A lipid-type substrate GPX activity detection kit, a detection method and application

Abstract

The application provides a lipid-type substrate GPX activity detection kit, a detection method and application, and the detection kit comprises the following reagents: AEA-M, dodecyl glucopyranoside, cholestanol hemisuccinate trimethylolamine salt, type 1 lipoxygenase and 2,3-naphthyl dialdehyde, wherein the AEA-M is synthesized by arachidonic acid and 1-amino-2,5-anhydro-1-deoxy-D-mannitol through an enzyme method. The application is suitable for detecting the activity of a lipid-type substrate GPX enzyme, the reagent reaction system has strong anti-turbidity capacity, the substrate has good solubility and good low-temperature stability, and is suitable for cell samples, high-fat content tissue samples and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a lipid-type substrate GPX activity detection kit, a detection method and application. BACKGROUND

[0002] Glutathione peroxidase (GPX) is a biological antioxidant enzyme that eliminates oxygen free radicals and other substances generated in the body through enzymatic reaction to maintain a reduced environment. Current research has found that there are eight types of GPX, and different types of GPX have different catalytic activities on different oxidative substrates. In the study of ferroptosis, iron ions induce lipid peroxidation, leading to cell membrane rupture and further cell death. Glutathione peroxidase 4, which is sensitive to lipid-type peroxidation substrates, can eliminate oxidized lipids and thus inhibit cell death. Therefore, it is of great significance to study the enzyme activity of glutathione peroxidase sensitive to lipid-type substrates.

[0003] GPX catalyzes the generation of oxidized glutathione from reduced glutathione and oxidative substrates. The currently marketed GPX enzyme activity detection products generally use organic hydroxyl peroxide as the substrate and glutathione reductase as the enzyme coupling reaction to catalyze the reduction of coenzyme NADH. The enzyme activity is reflected by the rate of decrease in OD value at 340 nm wavelength. The method measures the total enzyme activity of glutathione peroxidase, and cannot reflect the elimination ability of GPX on lipid oxidants. Oxidized substrates and coenzymes are not easy to store at 2-8℃ and are subject to natural degradation. Therefore, there is an urgent need for a lipid-type substrate GPX enzyme activity detection product with high specificity, high detection sensitivity and good stability. SUMMARY

[0004] Therefore, the present application provides a lipid-type substrate GPX activity detection kit, a detection method and application. The kit has only four components, is easy to operate, has few detection steps, and has good solubility of lipid turbid substrates in the reaction system. The reagent has good stability, can be detected at room temperature for a long time, and has high sensitivity and can be used for cell sample detection.

[0005] AEA-M is a lipid-type substrate that can be oxidized by type 1 lipoxygenase to generate oxidized AEA-M, which can be reduced by glutathione peroxidase. 2,3-naphthyl dialdehyde can react with reduced glutathione to generate a fluorescent substance, which can be detected at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Dodecyl pyranose glucoside, cholestanol hemisuccinate trimethylammonium salt can promote the solubility of the substrate AEA-M in the reaction system due to its surfactant effect, and improve the clarity of the reaction system.

[0006] The technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a lipid-type substrate GPX activity detection kit, which comprises a substrate solution containing AEA-M, an enzymatic solution, a fluorescent substrate solution and a standard solution;

[0008] The substrate solution comprises AEA-M, the enzymatic solution comprises type 1 lipoxygenase and reduced glutathione, and the fluorescent substrate solution comprises 2,3-naphthalene dialdehyde, and the structural formula of AEA-M is:

[0009] On the basis of the above scheme, the preparation method of AEA-M comprises the following steps: preferably, 0.304 g of arachidonic acid and 0.326 g of 1-amino-2,5-anhydro-1-deoxy-D-mannitol are weighed into a 10 mL round-bottom flask, 4 mL of n-hexane is added, 5 mg of lipase is added, and the reaction is carried out at 40°C for 6 h under nitrogen protection. After the reaction is completed, the reaction solution is washed with pure water for 3-4 times, and the solvent is removed by rotary evaporation to obtain yellow oily AEA-M.

[0010] On the basis of the above scheme, preferably, the substrate solution further comprises dodecyl glucopyranoside, cholesteryl hemisuccinate trimethylaminomethane salt, potassium azide and Triton X-100.

[0011] On the basis of the above scheme, preferably, in the substrate solution, the concentration of dodecyl glucopyranoside is 1.25 wt%-3.5 wt%, the concentration of cholesteryl hemisuccinate trimethylaminomethane salt is 0.7 wt%-3.6 wt%, the concentration of potassium azide is 1-5 mmol / L, the concentration of Triton X-100 is 0.5 wt%-2.5 wt%, and the concentration of AEA-M is 2-4 mmol / L, and the solvent is Tris-HCl buffer with pH 7-9 and 20-150 mmol / L. Further preferably, the concentration of dodecyl glucopyranoside is 2.5 wt%, the concentration of cholesteryl hemisuccinate trimethylaminomethane salt is 2.1 wt%, the concentration of potassium azide is 2.5 mmol / L, the concentration of Triton X-100 is 0.8 wt%, the concentration of AEA-M is 3 mmol / L, and the solvent is Tris-HCl buffer with pH 8.4 and 100 mmol / L.

[0012] On the basis of the above scheme, preferably, the enzymatic solution further comprises trehalose, bovine serum albumin and glycerol;

[0013] On the basis of the above scheme, preferably, in the enzymatic liquid, the concentration of trehalose is 1.6wt%-6.0wt%, the concentration of bovine serum albumin is 0.5wt%-1wt%, the concentration of glycerol is 0.5wt%-2.5wt%, the concentration of type 1 lipoxygenase is 50-250U / mL, the concentration of reduced glutathione is 2-5mmol / L, and the solvent is Tris-HCl buffer with pH 7-9 and 20-150mmol / L; further preferably, the concentration of trehalose is 4.3wt%, the concentration of bovine serum albumin is 0.6wt%, the concentration of glycerol is 1.2wt%, the concentration of type 1 lipoxygenase is 150U / mL, the concentration of reduced glutathione is 2.5mmol / L, and the solvent is Tris-HCl buffer with pH 8.4 and 100mmol / L.

[0014] On the basis of the above scheme, the fluorescent substrate liquid further comprises ammonium acetate, preferably, the concentration of ammonium acetate is 0.01wt%-0.03wt%, and the concentration of 2,3-naphthalene dialdehyde is 20-30mmol / L; further preferably, the concentration of ammonium acetate is 0.02wt%, and the concentration of 2,3-naphthalene dialdehyde is 25mmol / L.

[0015] In a second aspect, the present application provides a detection method of the kit as described above, comprising the following steps:

[0016] S1, processing of animal tissue samples or collected cell samples, preferably, homogenizing according to the proportion of tissue sample mass (g): physiological saline volume (mL) = 1:9 (such as 0.05g of tissue sample, adding 0.45mL of physiological saline), centrifuging at 4℃ and 10000xg for 10min, and taking the supernatant for detection; homogenizing according to the proportion of 1x10 6 cells (g): physiological saline volume (mL) = 1:0.2mL (such as 1x10 6 cells, adding 0.2mL of physiological saline), centrifuging at 4℃ and 10000xg for 10min, and taking the supernatant for detection;

[0017] S2, setting blank wells, standard wells and determination wells in the fluorescent enzyme-coated plate wells;

[0018] S3, adding double-distilled water to the blank wells, adding different concentrations of standard samples to the standard wells, and adding the supernatant in step S1 to the determination wells;

[0019] S4, adding equal volume of substrate liquid and enzymatic liquid to the determination wells in step S3, incubating the reaction, and preferably, the incubation reaction temperature is 37℃ and the time is 20min;

[0020] S5, adding the same volume of fluorescent substrate solution to the standard well, the blank well and the determination well in step S4 in sequence, incubating the reaction, preferably, the incubation temperature is 37 DEG C and the incubation time is 10 min;

[0021] S6, setting the excitation wavelength of the fluorescent enzyme label instrument to 350 nm and the emission wavelength to 450 nm, detecting the fluorescence value of the well of the fluorescence plate, and fitting the standard curve, preferably, fitting the standard curve according to the absolute fluorescence value of the standard well and the standard product concentration.

[0022] When the sample to be detected is a cell or a tissue sample, the enzyme activity is calculated by using the following formula:

[0023]

[0024] Wherein, F1: the fluorescence value of the determination well, F2: the fluorescence value of the blank well, f: the conversion coefficient of the standard well system and the determination well system, T: the incubation fixed time, Cpr: the protein concentration (gprot / L) of the sample to be detected when added to the detection system.

[0025] The definition of U is that the amount of enzyme required for catalyzing 1 μmol of reduced glutathione of the substrate per minute per gram of protein at a specific temperature in the reaction system is one activity unit.

[0026] In a third aspect, the application provides the use of the kit described above in detecting the GPX enzyme activity in a sample.

[0027] The lipid-type substrate GPX activity detection kit, the detection method and the application of the application have the following beneficial effects relative to the prior art:

[0028] (1) The kit of the application has high detection sensitivity and can be used for detecting cell samples; the kit has few reagent components and is easy to operate.

[0029] The application uses the reduced fatty substrate AEA-M, which can specifically detect the GPX enzyme activity of the fatty substrate, has strong thermal stability, good water solubility and can maintain long reaction activity at room temperature.

[0030] (2) The substrate solution dodecyl pyranoside, cholestan hemisuccinate and trimethylammonium methane salt used in the application promote the dissolution of the substrate AEA-M due to the surfactant effect, thereby ensuring the clarity of the system. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the accompanying drawings in the following description only represent some of the embodiments of the present application, and all other embodiments obtained by those of ordinary skill in the art without any creative effort based on these accompanying drawings also belong to the protection scope of the present application.

[0032] Figure 1 The principle diagram of the GPX enzyme activity detection of the present application;

[0033] Figure 2 The standard product fitting curve diagram of the present application;

[0034] Figure 3 The reference detection method and the present detection method result comparison diagram of the present application;

[0035] Figure 4 The reagent stability result comparison diagram of the present application;

[0036] Figure 5 The system turbidity optimization diagram of the present application. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments only represent some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without any creative effort also belong to the protection scope of the present application.

[0038] Example 1: Synthesis of lipid-type substrate AEA-M

[0039] A 0.304 g of arachidonic acid and 0.326 g of 1-amino-2,5-anhydro-1-deoxy-D- mannitol were weighed into a 10 mL round-bottom flask, 4 mL of n-hexane was added, 5 mg of lipase was added, and the reaction was carried out at 40 °C for 6 h under nitrogen protection. After the reaction was completed, the reaction solution was washed with pure water for 3-4 times, and the solvent was removed by vacuum rotary evaporation. The yellow oily product was obtained with a yield of 65% and a purity of 1H NMR (400 MHz, Chloroform-d). δ 8.0 (1H, s), 5.85 (1H, d), 5.51-5.41 (8H, m), 5.41 (2H, m), 4.71 (2H, m), 3.50 (2H, m), 2.8 (6H, dd, J1= 8.5 Hz, J2= 2.9 Hz), 2.34 (2H, t, J = 7.5 Hz), 2.16 (4H, m), 1.79 (2H, m), 1.29 (6H, m), 0.88 (3H, m). 13C NMR (101 MHz, Chloroform-d) δ 172.5, 130.3, 128.8, 127.7, 127.4, 102.4, 84.1, 76.4, 69.5, 40.1, 37.7, 36.6, 33.8, 32.8, 31.9, 29.6, 25.5, 21.8, 14.1.

[0040] Example 2: Preparation of a lipid-type GPX enzyme biopsy test kit

[0041] A lipid-type GPX enzyme biopsy test kit comprises the following reagents: a substrate solution, an enzymatic solution, a fluorescent substrate solution, a standard solution.

[0042] The substrate solution is prepared: AEA-M 3 mmol / L, dodecyl glucopyranoside 2.5 wt%, cholesteryl hemisuccinate tris-hydroxymethyl aminomethane salt 2.1 wt%, potassium azide 2.5 mmol / L, Triton X-100 0.8 wt%, and the solvent is Tris-HCl buffer with pH 8.4, 100 mmol / L.

[0043] The enzymatic solution is prepared: lipoxygenase type 1 150 U / mL, trehalose 4.3 wt%, bovine serum albumin 0.6 wt%, glycerol 1.2 wt%, reduced glutathione 2.5 mmol / L, and the solvent is Tris-HCl buffer with pH 8.4, 100 mmol / L.

[0044] The fluorescent substrate solution is prepared: 2,3-naphthalene acetal 25 mmol / L, ammonium acetate wt 0.02%;

[0045] The standard solution is prepared: reduced glutathione 1 mmol / L.

[0046] Example 3: Preparation of a lipid-type GPX enzyme biopsy test kit

[0047] A lipid-type GPX enzyme biopsy test kit comprising the following reagents: a substrate solution, an enzymatic solution, a fluorescent substrate solution, a standard solution.

[0048] The substrate solution is prepared as follows: AEA-M 2 mmol / L, dodecyl glucopyranoside 1.25 wt%, cholesteryl hemisuccinate tris-hydroxymethyl aminomethane salt 0.7 wt%, potassium azide 1 mmol / L, Triton X-100 0.5 wt%, and a solvent of Tris-HCl buffer at pH 7 and 20 mmol / L.

[0049] The enzymatic solution is prepared as follows: lipoxygenase type 1 50 U / mL, trehalose 1.6 wt%, bovine serum albumin 0.5 wt%, glycerol 0.5 wt%, reduced glutathione 2 mmol / L, and a solvent of Tris-HCl buffer at pH 7 and 20 mmol / L.

[0050] The fluorescent substrate solution is prepared as follows: 2,3-naphthyl diacetal 20 mmol / L, ammonium acetate 0.01 wt%;

[0051] The standard solution is prepared as follows: reduced glutathione 1 mmol / L.

[0052] Example 4: Preparation of a lipid-type GPX enzyme biopsy test kit

[0053] A lipid-type GPX enzyme biopsy test kit comprising the following reagents: a substrate solution, an enzymatic solution, a fluorescent substrate solution, a standard solution.

[0054] The substrate solution is prepared as follows: AEA-M 4 mmol / L, dodecyl glucopyranoside 3.5 wt%, cholesteryl hemisuccinate tris-hydroxymethyl aminomethane salt 3.6 wt%, potassium azide 5 mmol / L, Triton X-100 2.5 wt%, and a solvent of Tris-HCl buffer at pH 9 and 150 mmol / L.

[0055] The enzymatic solution is prepared as follows: lipoxygenase type 1 250 U / mL, trehalose 6.0 wt%, bovine serum albumin 1.0 wt%, glycerol 2.5 wt%, reduced glutathione 5 mmol / L, and a solvent of Tris-HCl buffer at pH 9 and 150 mmol / L.

[0056] The fluorescent substrate solution is prepared as follows: 2,3-naphthyl diacetal 30 mmol / L, ammonium acetate 0.03 wt%;

[0057] The standard solution is prepared as follows: reduced glutathione 1 mmol / L.

[0058] Example 5: Detection method of a lipid-type GPX enzyme biopsy test kit

[0059] A detection method of a lipid-type GPX enzyme biopsy test kit, the specific steps are as follows:

[0060] (1) Experimental process

[0061] 1. Animal tissue sample or collected cell sample processing:

[0062] According to the ratio of tissue sample quality (g) : physiological saline volume (mL) = 1:9, homogenate (such as 0.05 g of tissue sample, add 0.45 mL of physiological saline, centrifuge at 10000xg for 10 min at 4°C, take the supernatant for detection. 1x10 6 Cells (g) : physiological saline volume (mL) = 1:0.2 mL (such as 1x10 6 Cells, add 0.2 mL of physiological saline), centrifuge at 10000xg for 10 min at 4°C, take the supernatant for detection.

[0063] 2. Set blank wells, standard wells and determination wells on the fluorescence enzyme-labeled plate, and set the positions of each well according to Table 1:

[0064] Table 1. Plate well position setting table

[0065] 1 2 3 4 5 6 7 8 9 10 11 12 A S1 S9 S17 S25 S33 S41 S49 S57 S65 S73 S81 S89 B S2 S10 S18 S26 S34 S42 S50 S58 S66 S74 S82 S90 C S3 S11 S19 S27 S35 S43 S51 S59 S67 S75 S83 S91 D S4 S12 S20 S28 S36 S44 S52 S60 S68 S76 S84 S92 E S5 S13 S21 S29 S37 S45 S53 S61 S69 S77 S85 S93 F S6 S14 S22 S30 S38 S46 S54 S62 S70 S78 S86 S94 G S7 S15 S23 S31 S39 S47 S55 S63 S71 S79 S87 S95 H S8 S16 S24 S32 S40 S48 S56 S64 S72 S80 S88 S96

[0066] Among them: S1-S2: blank wells; S3-S16: standard wells; S17-S96: determination wells, wherein S17-S43 correspond to Example 2, S44-S70 correspond to Example 3, and S71-S96 correspond to Example 4.

[0067] 3. Add 20 μL of double distilled water to the blank wells, add 120 μL of different concentrations of standard to the standard wells, and add 20 μL of sample homogenate supernatant to the determination wells.

[0068] 4. Add 50 μL of substrate solution and 50 μL of enzyme solution to the determination wells and blank wells in step 3 in turn, and incubate at 37°C for 20 min.

[0069] 5. Add 100 μL of fluorescent substrate solution to the standard wells, blank wells and determination wells in step 4, and incubate at 37°C for 10 min.

[0070] 6. Set the excitation wavelength of the fluorescence enzyme marker to 350 nm and the emission wavelength to 450 nm, and detect the fluorescence value of the plate wells.

[0071] Example 6:

[0072] Using the kit in Examples 2-4 and the detection method in Example 5, the detection instrument: FlexStation 3 of American Molecular Devices Multifunctional Enzyme Labeling Instrument; 6 samples are selected: 10% rat kidney tissue homogenate, 10% rat liver tissue homogenate, 10% rat brain tissue homogenate, 10% mouse kidney tissue homogenate, 293T cells, Hela cells, recombinant protein GPX-4 are detected, each standard sample is detected twice, and each sample is detected three times, and the results are as follows:

[0073] The fluorescence values of the standard samples are shown in Table 2:

[0074] Table 2 Fluorescence values of standard samples

[0075]

[0076] The standard sample fitting curve is shown in Figure 2 .

[0077] When the sample to be tested is a cell or tissue sample, the enzyme activity is calculated using the following formula:

[0078]

[0079] Wherein, F1: fluorescence value of the determination hole, F2: fluorescence value of the blank hole, f: conversion factor of the standard hole system and the determination hole system, T: incubation fixed time, Cpr: protein concentration of the sample to be tested when added to the detection system (gprot / L).

[0080] The definition of U is: the amount of enzyme required to catalyze the reduction of 1 μmol of reduced glutathione per minute per gram of protein at a specific temperature in the reaction system is one unit of activity.

[0081] The fluorescence values and enzyme activities of the samples are shown in Table 3:

[0082] Table 3 Fluorescence values and enzyme activities of samples

[0083]

[0084] From the above experimental results, it can be seen that the AEA-M detection method is applicable to various animal tissues and cell samples, and has diversified application scenarios.

[0085] Comparative Example 1

[0086] The glutathione peroxidase enzyme activity detection method introduced in the literature (International Dairy Journal 10.5-6 (2000): 347-351) was used to detect common samples in the laboratory, and the method was operated according to Reference Example 1 and Example 2, and the detection results are shown in Figure 3 .

[0087] The detection effect of the tissue sample is consistent with that of the commercially available product, the GPX-4 detection enzyme activity of the fat type substrate in the AEA method detection result is better, the non-lipid type GPX1 detection enzyme activity is lower; the value can still be determined when the cell sample is detected.

[0088] Comparative Example 2

[0089] According to the detection method of Reference Comparative Example 1, the reagent is stored at 2-8℃ for 2 months, and the enzyme activity of the liver tissue sample stored at-80℃ is detected, and the detection result is as shown in Table 2. Figure 4

[0090] The AEA-M detection method has better reagent thermal stability, and can maintain longer reaction activity at low temperature.

[0091] Comparative Example 3

[0092] After adding dodecyl glucopyranoside, cholestanol hemisuccinate and trimethylammonium methane salt in the detection system, the solubility of the lipid type substrate can be significantly improved, the system can be ensured to be clear and stable, and the anti-interference ability of the reagent can be improved, as shown in Table 3. Figure 5 The left two holes are added with dodecyl glucopyranoside, cholestanol hemisuccinate and trimethylammonium methane salt. Figure 5 The right two holes are not added.

[0093] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A lipid-type substrate-sensitive glutathione peroxidase GPX-4 activity assay kit characterized by, The reagents include a substrate solution, an enzymatic solution, a fluorescent substrate solution and a standard solution; The standard solution is reduced glutathione; The substrate solution includes AEA-M, the enzymatic solution includes type 1 lipoxygenase and reduced glutathione, and the fluorescent substrate solution includes 2,3-naphthalene dialdehyde, and the structural formula of the AEA-M is: ； The substrate solution further contains dodecyl glucopyranoside, cholesteryl hemisuccinate trihydroxymethylammonium salt, potassium azide and triton X-100.

2. The kit of claim 1, wherein The preparation method of the AEA-M includes the following steps: dissolving arachidonic acid and 1-amino-2,5-anhydro-1-deoxy-D-mannitol in n-hexane, adding lipase, reacting at 35-45 DEG C under nitrogen protection for 5-7 h, washing with pure water for 3-4 times after the reaction is completed, and removing the solvent by rotary evaporation to obtain yellow oily AEA-M.

3. The kit of claim 2, wherein The mass ratio of the arachidonic acid, 1-amino-2,5-anhydro-1-deoxy-D-mannitol and lipase is 300-310:320-330:

5.

4. The kit of claim 1, wherein In the substrate solution, the concentration of dodecyl glucopyranoside is 1.25wt%-3.5wt%, the concentration of cholesteryl hemisuccinate trihydroxymethylammonium salt is 0.7wt%-3.6wt%, the concentration of potassium azide is 1-5 mmol / L, the concentration of triton X-100 is 0.5wt%-2.5wt%, the concentration of AEA-M is 2-4 mmol / L, and the solvent is Tris-HCl buffer solution with pH 7-9 and 20-150 mmol / L.

5. The kit of claim 1, wherein The enzymatic solution further contains trehalose, bovine serum albumin and glycerol.

6. The kit of claim 5, wherein In the enzymatic solution, the concentration of trehalose is 1.6wt%-6.0wt%, the concentration of bovine serum albumin is 0.5wt%-1wt%, the concentration of glycerol is 0.5wt%-2.5wt%, the concentration of type 1 lipoxygenase is 50-250 U / mL, the concentration of reduced glutathione is 2-5 mmol / L, and the solvent is Tris-HCl buffer solution with pH 7-9 and 20-150 mmol / L.

7. The kit of claim 1, wherein The fluorescent substrate solution further contains ammonium acetate, and the concentration of ammonium acetate is 0.01wt%-0.03wt%, and the concentration of 2,3-naphthalene dialdehyde is 20-30 mmol / L.

8. Use of the kit according to any one of claims 1-7 for detecting the activity of glutathione peroxidase GPX-4 sensitive to lipid-type substrates for non-diagnostic purposes.

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