A method for determining glucose dehydrogenase activity

Abstract

The present invention discloses a method for measuring glucose dehydrogenase activity, comprising the following steps: S1. adding a glucose dehydrogenase solution to be tested to a PMS reaction solution; immediately after the reaction is complete, adding an acid solution to adjust the pH to ≤3.0 to terminate the glucose dehydrogenase reaction; centrifuging, removing the supernatant, and retaining the precipitate; S2. taking a DCIP detection solution, measuring the initial absorbance of the DCIP detection solution at 600 nm; adding the DCIP detection solution to the precipitate in S1 to obtain a mixed solution; after the reaction is complete, measuring the post-reaction absorbance of the mixed solution at 600 nm again; substituting the difference between the post-reaction absorbance and the initial absorbance into a DCIP standard curve to calculate the molar mass of DCIP involved in the reaction, and calculating the glucose dehydrogenase activity value. The present invention ensures the authenticity of the measured data and prevents excessive deviation in the experimental results due to other factors such as excessively fast or slow operation or equipment reaction time. The reaction direction is controlled to minimize side reactions and prevent them from affecting the experimental results.

Description

Technical Field

[0001] The present invention relates to the field of enzyme preparation detection and application, and in particular to a method for determining glucose dehydrogenase activity. Background Art

[0002] Glucose dehydrogenase, an enzyme found in the livers of higher animals (such as cattle, sheep, dogs, and cats) and in Acetobacter subaxydans, catalyzes the reaction of D-glucose + NAD(P)D-gluconate (lactone) + NAD(P)H. The animal-derived enzyme (EC.1.1.1.47) can utilize both NAD and NADP. While it also exhibits 25% activity on D-xylose, it has little activity on other natural hexoses and pentoses (in animals). The Acetobacter enzyme (EC1.1.1.119) is specific for NADP but can also catalyze D-mannose.

[0003] Currently, the most common method for detecting glucose dehydrogenase activity is the DCIP colorimetric method. The definition of DCIP colorimetric enzyme activity detection is: the amount of enzyme required to reduce 1μmol DCIP in the solution per minute is defined as one enzyme activity unit. The steps for detecting enzyme activity by DCIP colorimetric method are as follows: take 100μL of appropriately diluted enzyme solution, add 3ml of colorimetric solution (50mmol / L pH7.0 PB buffer, 0.03mmol / L DCIP, 1.6mmol / L PMS, 100mmol / L glucose solution), mix gently, adjust to zero with redistilled water, and record the change in absorbance at 600nm every 1min at 37℃ for a total of 2-3min. Use enzyme buffer instead of enzyme solution as a control. The methods used in different literature are consistent, and only the concentration of the components in the colorimetric solution formula varies.

[0004] Detection mechanism:

[0005] The detection principle of DCIP colorimetric method is as follows:

[0006]

[0007] 2MPH+DCIP→2PMS+re-DCIP

[0008] Among them, D-Glucose is glucose; PMS is 5-methylphenazine methyl sulfate; D-glucono-δ-lactone is glucono-δ-lactone; MPH is 1-methylphenazine; DCIP is 2,6-dichlorophenol indophenol; re-DCIP is reduced DCIP; GDH is glucose dehydrogenase.

[0009] The reaction mechanism of PMS conversion to MPH is shown in Equation 1; the reaction mechanism of DCIP conversion to colorless re-DCIP is shown in Equation 2;

[0010]

[0011] Detection using test kits is expensive and requires instrumentation. The current DCIP colorimetric method produces inaccurate results with significant deviations. Summary of the Invention

[0012] The technical problem to be solved by the present invention is to provide a method for measuring glucose dehydrogenase activity with accurate measurement results and small deviation in view of the shortcomings of the existing technology.

[0013] To solve the above technical problems, the technical solution adopted by the present invention is: a method for determining glucose dehydrogenase activity, comprising the following steps:

[0014] S1. Glucose dehydrogenase solution to be tested was added to the PMS reaction solution. After the reaction was completed, an acid solution was immediately added to adjust the pH to ≤ 3.0 to terminate the glucose dehydrogenase reaction. The reaction was centrifuged and the supernatant was removed to retain the precipitate.

[0015] S2. Take a DCIP detection solution, measure the initial absorbance of the DCIP detection solution at 600 nm, add the DCIP detection solution to the precipitate in S1 to obtain a mixed solution, and after the reaction is completed, measure the post-reaction absorbance of the mixed solution at 600 nm again. Substitute the difference between the post-reaction absorbance and the initial absorbance into the DCIP standard curve to calculate the molar mass of DCIP involved in the reaction and the enzymatic activity of glucose dehydrogenase.

[0016] During the measurement process using the existing DCIP colorimetric method, it was found that there were two problems in the reaction system:

[0017] Question 1: There are side reactions in the entire reaction system. When there is no PMS in the system, part of DCIP can still be reacted. That is, GDH will provide H for DCIP when reacting with glucose. + , the side reactions are as follows:

[0018]

[0019] Question 2: When the DCIP in the entire reaction system is consumed, the glucose in the system has not yet reacted completely, and the first step of the reaction can still continue. At this time, a large amount of MPH will remain in the reaction system. After consulting papers and experimental verification, MPH is hydrophobic and will exist in the form of precipitation in the aqueous solution, causing the solution to be turbid, thereby affecting the final absorbance measurement results.

[0020] The combined effect of these two issues makes the measurement results sensitive to time. The entire operation requires strict time control. If the measurement time exceeds the optimal detection time (i.e., when the system is reacting normally and the DCIP in the solution has not yet reacted completely), the MPH precipitate generated in the reaction system will cause the solution to become turbid. The blue DCIP has a certain absorbance at 600nm, and the MPH turbidity in the solution also has an absorbance at 600nm. When MPH is present in the solution, we will be unable to determine the authenticity of the measurement results, which will seriously affect the measurement results and cause them to deviate significantly from the actual data.

[0021] The present invention adds acid in time to terminate the glucose dehydrogenase reaction, which can effectively avoid the occurrence of the above side reactions, improve the accuracy of the measurement results, and reduce the deviation.

[0022] In a preferred embodiment of the present invention, the acid in S1 is glacial acetic acid. Glacial acetic acid has no oxidizing and reducing properties and will not affect the detection of the absorbance value of the reaction product at 600 nm.

[0023] In a preferred embodiment of the present invention, the concentration of the PMS reaction solution is ≤1 mmol / L, preferably, ≤0.050 mmol / L. When the concentration of the PMS reaction solution is ≤1 mmol / L, the effect of residual PMS solution on subsequent measurement results can be minimized.

[0024] In a preferred embodiment of the present invention, the pH of the PMS reaction solution is 6.5-7.5. This is partly because the optimal pH range for glucose dehydrogenase is 6.5-7.5, and partly because PMS is more stable under neutral conditions and is susceptible to oxidation at higher pH levels. PMS is relatively stable in weakly acidic conditions, exhibiting a normal pale yellow color, but is susceptible to oxidation (color change) in alkaline environments, with the color becoming darker as the pH increases, even tending to black.

[0025] In a preferred embodiment of the present invention, glacial acetic acid is used to adjust the pH of the PMS reaction solution. Glacial acetic acid has no oxidizing or reducing properties and will not affect the detection of the absorbance value of the reaction product at 600 nm.

[0026] In a preferred embodiment of the present invention, the removal of the supernatant and the retention of the precipitate in S1 are performed under an inert gas atmosphere. Because the MPH precipitate is easily oxidized by air, the use of an inert gas atmosphere prevents MPH oxidation and ensures the authenticity of the experimental results.

[0027] In a preferred embodiment of the present invention, the time for the reaction in S1 to be completed is 1 minute. The timely addition of acid to terminate the glucose dehydrogenase reaction can effectively avoid the occurrence of the above side reactions, improve the accuracy of the measurement results, and reduce the deviation.

[0028] In a preferred embodiment of the present invention, the volume ratio of the PMS reaction solution to the glucose dehydrogenase solution in S1 is 8-12:1.

[0029] In a preferred embodiment of the present invention, the specific steps of preparing the PMS reaction solution are PMS: glucose = 1:10-1:100 mmol / L, the concentration of Tris is 50-200 mmol / L, and after preparation, it is stored in the dark at 4°C.

[0030] In a preferred embodiment of the present invention, the specific steps of preparing the DCIP detection solution are as follows: the concentration ratio of DCIP to PMS is 1:2, the concentration of Tris is 50-200 mmol / L, the pH is adjusted to 6.5-7.5 with glacial acetic acid, and the solution is stored in the dark for future use.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention greatly reduces time sensitivity. When the reaction reaches the required time, acid is immediately added to terminate the enzyme reaction, so that the measurement data remains authentic and there will not be excessive deviation in the experimental results due to other factors such as too fast or too slow operation or equipment reaction time.

[0033] 2. Carry out two reactions with adverse effects separately, artificially control the direction of the reaction, and control the side reactions to the greatest extent possible to avoid their influence on the experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are photos of PMS reaction solutions with different concentrations used in the PMS concentration screening experiment of the present invention;

[0035] Among them, 1: 500mmol / L 2: 100mmol / L 3: 50mmol / L 4: 10mmol / L 5: 5mmol / L 6: 1mmol / L 7: 0.5mmol / L 8: 0.1mmol / L 9: blank control

[0036] Figure 2 These are photos of PMS reaction solutions with different pH values in the pH screening experiment of the reaction system of the present invention;

[0037] Among them, 1: pH=6.0 2: pH=6.5 3: pH=7.0 4: pH=7.5 5: pH=8.0 6: pH=8.5 7: pH=9.0 8: pH=9.5 9: pH=10.0

[0038] Figure 3 These are photos of PMS reaction solutions under different weak acid systems in the weak acid screening experiment of the present invention;

[0039] Among them, 1: dilute nitric acid 2: citric acid 3: dilute hydrochloric acid 4: dilute sulfuric acid 5: glacial acetic acid 6: blank control.

[0040] Figure 4 Schematic diagram for calculating the absorbance value into the DCIP standard curve. DETAILED DESCRIPTION

[0041] Example 1

[0042] 1. Prepare PMS reaction solution: PMS 0.6mmol / L, glucose 100mmol / L, Tris 50mmol / L. Adjust the pH of the reaction system to 6.5 with glacial acetic acid. After preparation, store in the dark at 4℃.

[0043] 2. Prepare DCIP detection solution: DCIP 0.3mmol / L, Tris 50mmol / L, adjust the solution to pH 7.5 with glacial acetic acid, and store in the dark until used.

[0044] 3. In a 15ml centrifuge tube, take 5ml of the PMS reaction solution and add 0.5ml of the enzyme solution of unknown concentration. Time the reaction for 1 minute. Immediately add glacial acetic acid to adjust the pH to 3.0 to terminate the reaction. Centrifuge at 12,000 rpm for 2 minutes. In another 15ml centrifuge tube, take 5ml of the PMS reaction solution and add 0.5ml of purified water as a control. Repeat the above procedure.

[0045] 4. After centrifugation, introduce carbon dioxide into the carbon dioxide incubator to isolate oxygen. Remove the two groups of centrifugal supernatants in the incubator and leave the precipitate in the centrifuge tube.

[0046] 5. Add 5 ml of DCIP reaction solution to each of the two sets of centrifuge tubes. After the reaction is complete, take out 300 μL from each tube and add it to a 96-well plate. Then take 300 μL of unreacted DCIP solution and add it to the 96-well plate. Use a Thermoscientific MULTISKAN FC microplate reader to measure the absorbance at 600 nm. The measurement results are as follows:

[0047] Table 1 Absorbance values of test samples, reference substances and unreacted DCIP

[0048] Group Samples to be tested Reference substances Unreacted DCIP Absorbance 0.752 1.351 1.363

[0049] To develop a DCIP standard curve, different concentrations of DCIP solutions were prepared on the same day. The standard curve equation was developed based on the measurement results as follows: y = 0.0692x - 0.0036 R 2=0.9988 (x=measured absorbance value, y=the amount of DCIP substance contained in the measured 300 μL in μmol).

[0050] Substituting the absorbance value measured by the test sample into the equation, the amount of DCIP remaining in the measured solution can be obtained, which is 0.0484 μmol. Dividing it by the volume of 300 μL, the DCIP concentration in the test sample is 0.161 mmol / L. The remaining DCIP amount in the test sample is 0.161 mmol / L*0.005L=0.000805 mmol. The total DCIP amount of the original solution is 0.0015 mmol. The difference between DCIP before and after the reaction is 0.0015 mmol-0.000805 mmol=0.000695 mmol. The protein concentration of the test solution sample is detected by the BCA protein concentration detection kit, and the protein concentration is approximately 0.5 mg / ml. Therefore, the enzyme activity in the test solution can be obtained as follows: 0.000695 mmol / 0.5 mg / ml*5 ml=2.78 U / mg.

[0051] To verify the accuracy of the experiment, the same sample was tested using the Glucose-6-phosphate dehydrogenase (G6PDH) activity detection kit from Solebo Company, and the operation was carried out according to the instructions:

[0052] 1. Preheat the UV spectrophotometer for more than 30 minutes, adjust the wavelength to 340nm, and zero with distilled water.

[0053] 2. Preheat the reagents in a 37°C water bath for 30 minutes.

[0054] 3. Sample addition table:

[0055] Reagent name (μL) Assay tube (μL) Blank tube (μL) Working fluid 950 950 sample 50 - distilled water - 50

[0056] 4. Measure the absorbance change at 340 nm over 5 minutes. Record the absorbance at 0 seconds as A1 and the absorbance at 300 seconds as A2. Note: ΔA measurement = A2 measurement - A1 measurement, and ΔA blank = A2 blank - A1 blank.

[0057] 5. Calculation of G6PDH activity in tissues:

[0058] Calculated according to sample protein concentration: Unit definition: One unit of enzyme activity is defined as the amount of 1 nmol NADPH generated per minute per mg of sample protein in the reaction system.

[0059] G6PDH (U / mg prot) = [(ΔA assay - ΔA blank) ÷ (ε × d) × 109 × V total] ÷ (V sample × Cpr) ÷ T = 643 × (ΔA assay - ΔA blank) ÷ Cpr

[0060] ε: NADPH molar extinction coefficient, 6.22×103 L / mol / cm; d: optical path of 1 mL quartz cuvette, 1 cm; Vreact: total reaction volume, 0.001 L; Vsample: added sample volume, 0.05 mL; T: reaction time, 5 min; Vextract: added extract volume, 1 mL; Cpr: sample protein concentration, mg / mL;

[0061] Experimental process:

[0062] To control the data range within the test range, the sample to be tested was diluted once and then operated according to the steps in the above instruction manual. The results were: ΔA assay tube = A2 assay - A1 assay = 1.586 - 0.481 = 1.105, ΔA blank tube = A2 blank - A1 blank = 0.043 - 0.031 = 0.012, and the enzyme activity was calculated according to the sample concentration:

[0063] G6PDH (U / mg prot) = 643 × (ΔA assay - ΔA blank) ÷ Cpr = 643 × (1.105 - 0.012) ÷ 0.25 mg / ml = 2811.196 U / mg prot

[0064] Because this assay defines enzyme activity as 1 nmol of NADPH generated per minute per mg of sample protein in the reaction system, the enzyme activity definition in this patent is converted to 2811.196 ÷ 1000 = 2.811 U / mg. The enzyme activity results obtained by the two methods are: this patent method = 2.78 U / mg; the kit method = 2.811 U / mg. The difference between the values measured by this patent method and the kit method is less than 0.05 U / mg, which is within the allowable range. The test method of this invention is accurate.

[0065] PMS concentration screening experiment

[0066] 1. The PMS concentration should be less than 1mmol / L. PMS has a certain color in aqueous solution. It is light yellow at low concentrations and darkens at high concentrations. Excessively high concentrations of PMS will have a certain impact on the subsequent experimental detection of the 600nm absorbance value.

[0067] like Figure 1 As shown, 1-8 correspond to different concentrations of PMS reaction solutions. The other component standards of the solution preparation are in accordance with the patent content (PMS: glucose 1:10, 50mmol / L Tris).

[0068] The absorbance values of the PMS reaction solutions with different concentrations at 600 nm detected by the enzyme marker are shown in Table 2:

[0069] Table 2 Absorbance values of PMS reaction solutions at different concentrations at 600 nm detected by enzyme marker

[0070]

[0071] Among them, 1: 500mmol / L 2: 100mmol / L 3: 50mmol / L 4: 10mmol / L 5: 5mmol / L 6: 1mmol / L 7: 0.5mmol / L 8: 0.1mmol / L 9: blank control

[0072] It can be seen that PMS at high concentrations exhibits a certain absorbance value at 600nm, and the absorbance value gradually decreases as the concentration decreases. The absorbance value presented in group No. 7 (PMS concentration is 0.5mmol / L) is almost the same as that of the blank control group No. 9. During the experiment, in order to minimize the impact of PMS solution residue on subsequent measurement results, PMS solutions with concentrations of No. 6 (PMS concentration is 1mmol / L) and below were selected as measurement solutions.

[0073] The reason for selecting PMS with a concentration of 1 mmol / L or less during the measurement process is mainly to provide sufficient reaction substrate, that is, the amount of PMS, while ensuring the measurement accuracy as much as possible. It can be seen that the blank background noise value is about 0.05 during measurement, and the sample value difference is about one decimal place and one digit during measurement. Therefore, it is believed that when the measured value is less than 0.1, it is not much different from the background noise, and has little impact on the actual measurement data. Therefore, PMS with a concentration of 1 mmol / L or less is selected as the reaction system. The specific selection can be made according to the actual situation.

[0074] Reaction system pH screening experiment

[0075] The pH of the reaction system is controlled at 6.5-7.5. On the one hand, this is because the optimal pH range of glucose dehydrogenase is 6.5-7.5. On the other hand, PMS is more stable under neutral conditions. When the pH is high, PMS is prone to oxidation. Figure 2 This is the phenomenon after PMS solutions under different pH conditions are left standing in a dark place for a period of time.

[0076] PMS tends to be stable in weakly acidic conditions and presents a normal light yellow color. It is easily oxidized (color changes) in alkaline environments, and the color becomes darker as the pH value increases, and even tends to change to black.

[0077] Weak acid screening experiment

[0078] Among the commonly used acid treatments in the laboratory, glacial acetic acid has neither oxidizing nor reducing properties. Other commonly used acids in the laboratory, such as hydrochloric acid, sulfuric acid, nitric acid, citric acid, etc., all have certain redox properties. In order not to affect the experimental results, glacial acetic acid is selected as the enzyme activity terminator.

[0079] The state of PMS solution under different acid treatments is as follows Figure 3 As shown, among them, 1. dilute nitric acid 2. citric acid 3. dilute hydrochloric acid 4. dilute sulfuric acid 5. glacial acetic acid 6. blank control (blank control is an equal amount of PMS solution).

[0080] These commonly used acid reagents all contain strong or weak oxidizing or reducing properties, causing PMS to undergo certain transformations in the solution. This change will affect the final measurement results, which is reflected in the change of absorbance at 600nm. The absorbance measurement results of the above four solutions at 600nm are as follows:

[0081] Table 3 Absorbance at 600 nm after adding different acids to PMS solution

[0082]

[0083] Enzyme termination pH screening experiment

[0084] During the experiment, we found that at pH 3.0, GDH almost stopped functioning, completely losing its enzymatic activity. Alkali also caused PMS to produce a black precipitate, seriously affecting the experimental results. Alkali alone does not cause PMS to produce a black substance; it is the pH change caused by the alkali that causes the PMS to produce a black substance.

[0085] In the original measurement method, the enzyme was not stopped during measurement, so the measurement time would result in a large data deviation. In this patented method, an attempt was made to stop the enzyme at the expected time to ensure data accuracy as much as possible. Therefore, an attempt was made to use acid to terminate the enzyme, i.e., a low pH value inactivates and denatures the protein. The following experimental mechanism is as follows: To prove that the enzyme has stopped working at a certain pH value, the following experimental content is designed. Under the same other conditions (i.e., ensuring the enzyme amount, reaction temperature, substrate concentration, etc.), only the enzyme is treated with acid, and the enzyme reaction is observed. The final result (the amount of DCIP after the reaction) is used to determine whether the enzyme is inactivated after acid treatment.

[0086] To prove the above content, the following experiment was designed (this experiment uses the DCIP colorimetric method to qualitatively verify whether there is enzyme activity, that is, the reaction of DCIP and PMS mixed solution reflects enzyme activity): 8 groups of a certain amount of 0.1mg / ml glucose dehydrogenase enzyme solution were taken respectively, and the 8 groups of enzyme solutions were acid-treated with glacial acetic acid. The final pH of the treatment was 1 = blank control, 2 = 3.0, 3 = 3.5, 4 = 4.0, 5 = 4.5, 6 = 5.0, 7 = 5.5, and 8 = 6.0. After the acid treatment was completed, sodium hydroxide was immediately used to neutralize the pH value to between 6.5 and 7.5. Then, a certain amount of DCIP detection solution (a mixed buffer system of glucose, PMS, and DCIP) was added to the 8 groups of treated enzyme solutions for qualitative verification of the presence of enzyme activity. After reacting for 1 minute, the absorbance at 600nm was measured immediately using a microplate reader. The measurement results are shown in Table 4:

[0087] Table 4 Absorbance at 600 nm after enzyme termination reaction at different pH values

[0088]

[0089] It can be seen from the data in the table that, from the 8th group to the 3rd group of experiments, as the pH value decreases, the absorbance value at 600nm under the same reaction conditions gradually increases, indicating that the amount of DCIP remaining in the system gradually increases, indicating that the decrease in pH value affects the activity of the enzyme and thus affects the amount of DCIP reacted. The absorbance value at pH = 3.0 is almost the same as that of the enzyme-free system (Group 1), indicating that under this pH condition, the enzyme hardly works, so almost no DCIP is reacted, so the value hardly changes.

[0090] According to the analysis of the results, the enzyme hardly works at a pH of 3.0, and the enzyme activity is affected to a certain extent above 3.0, but it still works. Therefore, it is recommended to adjust the pH to 3.0 or below to terminate the enzyme reaction.

Claims

1. A method for determining glucose dehydrogenase activity, characterized in that The following steps are involved: S1. Glucose dehydrogenase solution to be tested was added to the PMS reaction solution. After the reaction was completed, an acid solution was immediately added to adjust the pH to ≤ 3.0 to terminate the glucose dehydrogenase reaction. The reaction was centrifuged and the supernatant was removed to retain the precipitate. S2. Take a DCIP test solution, measure the initial absorbance of the DCIP test solution at 600 nm, add the DCIP test solution to the precipitate in S1 to obtain a mixed solution, and after the reaction is completed, measure the absorbance of the mixed solution at 600 nm again. Substitute the difference between the absorbance after the reaction and the initial absorbance into the DCIP standard curve to calculate the molar mass of DCIP involved in the reaction and calculate the glucose dehydrogenase activity value; In S1, the supernatant is removed and the precipitate is retained under the protection of inert gas; The volume ratio of the PMS reaction solution to the glucose dehydrogenase solution in S1 is 8-12:1; The acid in S1 is glacial acetic acid; The concentration of the PMS reaction solution is 0.1~1mmol / L; The pH of the PMS reaction solution is 6.5-7.

5.

2. The method for determining glucose dehydrogenase activity according to claim 1, wherein The pH of the PMS reaction solution was adjusted with glacial acetic acid.

3. The method for determining glucose dehydrogenase activity according to any one of claims 1 to 2, wherein The time for the reaction to be completed in S1 was 1 minute.

4. The method for determining glucose dehydrogenase activity according to any one of claims 1 to 2, wherein The specific steps of preparing the PMS reaction solution are as follows: PMS: glucose = 1:10-1:100 mmol / L, the concentration of Tris is 50-200 mmol / L, and after preparation, it is stored in the dark at 4°C.

5. The method for determining glucose dehydrogenase activity according to any one of claims 1 to 2, wherein The specific steps of preparing the DCIP detection solution are as follows: the concentration ratio of DCIP to PMS is 1:2, the concentration of Tris is 50-200 mmol / L, the pH is adjusted to 6.5-7.5 with glacial acetic acid, and the solution is stored in the dark for future use.

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