Method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase

Abstract

The invention belongs to the technical field of enzyme activity determination, and particularly relates to a method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase, which comprises the following steps of: improving an extracting solution of a nitrogen metabolism enzyme, taking a Tris buffer solution containing MgSO4, EGTA and dithiothreitol as a crude enzyme extracting solution, and extracting the crude enzyme to obtain the nitrate reductase, the glutamate synthetase and the glutamate dehydrogenase. The determination method of nitrate reductase, glutamate synthase and glutamate dehydrogenase is further improved, and the method is simple to operate, time-saving and suitable for large-scale determination while the accuracy is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme activity determination, and particularly relates to a method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase. Background Art

[0002] During plant growth and development, if nitrate reductase (NR), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) are affected by the external environment, the synthesis of amino acids and proteins in the plant body will be correspondingly hindered, thereby adversely affecting plant growth and development. Because these key nitrogen metabolism enzymes play an irreplaceable role in nitrogen assimilation, changes in their activity often directly reflect the plant's nitrogen utilization status. Therefore, by monitoring changes in NR, GOGAT, and GDH activity, it is possible to timely understand the dynamic response of plants to nitrogen metabolism and help understand the nitrogen nutritional status of plants, providing a scientific basis for rationally regulating fertilization strategies and optimizing the crop growth environment.

[0003] However, in actual operation, there are differences in the crude enzyme extracts of NR, GOGAT and GDH. When determining the NR, GOGAT and GDH of a sample, it is often necessary to prepare multiple extracts, which makes the extraction of crude enzyme solutions time-consuming and labor-intensive.

[0004] The main method for determining the activity of GOGAT and GDH is the continuous determination method. The principle of this method is that when GOGAT and GDH are present in the solution, reduced coenzyme I (NADH) and substrate are added to the solution, and a reaction occurs to oxidize NADH to NAD. + Because NADH has a maximum absorbance at 340nm, the activity of GOGAT and GDH can be determined by continuously measuring the decrease in the absorbance of the solution at 340nm. This method is simple to operate, but the reading time at 340nm is too long. A reading is taken every 30 seconds, and a total of 6 readings are taken, taking 2 minutes and 30 seconds for a single sample. It is not suitable for large-scale determination. In addition, the content of GOGAT and GDH in the crude enzyme is low. If too little crude enzyme solution is added in a single determination, the change is too small, while adding too much will cause the color to darken and affect the reading.

[0005] The commonly used method for measuring NR activity is the nitrite generation method. The principle of this method is that when NR is present in a solution, NADH and KNO3 are added to it, a reaction occurs to convert nitrate into nitrite. Nitrite reacts with sulfonamide and naphthylethyleneamine to form a diazo reaction, turning the solution red. The solution has a maximum absorbance at 540nm. This method is simple to operate and highly accurate, but each measurement requires the preparation of sulfonamide solution and naphthylethyleneamine solution, and color development (30 minutes) is required, which is time-consuming. In the nitric acid reduction reaction, another substrate, NADH, does not require the addition of a color developer and has a maximum absorbance at 340nm. By comparing NADH consumption before and after the reaction, the activity of nitrate reductase can also be reflected. Summary of the Invention

[0006] The present invention provides a method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase.

[0007] The technical solutions of the present invention are as follows:

[0008] The present invention provides a method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase, comprising:

[0009] S1: Prepare a Tris buffer containing MgSO4, EGTA and dithiothreitol as a crude enzyme extract; wherein:

[0010] The concentration of MgSO4 is 2-5mmol·L -1 ;

[0011] The concentration of EGTA is 0.5-1 mmol·L -1 ;

[0012] The concentration of dithiothreitol is 2-4 mmol·L -1 ;

[0013] S2: The ground leaves were mixed with the crude enzyme extract and centrifuged, and the supernatant was the crude enzyme solution;

[0014] S3: Using reaction buffer, reaction substrate solution, crude enzyme solution, and reduced coenzyme I starting solution as the reaction system solution, use a UV spectrophotometer to measure the colorimetry and record the values ​​of the reaction system solution at 340 nm before and after a light-proof water bath. Calculate the enzyme activities of nitrate reductase, glutamate synthetase, and glutamate dehydrogenase, respectively, based on the extinction of reduced coenzyme I per gram of fresh weight per hour.

[0015] The concentration of S1 and MgSO4 is 4 mmol·L -1 ; The concentration of EGTA is 0.5 mmol·L -1 ; The concentration of dithiothreitol is 2mmol·L -1 .

[0016] The Tris buffer of S1 is 0.05 mol·L with a pH of 7.5. -1 Tris buffer.

[0017] The volume ratio of the S3 reaction system solution, reaction buffer solution, reaction substrate solution, crude enzyme solution and reduced coenzyme I starting solution is 1 mL: 0.6 mL: 0.1 mL: 0.3 mL.

[0018] The S3 reaction system solution is prepared by sequentially adding reaction buffer, reaction substrate solution, crude enzyme solution, and reduced coenzyme I starting solution.

[0019] Use an ultraviolet spectrophotometer to measure the color and record the values ​​of the reaction system solution at 340nm before and after the dark water bath, specifically:

[0020] First, adjust the absorbance of the quartz cuvette to zero with reaction buffer, then take the reaction system solution into the quartz cuvette and read the data at 340nm, which is recorded as the initial absorbance. After the colorimetry is completed, place the remaining reaction system solution in a light-proof water bath, take it out and place it in an environment of 0-4℃, take the reaction system solution into the quartz cuvette and read the data at 340nm, which is recorded as the termination absorbance.

[0021] The calculation formula of the S3 enzyme activity is:

[0022] Enzyme activity (U·g -1 ·h -1 ·FW)=(UA-UB)×V / Va / W / h;

[0023] Where UA is the initial absorbance; UB is the final absorbance; V is the volume of crude enzyme extract; Va is the amount of crude enzyme solution used in the determination; W is the sample weight; and h is the reaction time.

[0024] Beneficial effects

[0025] The present invention improves the extract of nitrogen metabolic enzymes, uses Tris buffer containing MgSO4, EGTA and dithiothreitol as the crude enzyme extract, and further improves the determination method of nitrate reductase, glutamate synthetase and glutamate dehydrogenase. While ensuring the accuracy of enzyme activity detection, this method is simple to operate, saves time, and is suitable for large-scale determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure shows the relationship between the number of samples and the time consumed for the traditional determination method and the determination method of the present application. DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0028] The present invention provides a method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase, comprising:

[0029] S1: Prepare Tris buffer containing MgSO4, EGTA and dithiothreitol as crude enzyme extract;

[0030] Preferably, Tris buffer is 0.05 mol·L at pH 7.5. -1 Tris buffer.

[0031] In crude enzyme extract:

[0032] The concentration of MgSO4 is 2-5mmol·L -1 ;

[0033] The concentration of EGTA is 0.5-1 mmol·L -1 ;

[0034] The concentration of dithiothreitol is 2-4 mmol·L -1 .

[0035] Preferably, the concentration of MgSO4 is 4 mmol·L -1 ; The concentration of EGTA is 0.5 mmol·L -1 ; The concentration of dithiothreitol is 2mmol·L -1 .

[0036] Mg 2+ It can stabilize the activity of glutamine synthetase and participate in the catalytic reaction of glutamine synthetase. The extract can also extract glutamine synthetase at the same time.

[0037] EGTA can bind free Ca 2+ 、Fe 2+ 、Fe 3+ ions, inhibiting the degradation of enzymes initiated by these ions. For example, Ca 2+ It can activate endogenous proteases, leading to the degradation of NR. EGTA has different affinities for different metal ions (determined by the stability constant log K f The binding order of EGTA and metal ions is Fe 3+ (log K f =11.6), Ca 2+ (log K f =11.0), Fe 2+ (logK f =9.5), Mg 2+ (log K f =5.2).

[0038] Dithiothreitol (DTT) is a strong reducing agent that can prevent the sulfhydryl groups in enzyme proteins from being oxidized into disulfide bonds, thereby protecting the natural conformation and activity of the enzyme protein.

[0039] S2: The ground leaves are mixed with the crude enzyme extract and centrifuged. The supernatant is the crude enzyme solution. The operation can be as follows:

[0040] The leaves were ground with liquid nitrogen, 0.5 g was weighed and placed in a 10 mL centrifuge tube, 5 mL of crude enzyme extract was added, and the mixture was centrifuged at 10,000 r·min. -1 Centrifuge at 4℃ for 20min, aspirate the supernatant as the crude enzyme solution, and store the crude enzyme solution in a refrigerator at 4℃ away from light.

[0041] S3: Using reaction buffer, reaction substrate solution, crude enzyme solution, and reduced coenzyme I starting solution as the reaction system solution, use a UV spectrophotometer to measure the colorimetry and record the values ​​of the reaction system solution at 340 nm before and after a light-proof water bath. Calculate the enzyme activities of nitrate reductase, glutamate synthetase, and glutamate dehydrogenase, respectively, based on the extinction of reduced coenzyme I per gram of fresh weight per hour.

[0042] The specific operations are as follows:

[0043] First, about the reaction buffer:

[0044] The reaction buffer used for nitrate reductase (NR) and glutamate synthase (GOGAT) was 25 mmol / L -1 Tris buffer (adjust pH to 7.5). The reaction buffer used for glutamate dehydrogenase (GDH) is 40 mmol / L -1 Tris-HCl buffer.

[0045] About the reaction substrate solution:

[0046] The reaction substrate solution of NR is 10 g·L -1 of KNO3 solution.

[0047] The reaction substrate solution of GOGAT is 10 mmol·L -1 Glutamine solution (containing 1g·L -1 α-isoglutaric acid, 0.1 g·L -1 of KCl).

[0048] The reaction substrate solution of GDH is 20 g·L -1 NH4Cl solution (containing 80 mg L -1 of α-isoglutaric acid).

[0049] About the reaction starting solution:

[0050] The starting solution used in the reaction was 0.6 mg mL -1Prepare a reduced coenzyme I (NADH) solution, weigh an appropriate amount of NADH and dissolve it in the reaction buffer (this solution should be kept away from light and stored at 4°C).

[0051] Then, the nitrogen metabolism enzyme (nitrate reductase (NR), glutamate synthetase (GOGAT), glutamate dehydrogenase (GDH)) assay should be performed in an ice box at 0-4°C. Take a test tube and add 1mL of reaction buffer, 0.6mL of reaction substrate solution, 0.1mL of crude enzyme solution, and 0.3mL of NADH starting solution in sequence. After adding the starting solution, shake well to use as the reaction system solution. Use a UV spectrophotometer for colorimetry. First, adjust the absorbance of the quartz cuvette to zero with reaction buffer. Then, take the reaction system solution into the quartz cuvette and read the data at 340nm. Record the data as the initial absorbance. After the colorimetry is completed, the remaining reaction system solution is placed in a light-proof water bath. After the end, take it out and place it in a 0-4°C environment. Take the reaction system solution into the quartz cuvette and read the data at 340nm. Record the data as the end absorbance. The enzyme activities of nitrate reductase, glutamate synthetase, and glutamate dehydrogenase are calculated using the extinction of NADH per gram of fresh weight per hour. The calculation method is as follows:

[0052] Enzyme activity (U·g -1 ·h -1 ·FW)=(UA-UB)×V / Va / W / h;

[0053] Where UA is the initial absorbance; UB is the final absorbance; V is the volume of crude enzyme extract; Va is the amount of crude enzyme solution used in the determination; W is the sample weight; and h is the reaction time.

[0054] The present invention improves the extract of nitrogen metabolic enzymes and uses Tris buffer containing MgSO4, EGTA and dithiothreitol as the crude enzyme extract, and further improves the determination method of nitrate reductase, glutamate synthetase and glutamate dehydrogenase. While ensuring accuracy, this method is simple to operate, saves time, and is suitable for large-scale determination.

[0055] Example 1

[0056] The experiment was conducted at the State Key Laboratory of Wheat Breeding at A University, and three crops were selected: corn (Zhengdan 958), wheat (Taimai 198), and peanut (Shanhua 9).

[0057] (1) Pretreatment: Soak the seeds in 0.3% hydrogen peroxide for 6 h, then place them in a germination tray for 3 days. After germination, place them in a 12-well hydroponic box (6 plants per box) and treat them with deionized water for 3 days, and then use Hoagland's nutrient solution for normal culture for 9 days.

[0058] (2) Experimental treatment: After pretreatment, each crop was divided into two groups. One group was managed with normal nitrogen (1mmol·L-1 Ca(NO3), treatment number N), and a blank treatment (using CaCl2 to supplement Ca, treatment number K), and culture was continued for 6 days.

[0059] (3) Preparation of crude enzyme extract: The crude enzyme extract is 0.05 mol·L at pH = 7.5. -1 Tris buffer (containing MgSO4, EGTA, and DTT). The concentration of MgSO4 is 4 mmol·L -1 ; The concentration of EGTA is 0.5 mmol·L -1 ; The concentration of DTT is 2 mmol·L -1 .

[0060] (4) Sampling: After the experimental treatment, the second unfolded leaf of the main stem of all crops was taken, wrapped in aluminum foil, frozen in liquid nitrogen, and stored in a -80℃ refrigerator.

[0061] (5) Extraction of crude enzyme solution: The leaves treated with different methods were ground with liquid nitrogen, 0.5 g was weighed and placed in a 10 mL centrifuge tube, 5 mL of crude enzyme extract was added, and the mixture was centrifuged at 10,000 r / min. -1 Centrifuge at 4°C for 20 min, aspirate the supernatant and store in a 4°C refrigerator away from light.

[0062] (6) NR activity determination: Three 2 mL centrifuge tubes were taken for each treatment of each crop, and 1 mL of reaction buffer, 0.6 mL of reaction substrate solution, 0.1 mL of crude enzyme solution, and 0.3 mL of NADH starting solution (0.6 mg mL -1 Reduced coenzyme I (NADH) solution), shake well, and place in an ice box as the reaction system solution.

[0063] Determination: Preheat the UV spectrophotometer (30 minutes) and adjust to 340 nm. Zero the spectrophotometer with the reaction substrate solution, then add 1 mL of the reaction solution sequentially and record the readings (UA). After the measurement, place the remaining reaction solution in a 28°C water bath in the dark for 20 minutes. After the measurement, remove the solution and place it in an ice box. Take 1 mL of the solution and continue reading, recording the data (UB).

[0064] By formula: enzyme activity (U g -1 ·h -1 ·FW) = (UA-UB) × V / Va / W / h, calculate enzyme activity;

[0065] Where UA is the initial absorbance; UB is the final absorbance; V is the volume of crude enzyme extract; Va is the amount of crude enzyme solution used in the determination; W is the sample weight; and h is the reaction time.

[0066] The assays for GOGAT and GDH activities were the same as those for NR, except that the reaction substrates were different.

[0067] About reaction buffer:

[0068] The reaction buffer used for nitrate reductase (NR) and glutamate synthase (GOGAT) was 25 mmol / L -1 Tris buffer (adjust pH to 7.5). The reaction buffer used for glutamate dehydrogenase (GDH) is 40 mmol / L -1 Tris-HCl buffer.

[0069] About the reaction substrate solution:

[0070] The reaction substrate solution of NR is 10 g·L -1 of KNO3 solution.

[0071] The reaction substrate solution of GOGAT is 10 mmol·L -1 Glutamine solution (containing 1g·L -1 α-isoglutaric acid, 0.1 g·L -1 of KCl).

[0072] The reaction substrate solution of GDH is 20 g·L -1 NH4Cl solution (containing 80 mg L -1 of α-isoglutaric acid).

[0073] (7) Data processing: Office 2021 software was used to create tables and perform word processing, and IBM SPSS Statistics 26 was used to perform independent sample t-test to mark the significance of the data (P < 0.05).

[0074] The experimental results are shown in Table 1-3:

[0075] The data in the table show that compared to wheat, corn, and peanuts treated with normal nitrogen (N), the activities of NR, GOGAT, and GDH in the blank (K) treatment were significantly reduced. The smaller standard deviations indicate that the analytical method has minimal bias, high precision, and good reproducibility.

[0076] Table 1 Effects of blank treatment (K) and normal nitrogen treatment (N) on NR activity in wheat, corn and peanut

[0077]

[0078] Table 2 Effects of blank treatment (K) and normal nitrogen treatment (N) on GOGAT activity in wheat, corn and peanut

[0079]

[0080] Table 3 Effects of blank treatment (K) and normal nitrogen treatment (N) on GDH activity in wheat, corn and peanut

[0081]

[0082]

[0083] Example 2

[0084] The time consumption of the traditional NR, GOGAT, and GDH determination methods and the improved determination methods were compared using mathematical equations.

[0085] In the traditional NR determination, there are five steps: assembly of the reaction system (A, 30 seconds for a single sample), catalytic reaction (B, 20 minutes), addition of color developer (C, 15 seconds for a single sample), color development reaction (D, 30 minutes), and colorimetry (E, 15 seconds for a single sample). The equation is y(min) = x + 50 (x is the number of samples).

[0086] The improved NR assay in this application is divided into four steps: assembly of the reaction system (A, single sample takes 30 s), colorimetry (B, single sample 15 s), catalytic reaction (C, takes 20 min), and colorimetry (D, single sample 15 s). The equation is y(min) = x + 20.

[0087] We found that regardless of the number of samples, the improved NR determination method of the present application always saves 30 minutes compared with the traditional NR determination method (e.g. Figure 1 Middle A).

[0088] In the traditional GOGAT and GDH assays, there are two steps: assembly of the reaction system (A, 30 seconds for a single sample) and extinction colorimetry (B, 2 minutes and 30 seconds for a single sample). The equation is y(min) = 3x.

[0089] The improved GOGAT and GDH assays in this application are divided into four steps: assembly of the reaction system (A, 30 s for a single sample), colorimetry (B, 15 s for a single sample), catalytic reaction (C, 20 min), and colorimetry (D, 15 s for a single sample). The equation is y(min) = x+20.

[0090] We found that when the number of samples was 10, the traditional GOGAT and GDH determination methods and the improved GOGAT and GDH determination methods took the same amount of time. When the number of samples was greater than 10, the improved determination method took less time, and this time-saving advantage became more significant as the number of samples increased (e.g. Figure 1 Middle B).

[0091] Therefore, it can be seen from the above that the improved method of the present application can save time and is suitable for large-scale operations.

Claims

1. A method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase, characterized in that: include: S1: Prepare Tris buffer containing MgSO4, EGTA and dithiothreitol as crude enzyme extract; in: The concentration of MgSO4 is 2-5mmol·L -1 ; The concentration of EGTA is 0.5-1 mmol·L -1 ; The concentration of dithiothreitol is 2-4 mmol·L -1 ; S2: The ground leaves were mixed with the crude enzyme extract and centrifuged, and the supernatant was the crude enzyme solution; S3: Using reaction buffer, reaction substrate solution, crude enzyme solution, and reduced coenzyme I starting solution as the reaction system solution, use a UV spectrophotometer to measure the colorimetry and record the values ​​of the reaction system solution at 340 nm before and after a light-proof water bath. Calculate the enzyme activities of nitrate reductase, glutamate synthetase, and glutamate dehydrogenase, respectively, based on the extinction of reduced coenzyme I per gram of fresh weight per hour.

2. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: The concentration of S1 and MgSO4 is 4 mmol·L -1 ; The concentration of EGTA is 0.5 mmol·L -1 ; The concentration of dithiothreitol is 2mmol·L -1 .

3. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: The Tris buffer of S1 is 0.05 mol·L with a pH of 7.

5. -1 Tris buffer.

4. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: The volume ratio of the S3 reaction system solution, reaction buffer solution, reaction substrate solution, crude enzyme solution and reduced coenzyme I starting solution is 1 mL: 0.6 mL: 0.1 mL: 0.3 mL.

5. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: The S3 reaction system solution is prepared by sequentially adding reaction buffer, reaction substrate solution, crude enzyme solution, and reduced coenzyme I starting solution.

6. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: Use an ultraviolet spectrophotometer to measure the color and record the values ​​of the reaction system solution at 340nm before and after the dark water bath, specifically: First, adjust the absorbance of the quartz cuvette to zero with reaction buffer, then take the reaction system solution into the quartz cuvette and read the data at 340nm, which is recorded as the initial absorbance. After the colorimetry is completed, place the remaining reaction system solution in a light-proof water bath, take it out and place it in an environment of 0-4℃, take the reaction system solution into the quartz cuvette and read the data at 340nm, which is recorded as the termination absorbance.

7. The method for determining nitrate reductase, glutamate synthetase and glutamate dehydrogenase according to claim 1, wherein: The calculation formula of the S3 enzyme activity is: Enzyme activity (U·g -1 ·h -1 ·FW)=(UA-UB)×V / Va / W / h; Where UA is the initial absorbance; UB is the final absorbance; V is the volume of crude enzyme extract; Va is the amount of crude enzyme solution used in the determination; W is the sample weight; and h is the reaction time.

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