution and 35 mmol / L Tris-HCl buffer at pH 7, and add Na2CO3 to make the CO3 in the Tris-HCl buffer 2- A 1.0 mg / ml carbonic anhydrase solution was prepared at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L;

[0065] (2) At 35°C, 2010 μL of different CO32- After mixing 35mmol / L pH=7 Tris-HCl buffer with 250μL of 1.0mg / ml carbonic anhydrase solution, 240μL of 15mmol / L indoleacetic acid ester was added to the 96-well plate, and the absorbance was measured at a wavelength of 375nm after 120min of reaction. The control group was used without enzyme addition.

[0066] (3) According to the concentration-absorbance equation, CO3 2- The mass N of the indole-based substance formed by CA catalysis at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L was 922, 1106, 1405, 956, 438, and 346 μmol, respectively. The enzyme activity A was calculated according to the formula:

[0067] C=OD 375 / 5.6

[0068] N=C*V R

[0069]

[0070] Among them, OD 375 is the absorbance of different concentrations of indole solution at 375nm, C is the concentration of indole solution (mmol / L), the extinction coefficient ε is 5600 / mol, and the optical path is 1mm. U is the enzyme activity unit of enzyme activity A, 1U means that carbonic anhydrase catalyzes the generation of 1μmol of indole in 1 minute, N is the amount of substance (μmol) catalyzed by CA to generate indole acetate, t is 120min, V R 2.5ml, V E is 0.25ml.

[0071] Calculated different CO3 2- The enzyme activity changes under different concentrations. Figure 5 As shown in (b), CO3 2- The enzyme activities at concentrations of 0, 20, 40, 60, 80, and 100 mmol / L were 0.080, 0.096, 0.122, 0.083, 0.038, and 0.030 U, respectively. 2+ Carbonic anhydrase activity was measured by p-nitrophenyl acetate at Figure 5 (b) The carbonic anhydrase activity measured by the two methods showed the same trend. 2- Effects on CA activity Figure 5 As shown in (b), CO3 2- The effect on carbonic anhydrase activity is manifested as the 2- The increase in concentration showed an increase first and then inhibition, C(CO32- ) = 0-60mM, CO3 2- It can improve the activity of carbonic anhydrase in catalyzing CO2 hydration reaction and 2- )=40mM, CA activity reaches the maximum value; CO3 2- When the concentration is greater than 60mM, It will inhibit the catalytic activity of CA, and the inhibitory effect will be more obvious as the concentration increases.

[0072] The analysis based on the hydrolysis of indoleacetic acid esters of the present invention is more convenient, reducing the low sensitivity problems caused by small differences, autolysis problems, and low reproducibility. The reaction solution volume is very small, which meets the requirements of high-throughput screening of 96 microplates, and can also save analysis costs. The indole group produced by carbonic anhydrase catalysis measured in the embodiment of the present invention is in the μmol order, indicating that the method can measure the concentration change in the μmol order and is extremely sensitive to the concentration change.

Claims

1. A method for determining carbonic anhydrase activity based on indoleacetic acid ester, characterized in that: The steps include: (1) preparing a catalytic substrate indoleacetic acid ester solution, a Tris-HCl buffer solution and a carbonic anhydrase solution; (2) mixing a carbonic anhydrase solution and a Tris-HCl buffer, and then adding an indoleacetic acid ester solution; after a sufficient reaction time, measuring the absorbance at a wavelength of 375 nm using a spectrophotometer; (3) Calculate the amount of indoleacetic acid catalyzed by CA to generate indole according to the indole concentration-absorbance equation, and obtain the CA enzyme activity according to the enzyme activity calculation equation; In step (3), the indole concentration-absorbance equation is: C=OD 375 / (ε*L); Where C is the concentration of indole solution mmol / L, OD 375 is the absorbance of indole solutions of different concentrations at 375 nm, ε is the indole extinction coefficient of 5600 / mol, and L is the optical path length, i.e., the height of the wells of a 96-well plate; In step (3), the CA enzyme activity A is calculated as: The unit of enzyme activity A is U, N is the amount of substance that CA catalyzes indole acetate to generate indole groups in μmol, t is the reaction time in min, and V is R is the volume of the reaction solution L, V E is the volume of carbonic anhydrase solution, L; The calculation formula of N is: N=C*V R 。 2. The method for determining carbonic anhydrase activity based on indoleacetic acid ester according to claim 1, characterized in that: In step (1), the concentration of the indoleacetic acid ester solution is 10-15 mmol / L, the concentration of the Tris-HCl buffer is 30-40 mM, and the pH is 7.0-7.

5.

3. The method for determining carbonic anhydrase activity based on indoleacetic acid ester according to claim 1, characterized in that: In step (1), the concentration of the carbonic anhydrase solution is 1.0-1.5 mg / mL.

4. The method for determining carbonic anhydrase activity based on indoleacetic acid ester according to claim 1, characterized in that: In step (2), the amount of indoleacetic acid ester solution is 200-240 μL, the amount of Tris-HCl buffer is 2010-2250 μL, and the amount of enzyme solution is 250-300 μL.

5. The method for determining carbonic anhydrase activity based on indoleacetic acid ester according to claim 1, characterized in that: In step (2), the reaction temperature is 35-40° C. and the reaction time is 100-130 min.

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