Electromagnetic base solution diluent, diluent preparation method and method for treating phosphorylated protein by using diluent

Abstract

The invention belongs to the technical field of biochemistry, and discloses an electromagnetic base solution diluent, a preparation method of the electromagnetic base solution diluent and application of the electromagnetic base solution diluent in protein phosphorylation treatment. The electromagnetic base liquid diluent comprises an electromagnetic base liquid and deionized water, the electromagnetic base liquid prepared in the patent US 11691906B1 is adopted as the electromagnetic base liquid, and the molar ratio of the deionized water to the electromagnetic base liquid is 1000 + / -50: 1; the numerical value of a hydrogen bond network characteristic peak generated by stretching vibration of Raman spectrum O-H of the diluent is 3000-3600 cm, the pH value range is 11.5-12, and the ORP value is-150 + / -10 mV. The phosphorylation level is adjusted through an accurate electron transfer process, and the method is milder and controllable; the reaction can be efficiently carried out under mild conditions, the energy consumption is low, and the reaction time is short; the method shows higher selectivity and better protein activity retention rate.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and in particular relates to an electromagnetic base liquid diluent, a preparation method of the electromagnetic base liquid diluent and an application of the electromagnetic base liquid diluent in protein phosphorylation treatment. Background Art

[0002] Protein phosphorylation is an important post-translational modification that plays a key role in cell signal transduction and protein function regulation. Abnormal protein phosphorylation is associated with a variety of diseases, especially in neurodegenerative diseases, where overphosphorylated Tau proteins form pathological aggregates. In the prior art, methods for treating abnormally phosphorylated proteins mainly include the use of phosphatase inhibitors or chemical modifiers, but these methods often have problems such as poor selectivity and large side effects. Summary of the invention

[0003] The purpose of the present invention is to provide an electromagnetic base liquid diluent and a method for selectively regulating protein phosphorylation by using the diluent.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: an electromagnetic base liquid diluent, comprising an electromagnetic base liquid and deionized water, wherein the electromagnetic base liquid adopts the electromagnetic base liquid prepared in US 11691906B1 patent, and the molar ratio of deionized water to the electromagnetic base liquid is 1000±50:1; the Raman spectrum of the diluent has a hydrogen bond network characteristic peak value generated by OH stretching vibration at 3000-3600cm⁻¹, a pH range of 11.5-12, and an ORP value of -150±10 mV.

[0005] The stable electromagnetic base fluid described in US 11691906B1 is essentially a stable hydrated electron base fluid, which is a good reducing agent, but it cannot be directly applied to protein phosphorylation. The present invention has found through experiments that in an aqueous solution environment, the hydrated electrons (eaq-) in the electromagnetic base fluid are surrounded by water molecules to form a special solvation structure. According to Raman spectroscopy analysis, when the solution is diluted 1000±50 times, a more stable hydrogen bond network can be formed. This reconstructed hydrogen bond network provides a unique reaction environment for the electron transfer process. The reconstructed hydrogen bond network has the following characteristics: an ordered long-range hydrogen bond network structure is formed, which provides a stable quantum channel for electrons, and the characteristic peak of the hydrogen bond network is in the range of 3000-3600 cm⁻¹ (Raman spectrum OH stretching vibration). This structure is a special metastable state formed between water molecules. The network is in a precisely controlled energy window with a reduction potential of about -150mV. Through this reconstruction, a solvation environment suitable for electron transfer is formed.

[0006] A method for preparing an electromagnetic base liquid diluent comprises the following steps: step 1: diluting the electromagnetic base liquid with deionized water under an inert atmosphere, with a dilution factor of 1000±50; step 2: stopping the dilution when the characteristic peak value of the hydrogen bond network generated by the OH stretching vibration of the Raman spectrum is between 3000 and 3600 cm⁻¹, the pH value range is between 11.5 and 12, and the ORP value is between -150±10 mV.

[0007] The method for treating phosphorylated protein with electromagnetic base liquid diluent comprises the following steps: step 1: mixing and stirring the phosphorylated protein with the electromagnetic base liquid diluent to fully dissolve the phosphorylated protein in the electromagnetic base liquid diluent; monitoring the pH value and ORP value of the mixed solution in real time, wherein the pH value ranges from 11.5 to 12 and the ORP value is -150±10 mV; and step 2: obtaining the treated protein by separation and purification.

[0008] The reconstructed hydrogen bond network environment provides a stable solvation environment for hydrated electrons, allowing them to maintain a high reduction activity; and the hydrated electrons preferentially interact with the phosphate groups with high electron affinity, making the reduction reaction have the characteristics of selective recognition; and this environment is conducive to electron transfer. Through the quantum tunneling effect, the hydrated electrons are transferred to the phosphate group to form an unstable intermediate; electron transfer causes the electron cloud density of the phosphate group to change, affecting its interaction with the protein, thereby achieving the regulation of phosphorylation modification.

[0009] Preferably, the mixing and stirring parameters in step 1 are: reaction temperature: 37±0.5°C; mass ratio of electromagnetic base liquid diluent to phosphorylated protein: 1:3 to 1:10; stirring time: 30-60 minutes; stirring rate: 100-200 rpm.

[0010] Specifically, the separation and purification in step 2 is carried out by dialysis, ultrafiltration, and fast protein liquid chromatography FPLC.

[0011] The advantages of the present invention are: 1. Difference in treatment mechanism: Traditional enzymatic treatment relies on the catalytic action of phosphatase, which may bring more non-specific effects. The diluent of the present invention adjusts the phosphorylation level through a precise electron transfer process, which is more gentle and controllable. 2. Comparison of reaction conditions: Traditional methods often require a long reaction time, are inefficient, and have more stringent requirements on reaction conditions. The present invention can be carried out efficiently under mild conditions, with less energy consumption and short reaction time. 3. Treatment effect: The present invention exhibits higher selectivity and better protein activity retention rate. The selectivity of traditional methods is relatively low, and may have a greater impact on the overall structure of the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1It is a comparison chart of the chemical shift changes of characteristic hydrogen in the electromagnetic base liquid dilution at different dilution concentrations.

[0013] Figure 2 This is a comparison chart of the Raman spectrum analysis of the electromagnetic base liquid dilution at different dilution concentrations.

[0014] Figure 3 It is a reaction mechanism diagram of the present invention.

[0015] Figure 4 It is a comparison diagram of the treatment effects of the present invention. DETAILED DESCRIPTION

[0016] To facilitate the understanding of the present invention, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments.

[0017] like Figure 1 As shown, the instrument used for the test is Bruker AVANCEIIIHD400, and the test item is nuclear magnetic resonance. First, we prepare the electromagnetic base liquid sample and use deionized water as the dilution solvent to prepare samples A, B, C, and D of different concentrations.

[0018] A: stock solution B: stock solution: water = 1:100 C: stock solution: water = 1:500 D: stock solution: water = 1:1000 Through the 1H of four samples NMR, we can see that the chemical shift of characteristic hydrogen of sample A in the original solution is 4.58ppm. After dilution with deionized water, the characteristic hydrogen in the sample moves to the high field in turn. However, when the dilution factor reaches 1000, a rebound occurs, and the chemical shift of characteristic hydrogen moves to the low field to 4.23ppm. It can be seen that sample A is the original solution, and there is a strong hydrogen bond interaction between ions and water molecules. When diluted with water, the newly added water molecules will form new hydrogen bonds with the original ions and water molecules. This process destroys the original hydrogen bonds, resulting in a decrease in the number of hydrogen bonds, that is, sample B and sample C. However, as the number of water molecules in the system gradually increases, more new hydrogen bonds will be formed between water molecules and between water molecules and ions, causing the total number of hydrogen bonds in the system to gradually increase until a new equilibrium is reached. At this time, the strength of water-water hydrogen bonds increases, more hydrogen bonds are formed between water molecules, the electron cloud around the hydrogen nucleus is pulled away by hydrogen bonds, the electron cloud density decreases, the deshielding effect increases, the chemical shift moves to the low field, and the chemical shift reversal phenomenon occurs. The hydrogen bonding effect in the system is enhanced, and the system is more stable.

[0019] like Figure 2As shown, the instrument used in the test is a Raman spectrometer, and the test item is Raman spectroscopy. First, we prepare the electromagnetic base liquid sample and use deionized water as the dilution solvent to prepare samples A, B, C, and D of different concentrations.

[0020] A: stock solution B: stock solution: water = 1:100 C: stock solution: water = 1:500 D: stock solution: water = 1:1000 In the Raman spectrum of sample A, the wave numbers are 2982 and 2938 cm -1 The double peaks are respectively attributed to the antisymmetric stretching vibration and symmetric stretching vibration peaks of the methyl CH in the sample, 2887 cm -1 The peak is the CH stretching vibration peak of the methylene group, 1646 cm -1 The weak peaks at 1026 and 928 cm are HOH deformation vibration peaks. -1 The peak is the CO stretching vibration peak, 774 cm -1 The peak is the CH plane swing vibration peak in the long-chain hydrocarbon group, 598 cm -1 The peaks may be generated by charge-assisted hydrogen bonds or electrostatic interactions between anions and cations. The Raman spectra of samples B, C, and D are basically consistent with those of sample A, and the positions of each characteristic peak remain unchanged. However, the stretching vibration peak of the saturated hydrocarbon group basically disappears after dilution, indicating that the organic components in the sample are diluted. The peak at 1646 cm -1 The HOH deformation vibration peak at 1026 cm -1 The CO stretching vibration peak at 1096 cm -1 This indicates that the electron cloud on the CO bond tends to concentrate on the CO bond itself, reducing the attraction to the hydrogen atoms in the hydrogen bond, resulting in a decrease in the strength of the hydrogen bond. However, the degree of decrease does not show a positive correlation with the decrease in system concentration.

[0021] like Figure 3 Shown is a reaction mechanism diagram of the present invention.

[0022] From the perspective of quantum chemistry, the electron transfer process can be described by Marcus theory. The rate constant k of the process of electron transfer from hydrated electrons to phosphate groups can be expressed as: k = (2π / ħ)|V|²(4πλkBT)^(-1 / 2)exp[-(ΔG° + λ)² / 4λkBT] in: ħ - Reduced Planck constant V - Electronic coupling matrix element λ - reorganization energy ΔG° - standard free energy change of the reaction kB - Boltzmann constant T - Temperature Under dilute conditions, the electron transfer process can be written in the following basic steps: eaq-(H2O)n + RO-PO3²- → [eaq-···RO-PO3²-](H2O)n → RO-PO3³-·+ H2O In this process, hydrated electrons first form a precursor complex with the phosphate group, and then electron transfer occurs. The reconstructed hydrogen bond network plays a key role in this process: it provides an ordered solvation environment for electron transfer and reduces the activation energy barrier of the reaction.

[0023] A specific chemical equation can be represented as a series of equilibrium and electron transfer steps: 1. Solvation equilibrium: eaq- + nH2O ⇌ eaq-(H2O)n K1 2. Precursor complex formation: eaq-(H2O)n + RO-PO3²- ⇌ [eaq-···RO-PO3²-](H2O)n K2 3. Electron transfer: [eaq-···RO-PO3²-](H2O)n → RO-PO3³-· + H2O k3 4. Subsequent rearrangement: RO-PO3³-· → RO· + PO3²- k4 Where K1 and K2 are equilibrium constants, and k3 and k4 are rate constants. At pH 12, these reactions are affected by OH- ions: OH- + RO-PO3²- ⇌ RO-PO3³- + H2O KOH Such alkaline conditions favor the deprotonation of the phosphate group, making it more receptive to electrons.

[0024] Under 1000-fold dilution conditions, the overall rate equation of the reaction can be expressed as: -d[eaq-] / dt = kobs[eaq-][RO-PO3²-] where kobs is the observed rate constant, which is related to the various equilibrium and rate constants mentioned above: kobs = K1K2k3 / (1 + K1[H2O]) This expression shows that the reaction rate is affected by the solvation equilibrium, precursor complex formation and electron transfer steps. The reconstructed hydrogen bond network after dilution regulates the reaction rate by affecting K1 and K2.

[0025] The free energy change ΔG of the electron transfer process can be estimated using the redox potential: ΔG = -nF(E°acceptor - E°donor) Where F is the Faraday constant, n is the number of transferred electrons, and E° is the standard redox potential.

[0026] This mild electron transfer environment is particularly suitable for processing phosphorylated proteins because: 1. The reaction rate is moderate, avoiding excessively fast non-selective reactions; 2. The hydrogen bond network provides a stable solvation environment; 3. The electron transfer process is more controllable, which is conducive to maintaining the overall structure of the protein; Through this complete reaction mechanism, we can understand why a 1000-fold diluted aqueous electron solution can effectively act on phosphorylated proteins while maintaining the selectivity and controllability of the reaction. This provides a theoretical basis for the development of more effective treatment methods.

[0027] Example 1: Treatment of phosphorylated GSK-3β protein Step 1: Under an inert atmosphere, the electromagnetic base liquid is diluted with deionized water at a dilution factor of 950 times (i.e., the molar ratio of deionized water to electromagnetic base liquid is 950:1); the electromagnetic base liquid is prepared using the patented method of US 11691906B1. The inert atmosphere helps prevent external oxygen from affecting the ORP value.

[0028] Step 2: When the characteristic peak value of the hydrogen bond network generated by the OH stretching vibration in the Raman spectrum is at 3000 cm⁻¹, the dilution is stopped to obtain an electromagnetic base liquid dilution with a pH value of 12 and an ORP value of -140 mV.

[0029] Step 3: Use a water bath, and control the water bath temperature at 37°C. This temperature can ensure the stability of the pH and ORP values ​​during the reaction. Pour the electromagnetic base liquid diluent and phosphorylated protein in a mass ratio of 1:3 into the container, stir at a rate of 200rpm for 30 minutes, and monitor the pH and ORP values ​​of the mixed liquid in real time during the stirring process; although only the diluent and protein are mixed, these parameters still need to be monitored because they may change naturally over time, especially during the interaction between proteins and hydrated electrons. If changes occur, the changes can be controlled by changing the stirring speed. However, in this experiment, the pH and ORP values ​​remained stable throughout the stirring process, that is, the pH value was 12 and the ORP value was -140 mV.

[0030] Step 4: obtain the treated protein by separation and purification through dialysis.

[0031] like Figure 4 As shown, the traditional enzymatic treatment has a reaction time of 24 hours, a selectivity of 70%, and a protein activity retention rate of 60%. However, the reaction time of this experiment is half an hour, the selectivity is 90%, and the activity retention rate of the protein obtained after treatment is 85%.

[0032] Example 2: Treatment of phosphorylated Tau protein Step 1: Under an inert atmosphere, the electromagnetic base liquid is diluted with deionized water at a dilution factor of 1050 times (i.e., the molar ratio of deionized water to the electromagnetic base liquid is 1050:1); the electromagnetic base liquid is prepared using the patented method of US 11691906B1. The inert atmosphere helps prevent external oxygen from affecting the ORP value.

[0033] Step 2: When the characteristic peak value of the hydrogen bond network generated by the OH stretching vibration in the Raman spectrum is at 3400 cm⁻¹, the dilution is stopped to obtain an electromagnetic base liquid dilution with a pH value of 11.7 and an ORP value of -150 mV.

[0034] Step 3: Use a water bath with the temperature controlled at 36.5°C, which can ensure the stability of the pH and ORP values ​​during the reaction. Pour the electromagnetic base liquid diluent and phosphorylated protein in a mass ratio of 1:5 into the container and stir at a rate of 110 rpm for 47 minutes. Monitor the pH and ORP values ​​of the mixture in real time during the stirring process. In this experiment, the pH and ORP values ​​remained stable throughout the stirring process, that is, the pH value was 11.7 and the ORP value was -150 mV.

[0035] Step 4: Separate and purify the treated protein by ultrafiltration.

[0036] The protein activity retention rate obtained in this experiment was 87%, and the selectivity of hydrated electrons for phosphorylated proteins was 94%.

[0037] Example 3: Treatment of phosphorylated p53 protein Step 1: Under an inert atmosphere, the electromagnetic base liquid is diluted with deionized water by 1000 times (i.e., the molar ratio of ionized water to electromagnetic base liquid is 1000:1); the electromagnetic base liquid is prepared by the patented method of US 11691906B1. The inert atmosphere helps prevent external oxygen from affecting the ORP value.

[0038] Step 2: When the characteristic peak value of the hydrogen bond network generated by the OH stretching vibration in the Raman spectrum is at 3600 cm⁻¹, the dilution is stopped to obtain an electromagnetic base liquid dilution with a pH value of 11.5 and an ORP value of -160 mV.

[0039] Step 3: Use a water bath with the temperature controlled at 37.5°C, which can ensure the stability of the pH and ORP values ​​during the reaction. Pour the electromagnetic base liquid dilution and phosphorylated protein in a mass ratio of 1:10 into the container and stir at a rate of 100 rpm for 60 minutes. Monitor the pH and ORP values ​​of the mixture in real time during the stirring process. In this experiment, the pH and ORP values ​​remained stable throughout the stirring process, that is, the pH value was 11.5 and the ORP value was -160 mV.

[0040] Step 4: Separate and purify the treated protein by FPLC.

[0041] The treated p53 protein was collected by using an anion exchange column (Q Sepharose) with 20mM Tris-HCl (pH 8.0) as buffer and 0-500mM NaCl gradient elution at a flow rate of 1ml / min. Result verification: The phosphorylation level of p53 protein was detected by mass spectrometry analysis and phosphorylation-specific antibodies. The results showed that the phosphorylation levels of Ser15 and Ser20, which are related to DNA damage response, decreased by 95% and 93%, respectively, while the phosphorylation level of Ser46, which is related to transcriptional activation, decreased by only 7%, indicating that this method has a high site selectivity. The DNA binding activity assay of the treated p53 protein showed that the DNA binding affinity was increased by 3.2 times and the transcriptional activation ability was increased by 2.8 times compared with the untreated phosphorylated p53. Protein thermal stability analysis showed that the Tm value of the treated p53 protein increased from 42.3℃ to 47.8℃, indicating that the structural stability was significantly enhanced.

[0042]

[0043] In summary, the present invention has the following advantages: 1. Difference in treatment mechanism: Traditional enzymatic treatment relies on the catalytic action of phosphatase, which may bring more non-specific effects. The diluent of the present invention adjusts the phosphorylation level through a precise electron transfer process, which is more gentle and controllable. 2. Comparison of reaction conditions: Traditional methods often require a long reaction time, are inefficient, and have more stringent requirements on reaction conditions. The present invention can be carried out efficiently under mild conditions, with less energy consumption and short reaction time. 3. Treatment effect: The present invention exhibits higher selectivity and better protein activity retention rate. The selectivity of traditional methods is relatively low, and may have a greater impact on the overall structure of the protein.

Claims

1. An electromagnetic base liquid diluent, characterized in that: It includes an electromagnetic base liquid and deionized water, wherein the electromagnetic base liquid is the electromagnetic base liquid prepared in US11691906B1, and the molar ratio of deionized water to the electromagnetic base liquid is 1000±50:1; the Raman spectrum of the diluted liquid has a hydrogen bond network characteristic peak value generated by OH stretching vibration at 3000-3600cm⁻¹, a pH range of 11.5-12, and an ORP value of -150±10 mV.

2. A method for preparing an electromagnetic base liquid diluent, characterized in that: The following steps are involved: Step 1: Under an inert atmosphere, dilute the electromagnetic base liquid with deionized water at a dilution factor of 1000±50; Step 2: When the characteristic peak value of the hydrogen bond network generated by the OH stretching vibration in the Raman spectrum is between 3000-3600cm⁻¹, the pH value range is 11.5-12, and the ORP value is -150±10 mV, stop dilution.

3. A method for treating phosphorylated protein using the electromagnetic base liquid diluent according to claim 1, characterized in that: Step 1: Mix and stir the phosphorylated protein with the electromagnetic base liquid diluent to fully dissolve the phosphorylated protein in the electromagnetic base liquid diluent; monitor the pH value and ORP value of the mixed solution in real time, the pH value range is 11.5-12, and the ORP value is -150±10mV; Step 2: Obtain the processed protein by separation and purification.

4. The method for treating phosphorylated protein with an electromagnetic base liquid diluent according to claim 3, characterized in that: The parameters of mixing and stirring in step 1 are: Reaction temperature: 37±0.5℃; Mass ratio of electromagnetic base diluent to phosphorylated protein: 1:3 to 1:10; Mixing time: 30-60 minutes Stirring rate: 100-200rpm.

5. The method for treating phosphorylated protein with an electromagnetic base liquid diluent according to claim 3, characterized in that: The separation and purification in step 2 is carried out by dialysis, ultrafiltration, and fast protein liquid chromatography FPLC.

Images