ODC1 enzyme activity detection method based on CB6 probe

Abstract

The invention relates to a method for determining enzyme activity of ODC1 (ornithine decarboxylase 1), which comprises the following steps: digesting, centrifuging and resuspending cells, and taking a part of sample to detect protein concentration; the method comprises the following steps: cracking cells by using a specially-made enzyme activity cell lysis solution and a protein lysis solution, and ensuring full cracking through ultrasonication; carrying out quantitative analysis on the protein in the lysate by adopting a BCA kit; in the enzyme activity detection stage, a cell lysis solution and an enzyme activity detection working solution containing a specific fluorescent probe (CB6) are mixed and react at 37 DEG C for a certain time, and then the change of fluorescence intensity is determined by a multifunctional fluorescence microplate reader, so that the enzyme activity of the ODC1 is evaluated. According to the method, accurate determination of the ODC1 enzyme activity is realized by optimizing cell treatment, protein quantification and enzyme activity detection processes. The method has the advantages of simplicity and convenience in operation, high sensitivity, good specificity and the like, and is suitable for the fields of basic research, drug screening and the like related to the ODC1 enzyme activity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a highly sensitive method for measuring ODC1 enzyme activity and its application. Background Technology

[0002] ODC1 is a key enzyme in living organisms, participating in the polyamine synthesis pathway and playing a crucial role in cell proliferation, differentiation, and survival. Since abnormal ODC1 enzyme activity is closely related to the development of various diseases, developing an accurate and sensitive method for measuring ODC1 enzyme activity is of great significance for both basic research and clinical applications.

[0003] Traditional methods for measuring ODC1 enzyme activity often rely on radioactive isotope labeling or complex biochemical reactions, which suffer from cumbersome operation, low sensitivity, and poor safety. In recent years, fluorescence detection technology has been widely used in enzyme activity assays due to its high sensitivity, high specificity, and ease of operation. However, fluorescence detection methods for ODC1 enzyme activity still require further optimization to improve accuracy and reliability. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, this invention aims to develop a highly sensitive method for measuring ODC1 enzyme activity based on fluorescence quenching. This method has many advantages, including accuracy, speed, good repeatability, high sensitivity, and stable linear data, providing a practical solution for ODC1 enzyme activity measurement.

[0005] This invention provides a method for effectively detecting ODC1 enzyme activity, comprising the following steps: The first step involves digesting the cells with trypsin and centrifuging them. After resuspending them in PBS, the cells are centrifuged again to discard the supernatant. The cell pellet is then lysed with lysis buffer. The second step is to centrifuge the lysate and collect the supernatant, then use the BCA method to determine the protein concentration. The third step is to add the enzyme activity detection working solution into the test plate, add the sample after equilibration, equilibrate the reaction again, and then use a multi-functional fluorescent microplate reader to measure the fluorescence intensity. The fourth step is to calculate the ODC1 enzyme activity.

[0006] Specifically, the concentration of trypsin used for cell treatment is 0.25%; preferably, the cell digestion time is 2 minutes. In a preferred embodiment, the centrifugation conditions in the first step are 4°C, 3000 rpm for 5 minutes. More preferably, the lysis buffer in the first step consists of 965 µL Tris (10 mM), 10 µL EDTA (100 mM), and 25 µL DTT (100 mM), and must be prepared fresh for use.

[0007] Preferably, the enzyme activity detection working solution in the third step consists of 225 µL Tris (10 mM), 6 µL DSMI (260 µM), and 4 µL CB6 (130 µM), and must be prepared and used immediately.

[0008] Specifically, the assay plate in the third step is a 96-well black ELISA plate.

[0009] Preferably, in the third step, each well sample consists of 25 µL of cell lysis buffer, 2 µL of ornithine solution (1 mM), and 2 µL of PLP (10 µM).

[0010] In the specific implementation, the excitation wavelength and emission wavelength of the multifunctional fluorescent microplate reader in the third step are 460 nm and 590 nm, respectively.

[0011] In one embodiment, the method for the fourth step, the ODC1 enzyme activity assay, is as follows: One unit of ODC1 enzyme activity is defined as follows: at 25°C and pH 7.5, ODC1 enzyme catalyzes the formation of putrescine from ornithine. Putrescine displaces the substrate bound to the CB6 probe, leading to a change in fluorescence intensity. The fluorescence intensity value indirectly reflects the ODC1 enzyme activity. The final fluorescence intensity X of the CB6-bound substrate is calculated using the following formula: X=(A×0.0417) / (B-0.0622) In the formula: A: Absorbance of the sample after reacting at 460 nm for 1 minute B: Absorbance of the sample after reacting at 562 nm for 1 minute 0.0417: Slope of the BCA standard curve equation 0.0622: Intercept of the BCA standard curve equation.

[0012] This invention presents a stable, rapid, efficient, highly sensitive, and highly specific method for measuring ODC1 enzyme activity, obtained through research and analysis. The obtained data exhibits good linearity. It is suitable for basic research and drug screening related to ODC1 enzyme activity. Attached Figure Description

[0013] Figure 1 : ODC1 enzyme activity sensitivity test graph. Detailed Implementation Example 1 is described with reference to a specific case. The optimized ODC1 enzyme activity detection method was experimentally verified, and the specific procedures are as follows: 1. Cell treatment After 36 hours of culture following cell transfection, the cells were digested with 0.25% trypsin for 2 minutes. An equal volume of cell culture medium was then added to terminate the digestion process. The cells were then centrifuged at 3000 rpm for 5 minutes using a refrigerated centrifuge, and the supernatant was discarded.

[0014] 2. Protein concentration detection The cell pellet was resuspended in 200 µL of pre-cooled PBS, and 5% of the sample was taken for protein concentration detection using the BCA method to prepare for subsequent quantitative analysis.

[0015] 3. ODC1 enzyme activity assay Add enzyme activity assay working solution to a 96-well black microplate and equilibrate at 37°C for 10 minutes. After equilibration, add 25 µL of cell lysis buffer to each well, followed by 2 µL of freshly prepared ornithine solution (1 mM) and 2 µL of PLP solution (10 µM). Incubate the 96-well plate at 37°C for 50 minutes, then measure the fluorescence intensity using a multi-functional fluorescent microplate reader.

[0016] 4. Calculation of ODC1 enzyme activity The final CB6 binding substrate fluorescence intensity X is calculated using the following formula:

[0017] In the formula: A: Absorbance of the sample after reacting at 460 nm for 1 minute B: Absorbance of the sample after reacting at 562 nm for 1 minute 0.0417: Slope of the BCA standard curve equation 0.0622: Intercept of the BCA standard curve equation Measurement error: 10 -4 The results showed that the degree of CB6 replacement increased with increasing ornithine concentration. Figure 1 A). Simultaneously, with increasing putrescine concentration, the degree of CB6 replacement increases ( Figure 1 B). Finally, in HCT116 cells, knockdown or overexpression of ODC1 resulted in significant changes in the fluorescence intensity of CB6 binding to DSMI. Figure 1 (C) The fluorescence intensity of CB6 binding to DSMI in the control group was 72150 AU, the fluorescence intensity of CB6 binding to DSMI in the ODC1 knockdown group was 166730 AU, and the fluorescence intensity of CB6 binding to DSMI in the ODC1 overexpression group was 43102 AU. With image observation, the absorbance changes were linear, and the response curve R... 2All values ​​are greater than 0.99, indicating a small variance, and the experimental values ​​in this group are stable and reliable.

Claims

1. A method for detecting ODC1 enzyme activity based on CB6 probe, characterized in that The method comprises the following steps: First step, the cells are digested with trypsin and centrifuged, resuspended with PBS and centrifuged again to discard the supernatant, and the cell pellet is added with lysis solution; Second step, after lysis, centrifugation is performed to take the supernatant, and the protein concentration is measured by BCA method; Third step, the enzyme activity detection working solution is added to the detection plate, and after equilibration, the sample is added, and after equilibration reaction, the fluorescence intensity is measured by multifunctional fluorescence enzyme label instrument; Fourth step, calculate the ODC1 enzyme activity.

2. The detection method of claim 1, wherein, The enzyme activity detection is realized by the fluorescence quenching principle of CB6 probe: ODC1 enzyme catalyzing ornithine to generate putrescine, putrescine replacing the substrate combined with the probe, resulting in change of fluorescence intensity, and the fluorescence intensity value indirectly reflects the ODC1 enzyme activity.

3. The detection method of claim 1, wherein, The trypsin concentration in the first step is 0.25%, and the digestion time is 2 minutes.

4. The detection method of claim 1, wherein, The centrifugation condition in the first step is 4°C, 3000 rpm for 5 minutes.

5. The method of claim 1, wherein, The volume of PBS in the first step is 200µL and needs to be pre-cooled at 4°C.

6. The detection method of claim 1, wherein, The composition of the lysis solution in the first step consists of 965µL Tris (10mM), 10µL EDTA (100mM) and 25µL DTT (100mM), which needs to be prepared and used immediately.

7. The method of claim 1, wherein The centrifugation condition in the second step is 4°C, 12000 rpm for 10 minutes.

8. The detection method of claim 1, wherein, In the second step, 5% of the sample is taken, and the protein concentration is detected by BCA kit. That is, 2µL of sample is added to each well, supplemented with PBS to 20µL, then 200µL of BCA working solution is added to each well, mixed gently, incubated at 37°C for 30 minutes, and the absorbance at 562nm wavelength is measured by enzyme label instrument. The sample OD value is substituted into the standard curve equation to calculate the sample protein concentration.

9. The method of claim 1, wherein, The composition of the enzyme activity detection working solution in the third step consists of 225µL Tris (10mM), 6µL DSMI (260µM) and 4µL CB6 (130µM), which needs to be prepared and used immediately.

10. The method of claim 1, wherein, The equilibration condition in the third step is 37°C for ten minutes.

11. The method of claim 1, wherein, The detection plate in the third step is a 96-well black enzyme label plate.

12. The detection method of claim 1, wherein, The sample composition in each well in the third step is 25µL of cell lysate, 2µL of ornithine solution (1mM) and 2µL of PLP (10µM).

13. The method of claim 1, wherein, The equilibration reaction condition in the third step is 37°C for fifty minutes.

14. The method of claim 1, wherein, The excitation wavelength and emission wavelength of the multifunctional fluorescence enzyme label instrument in the third step are 460nm and 590nm, respectively.

15. The method of claim 1, wherein, In the fourth step, the final CB6 combined substrate fluorescence intensity X is calculated as follows: X= (A x 0.0417) / (B-0.0622) In the formula: A: absorbance of sample at 460nm for 1 minute B: absorbance of sample at 562nm for 1 minute 0.0417: slope of BCA standard curve equation 0.0622: intercept of BCA standard curve equation.

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