Combined determination method for activity of multiple antioxidant enzymes

Abstract

The invention belongs to the technical field of enzyme activity determination. The combined determination method comprises the following steps: (1) adding plant tissues into liquid nitrogen, grinding, adding a phosphate buffer solution with the pH value of 7.8, continuously grinding until no crude fiber exists, and centrifuging to obtain an enzyme extracting solution; (2) determining the activity of SOD in the enzyme extracting solution by adopting a nitrogen blue tetrazole photochemical reduction method; (3) preparing a reaction system by using a phosphate buffer solution with the concentration of 50mmol. L <-1 > and the pH value of 5.5, and determining the POD activity in the enzyme extracting solution by adopting a guaiacol colorimetric method; and (4) determining by adopting an ultraviolet spectrophotometer method, immediately starting timing after H2O2 is added, reading once every 1min until the OD value is unchanged, and calculating the CAT activity by using the average value of OD change per minute. The method can solve the problems of tedious experimental steps, low efficiency, unstable data and the like due to respective extraction of three enzymes, ensures the accuracy of enzyme activity determination, simplifies the experimental steps, and improves the experimental efficiency and the data stability.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme activity determination, and in particular relates to a combined determination method for multiple antioxidant enzyme activities. Background Art

[0002] In plants, antioxidant enzymes mainly include superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-R), etc., which are called the cell's protective enzyme system. Among them, SOD, POD and CAT are the most important.

[0003] The suitable temperature, pH and reaction system conditions for the extraction of different enzymes are different. The traditional method for determining the activities of the three enzymes requires the enzymes to be extracted and measured separately. This makes the determination of the activities of the three antioxidant enzymes cumbersome and inefficient. At the same time, different scholars use different pH values ​​of buffer solutions for extracting POD and determining its activity: Wang Xuekui, Li Hesheng, Jang, and Yang Qing believe that the optimal pH for determining POD is 5.5, 6.4, and 7.0, respectively; and when determining CAT activity, problems such as unstable measurement values ​​and poor repeatability of results are often encountered.

[0004] Therefore, how to provide a method for extracting multiple antioxidant enzymes at one time and conducting combined determination is a technical problem that those skilled in the art urgently need to solve. Summary of the invention

[0005] In view of this, in order to eliminate these defects and improve the method of determining the three enzymes, the present invention provides a method for the combined determination of the activities of three antioxidant enzymes, which can solve the problems of separate extraction of the three enzymes, cumbersome experimental steps, low efficiency and unstable data, and simplifies the experimental steps while ensuring the accuracy of enzyme activity determination, and improves experimental efficiency and data stability.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The combined determination method of multiple antioxidant enzyme activities comprises the following steps:

[0008] (1) Extraction of enzyme solution: add liquid nitrogen to plant tissue and grind it, add pH 7.8 phosphate buffer and continue grinding until there is no crude fiber, and centrifuge to obtain enzyme extract;

[0009] (2) Determination of SOD activity: The SOD activity in the enzyme extract was determined by the nitroblue tetrazolium photoreduction method;

[0010] (3) Determination of POD activity: The reaction system was prepared with 50 mmol·L-1 pH 5.5 phosphate buffer, and the POD activity in the enzyme extract was determined by the guaiacol colorimetric method;

[0011] (4) Determination of CAT activity: UV spectrophotometer was used to measure the activity. 2 O 2 Then, start timing immediately and read the value every 1 minute until the OD value remains constant. The average value of the OD change per minute is used to calculate the CAT activity.

[0012] Preferably, the concentration of pH 7.8 phosphate buffer in step (1) is 50 mmol·L -1 , centrifugal speed 10000rpm, centrifugal time 20min.

[0013] Preferably, the SOD activity is determined by mixing the reaction medium, enzyme extract and riboflavin solution into a reaction solution, placing the mixture in an artificial intelligence climate box and illuminating the mixture, adding a zeroing reaction solution after the mixture is illuminated, and then measuring the OD value of the reaction solution at 560 nm to calculate the SOD activity; the zeroing reaction solution is a mixture of the reaction medium, riboflavin solution and phosphate buffer solution.

[0014] Preferably, the reaction medium comprises: 50 mmol·L -1 pH 7.8 phosphate buffer, containing 77.12 μmol·L - 1 NBT, 0.1mmol·L -1 EDTA, 13.37mmol·L -1 Methionine;

[0015] Riboflavin solution containing 0.10mmol·L -1 EDTA 50mmol·L -1 pH 7.8 phosphate buffer was prepared to a concentration of 80.2 μmol·L -1 Riboflavin solution.

[0016] Preferably, the POD activity is measured by using 50 mmol·L -1 pH 5.5 phosphate buffer, 50 mmol·L -1 Guaiacol, 2% H 2 O 2 , the enzyme extract was configured into a reaction system, shaken, and placed in a 34°C water bath for 3 min. Then, 30 μL of 20% trichloroacetic acid was immediately added and shaken. The reaction solution in the control tube was used as a blank. Colorimetry was performed at a wavelength of 470 nm, the OD value was read, and the POD activity was calculated.

[0017] Preferably, the POD activity determination reaction system contains phosphate buffer, guaiacol, H 2 O 2 , the volume ratio of enzyme extract is 2.99:1:1:0.1.

[0018] Preferably, the CAT activity is measured by adding 50 mmol·L -1 pH 7.8 phosphate buffer, enzyme extract and distilled water were mixed, preheated at 25°C, and 0.1 mol·L -1 H 2 O 2 , start timing immediately and quickly pour into a quartz colorimetric cup, measure the OD value at 240nm, read once every 1 minute until the OD value remains unchanged, and calculate the CAT activity using the average value of the OD change per minute.

[0019] Preferably, in the determination of CAT activity, the volume ratio of phosphate buffer, enzyme extract and distilled water is 0.2:1.5:1.0.

[0020] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following excellent effects:

[0021] The invention uses a SOD extraction method to extract once, and when determining the activity of POD, uses an optimal pH buffer solution to configure the reaction system; when determining CAT, prolongs the CAT determination time, so as to achieve one-time extraction and determination of three enzyme activities, and the results are not significantly different from the results of separate determination of POD and CAT. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] Step 1: Extraction of enzyme solution:

[0025] Remove the veins of the plant leaves, cut them into pieces, weigh 0.5g and put them into a mortar, add an appropriate amount of liquid nitrogen and grind them thoroughly. Take 6mL of 50mmol·L -1 Put pH 7.8 phosphate buffer in a test tube, take 2 mL from it and gradually add it to the mortar while grinding until there is no crude fiber. Transfer it to a centrifuge tube, and then wash the mortar with the remaining pH 7.8 phosphate buffer. Transfer the washing liquid to the centrifuge tube and centrifuge at 10000rpm for 20min. The supernatant is the enzyme extract.

[0026] Step 2: Determination of SOD activity:

[0027] 1. Enzyme activity determination: Take 3 small dry beakers and add reagents according to Table 1. Place No. 2 in the dark, and place No. 1 and No. 3 in an artificial intelligence climate box and illuminate for 10 minutes. After the illumination, quickly put them in a white porcelain plate and cover them to avoid light. Use the reaction solution in No. 2 to zero and measure the OD value of the reaction solution in No. 1 and No. 3 at 560nm.

[0028] Table 1 Reaction system for SOD activity determination

[0029]

[0030] Note: ① Reaction medium: 50mmol·L -1 pH 7.8 phosphate buffer, containing 77.12 μmol·L -1 NBT, 0.1mmol·L -1 EDTA (ethylenediaminetetraacetic acid), 13.37mmol·L -1 Met (methionine).

[0031] ②80.2 μmol·L -1 Riboflavin solution: use 0.10mmol·L -1 EDTA 50mmol·L -1 pH 7.8 phosphate buffer was prepared.

[0032] 2. Calculation of results

[0033] SOD activity was expressed as the number of enzyme activity units that inhibited 50% reduction of NBT within 1 hour per 1 g fresh sample.

[0034]

[0035] Where: ΔOD is the difference between the control and the measured OD value; V 1 is the total volume of enzyme extract (mL); D0 is the OD value of the control; W is the sample weight (g); t is the illumination time (h); V 2 is the volume of enzyme solution added (mL); 50% means 50% photochemical reduction of NBT.

[0036] Step 3: Determination of POD activity:

[0037] 1. Enzyme activity determination:

[0038] Take 2 test tubes, one for the determination tube and the other for the control tube, add reagents according to Table 2 to prepare the POD reaction system. Shake well, put in a 34℃ water bath for 3 minutes, immediately add 30μL of 20% trichloroacetic acid, shake well, use the reaction solution in the control tube as blank, compare colors at a wavelength of 470nm, and read the OD value.

[0039] Table 2 Reaction system for POD activity determination

[0040]

[0041] 2. Calculation of results

[0042] The OD change of 0.01 per minute is defined as 1 POD activity unit (U).

[0043]

[0044] Where: OD 470 is the OD value at 470nm; W is the sample weight (g); t is the reaction time (min); V 1 V is the volume of enzyme solution during extraction (mL); 2 is the volume of enzyme solution during the reaction (mL).

[0045] Step 4: Determination of CAT activity:

[0046] 1. Enzyme activity assay: Take 2 test tubes, one for the assay tube and one for the control tube, and add reagents according to the table below. After preheating at 25°C, add 0.3 mL of 0.1 mol·L -1 H 2 O 2 After adding each tube, start timing immediately and quickly pour into a quartz colorimetric cup. Measure the OD value at 240nm. Read once every 1 minute until the OD value remains unchanged. Calculate the enzyme activity using the average OD change per minute.

[0047] Table 3 Reaction system for CAT activity determination

[0048]

[0049] 2. Calculation of results

[0050] CAT activity was measured by 1g fresh sample within 1min. 240 It is expressed as the number of enzyme activity units expressed when the value is reduced by 0.1.

[0051]

[0052] Where: ΔOD 240 V is the difference between the extinction values ​​of the test tube and the control tube; T is the total volume of crude enzyme extract (mL); V 1 is the volume of crude enzyme solution used for determination (mL); t is the volume of H added 2 O 2 The time to the last reading (min); W is the fresh weight of the sample (g).

[0053] Comparative Example 1

[0054] Preparation of enzyme solution: Remove the veins of plant leaves, cut into pieces, weigh 0.5g and add into a mortar, take 6mL of 50mmol·L -1 Put pH 5.5 phosphate buffer into a test tube, take 2 mL from it and put it into a mortar, grind it thoroughly until there is no crude fiber, put it into a centrifuge tube, and then wash the mortar with the remaining pH 5.5 phosphate buffer. Transfer the washing liquid into a centrifuge tube and centrifuge it at 3000r / min for 10min. The supernatant is the enzyme extract.

[0055] POD activity determination: pH 5.5 phosphate buffer was used, and the rest was the same as in Example 1.

[0056] Calculation of results: Same as in Example 1.

[0057] The present invention compares the size and difference of POD activity measured by the method of comparative example 1 (single measurement) and POD activity measured by the enzyme extract of example 1 (combined measurement). The results show that when the combined measurement and the reaction system are prepared with 50mmol·L -1 The POD activity measured by pH 5.5 phosphate buffer was not significantly different from the single test result (Table 4). Therefore, in order to simplify the experimental steps and improve efficiency, the enzyme extract of Example 1 and pH 5.5 phosphate buffer were used to measure the POD activity.

[0058] Table 4 Comparison of POD activity between single and combined testing

[0059]

[0060] Comparative Example 2

[0061] Preparation of enzyme solution: Remove the veins of plant leaves, cut into pieces, weigh 0.5g and add into a mortar, take 6mL of 50mmol·L -1 Put pH 6.4 phosphate buffer into a test tube, take 2 mL from it and put it into a mortar, grind it thoroughly until there is no crude fiber, put it in a centrifuge tube, and then wash the mortar with the remaining pH 6.4 phosphate buffer. Transfer the washing liquid into a centrifuge tube and centrifuge it at 13000r / min for 30min. The supernatant is the enzyme extract.

[0062] POD activity determination: pH 6.4 phosphate buffer was used, and the rest was the same as in Example 1.

[0063] Calculation of results: Same as in Example 1.

[0064] Comparative Example 3

[0065] Preparation of enzyme solution: Remove the veins of plant leaves, cut them into pieces, weigh 0.5 g and add them into a mortar, then take 6 mL of 50 mmol / L -1Put pH 7.0 phosphate buffer into a test tube, take 2 mL from it and gradually add it to the mortar while grinding until there is no crude fiber. Transfer it to a centrifuge tube, and then wash the mortar with the remaining pH 7.0 phosphate buffer. Transfer the washing liquid to a centrifuge tube and centrifuge at 10000rpm for 20min. The supernatant is the enzyme extract.

[0066] POD activity determination: pH 7.0 phosphate buffer was used, and the rest was the same as in Example 1.

[0067] Calculation of results: Same as in Example 1.

[0068] The POD activity was determined separately by the three methods of Comparative Examples 1-3. The results are shown in Table 5. Except for celery, which had no significant difference between pH 5.5 and pH 6.4, the POD activity of other plants of the same species at other pH values ​​was significantly different, and the POD activity measured at pH 5.5 was the highest. It can be seen that the optimal pH value for POD activity determination is 5.5.

[0069] Table 5 Comparison of different methods for determining POD activity

[0070]

[0071] Note: Different capital letters indicate significant differences in pH values ​​of the same plant (P < 0.05), and different lowercase letters indicate significant differences in pH values ​​of the same plant (P < 0.05).

[0072] Comparative Example 4

[0073] Extraction of enzyme solution: Remove the veins of plant leaves, cut into pieces, weigh 0.5g and put into a mortar, add appropriate amount of liquid nitrogen and grind thoroughly. Take 6mL0.2mol·L -1 Put pH 7.8 into a test tube, take 2 mL and gradually add it to the mortar while grinding until there is no crude fiber, transfer it to a centrifuge tube, and then wash the mortar with the remaining pH 7.8 phosphate buffer, transfer the washing liquid to the centrifuge tube, let it stand for 10 minutes, centrifuge at 4000rpm for 15 minutes, the supernatant is the CAT extract, and store it at 5℃ for later use.

[0074] Determination of CAT activity

[0075] In addition, 0.2 mol·L -1 Except for pH 7.8 phosphate buffer, other steps are the same as those in Example 1.

[0076] Result calculation

[0077] CAT activity was expressed as the number of enzyme activity units expressed by a decrease of 0.1 in A240 per 1 g fresh sample within 1 min.

[0078]

[0079] Where: ΔA 240 V is the difference between the extinction values ​​of the test tube and the control tube; T is the total volume of crude enzyme extract (mL); V 1 is the volume of crude enzyme solution used for determination (mL); t is the volume of H added 2 O 2 The time to the last reading (min); W is the fresh weight of the sample (g).

[0080] Comparing the process and results of measuring CAT activity in Example 4 (single measurement) and Example 1 (combined measurement), it can be seen from Table 6 that after adding the enzyme extract, it takes 6 to 8 minutes for the OD value to stabilize. Therefore, the time for measuring the OD value in the present invention is extended until the OD value of the reaction system remains unchanged, and the CAT activity is expressed by the average value of the CAT activity per minute, so that a stable and accurate CAT activity can be obtained. In addition, there is no significant difference between the single measurement of CAT activity and the combined measurement of CAT activity, which solves the problem of unstable CAT activity measured by the enzyme extract of extracting three enzymes at one time.

[0081] Table 6 OD values ​​at different times after adding enzyme extract

[0082]

[0083]

[0084] Table 7 OD difference every 1 min

[0085]

[0086] Table 8 CAT activity at 1-min intervals and comparison of the two methods

[0087]

[0088]

[0089] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for the combined determination of multiple antioxidant enzyme activities, characterized in that: The following steps are involved: (1) Extraction of enzyme solution: add liquid nitrogen to plant tissue and grind it, add pH 7.8 phosphate buffer and continue grinding until there is no crude fiber, and centrifuge to obtain enzyme extract; (2) Determination of SOD activity: The SOD activity in the enzyme extract was determined by the nitroblue tetrazolium photoreduction method; (3) Determination of POD activity: 50 mmol·L -1 The reaction system was prepared with pH 5.5 phosphate buffer, and the POD activity in the enzyme extract was determined by guaiacol colorimetry. (4) Determination of CAT activity: The activity was determined by ultraviolet spectrophotometry. The timer was started immediately after the addition of H2O2 and the reading was taken every 1 minute until the OD value remained constant. The CAT activity was calculated using the average value of the OD change per minute.

2. The combined determination method of multiple antioxidant enzyme activities according to claim 1, characterized in that: The concentration of pH 7.8 phosphate buffer in step (1) is 50 mmol / L -1 , centrifugal speed 10000rpm, centrifugal time 20min.

3. The combined determination method of multiple antioxidant enzyme activities according to claim 1, characterized in that: Determination of SOD activity: The reaction medium, enzyme extract and riboflavin solution are mixed into a reaction solution and placed in an artificial intelligence climate box for illumination. After the reaction solution is illuminated, a zeroing reaction solution is added to protect the reaction solution from light and wither. The OD value of the reaction solution at 560nm is determined to calculate the SOD activity. The zeroing reaction solution is a mixture of the reaction medium, riboflavin solution and phosphate buffer solution.

4. The combined determination method of multiple antioxidant enzyme activities according to claim 3, characterized in that: The reaction medium includes: 50 mmol·L -1 pH 7.8 phosphate buffer, containing 77.12 μmol·L -1 NBT, 0.1mmol·L -1 EDTA, 13.37mmol·L -1 Methionine; Riboflavin solution containing 0.10mmol·L -1 EDTA 50mmol·L -1 pH 7.8 phosphate buffer was prepared to a concentration of 80.2 μmol·L -1 Riboflavin solution.

5. The combined determination method of multiple antioxidant enzyme activities according to claim 1, characterized in that: Determination of POD activity: 50 mmol·L -1 pH 5.5 phosphate buffer, 50 mmol·L -1 Guaiacol, 2% H2O2 and enzyme extract were prepared into a reaction system, shaken and kept in a 34°C water bath for 3 min. Trichloroacetic acid was immediately added and shaken. The reaction solution in the control tube was used as blank. Colorimetry was performed at a wavelength of 470 nm, the OD value was read, and the POD activity was calculated.

6. The combined determination method of multiple antioxidant enzyme activities according to claim 5, characterized in that: The volume ratio of phosphate buffer, guaiacol, H2O2, and enzyme extract in the reaction system for determination of POD activity was 2.99:1:1:0.

1.

7. The method for combined determination of multiple antioxidant enzyme activities according to claim 1, characterized in that: Determination of CAT activity: 50 mmol·L -1 pH 7.8 phosphate buffer, enzyme extract and distilled water were mixed, preheated at 25°C, and 0.1 mol·L -1 H2O2, start timing immediately and quickly pour into a quartz colorimetric cup, measure the OD value at 240nm, read once every 1min until the OD value remains unchanged, and calculate the CAT activity using the average value of the OD change per minute.

8. The method for combined determination of multiple antioxidant enzyme activities according to claim 7, characterized in that: In the determination of CAT activity, the volume ratio of phosphate buffer, enzyme extract and distilled water was 0.2:1.5:1.0.

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