Tyrosine-modified polymerized hemoglobin and preparation method thereof
By modifying tyrosine on polymerized hemoglobin, the problem of oxidative damage of red blood cell substitutes in the pathological state is solved, good antioxidant performance is achieved, and the occurrence of oxidative damage is reduced.
Patent Information
- Application Number
- CN202310680617.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-09
- Publication Date
- 2024-12-10
AI Technical Summary
In the pathological state, existing red blood cell substitutes cause oxidative damage due to the peroxidase activity of hemoglobin, and antioxidants such as vitamin C are costly and easily oxidized, making it difficult to effectively prevent oxidative damage.
By modifying tyrosine on polymerized hemoglobin, using tyrosine to react with hydrogen peroxide to consume hydrogen peroxide, weakening its oxidation effect on hemoglobin, and improving the solubility and modification efficiency of tyrosine through crosslinking agents, forming Tyrosine-modified polymeric hemoglobin with antioxidant properties.
It effectively reduces the oxidative damage of hemoglobin, improves the antioxidant performance, avoids the formation of pseudoperoxidase cycle, and significantly reduces the occurrence of oxidative damage.
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Figure CN119101146A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to polymerized hemoglobin and a preparation method thereof, in particular to tyrosine-modified polymerized hemoglobin. Background Art
[0002] Hemoglobin oxygen carrier is a red blood cell substitute developed with hemoglobin as the main raw material, and its main function is to carry oxygen. However, hemoglobin itself has pseudoperoxidase activity. Under pathological conditions, the hydrogen peroxide in the reactive oxygen species (ROS) it produces can react with hemoglobin (Hb), causing hemoglobin (HbFe 2+ ) is oxidized to methemoglobin (HbFe 3+ ) and HbFe 4+ , HbFe 4+ It has strong oxidizing properties and can cause various damages to the body. Driven by hydrogen peroxide, HbFe 3+ and HbFe 4+ It can circulate continuously, forming a pseudoperoxidase cycle, and also generating more HbFe2+ that does not have the ability to carry oxygen. 3+ Under pathological conditions, hydrogen peroxide has a high concentration, which can drive the pseudoperoxidase cycle and make HbFe 4+ It can be continuously produced, causing oxidative damage to the body.
[0003] At present, most red blood cell substitutes will cause the above-mentioned toxic side effects. The common solution is to use substances with antioxidant activity in combination with red blood cell substitutes. Vitamin C is a typical low molecular weight non-enzymatic antioxidant with strong antioxidant properties. However, vitamin C is easily oxidized when exposed to air, which aggravates oxidative damage. Hemoglobin catalase (CAT) and superoxide dismutase (SOD) have good antioxidant activity and help protect hemoglobin from free radical damage, but because human SOD and CAT are usually obtained by genetic recombination, the cost is relatively high. Summary of the invention
[0004] In view of this, one object of the present invention is to provide a tyrosine-modified polymerized hemoglobin. The tyrosine-modified polymerized hemoglobin has strong antioxidant properties. Another object of the present invention is to provide a method for preparing tyrosine-modified polymerized hemoglobin. The method can modify tyrosine on polymerized hemoglobin.
[0005] In one aspect, the present invention provides a tyrosine-modified polymerized hemoglobin, wherein the polymerized hemoglobin is modified with tyrosine; the hemoglobin monomers forming the polymerized hemoglobin are selected from one or more of hemoglobin or hemoglobin derivatives.
[0006] Ferrous hemoglobin has catalase activity, and tyrosine is an endogenous non-toxic compound and also a substrate of ferrous hemoglobin, which can catalyze the reaction between tyrosine and hydrogen peroxide, consume hydrogen peroxide, thereby weakening or eliminating the effect of hydrogen peroxide on oxidizing ferrous hemoglobin and preventing it from entering the pseudoperoxidase cycle. However, tyrosine is difficult to dissolve under physiological conditions, cannot be directly added for antioxidant effect, and is difficult to modify it on hemoglobin in large quantities. The present invention successfully modifies tyrosine on polymerized hemoglobin, so that the polymerized hemoglobin has good antioxidant properties.
[0007] According to the polymerized hemoglobin of the present invention, preferably, the hemoglobin derivative is selected from one or more of deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin and sulfhemoglobin.
[0008] In certain embodiments, the hemoglobin derivative is selected from one or more of deoxyhemoglobin and oxyhemoglobin.
[0009] Hemoglobin monomers can be derived from humans, animals or recombinant proteins. Animals are preferably mammals, including but not limited to cattle, pigs, sheep, etc.
[0010] According to the polymerized hemoglobin of the present invention, preferably, tyrosine is modified on the polymerized hemoglobin by a cross-linking agent, wherein the cross-linking agent is capable of reacting with tyrosine and -NH2 contained in the polymerized hemoglobin.
[0011] The cross-linking agent can be selected from one or more of polyaldehydes, ring-opened raffinose, and diaspirin.
[0012] In some embodiments, the crosslinking agent is a polyaldehyde. Preferably, the polyaldehyde is an alkylaldehyde or aromatic aldehyde containing 2 to 8 carbon atoms. A polyaldehyde refers to a chemical substance containing two or more aldehyde groups in a molecule. In some embodiments, it is a dialdehyde.
[0013] According to the polymerized hemoglobin of the present invention, preferably, the polyaldehyde is selected from one or more of glyoxal, malondialdehyde, succindialdehyde, glutaraldehyde, adipaldehyde and phthalaldehyde.
[0014] The polymerized hemoglobin according to the present invention is preferably polymerized from raw materials including hemoglobin monomers, a cross-linking agent and tyrosine.
[0015] The molar ratio of the hemoglobin monomer to the cross-linking agent is 1:(8-100); preferably 1:(10-60).
[0016] The molar ratio of tyrosine to the cross-linking agent is (0.7-25):1; preferably (1-20):1.
[0017] On the other hand, the present invention provides a method for preparing the above-mentioned tyrosine-modified polymerized hemoglobin, comprising the following steps: reacting a tyrosine alkaline solution with a cross-linking agent to form a first reaction product; reacting the first reaction product with a hemoglobin monomer. The types of the cross-linking agent and the hemoglobin monomer and the amounts of tyrosine, the cross-linking agent and the hemoglobin monomer are specifically as described above and will not be repeated here.
[0018] The present invention uses tyrosine alkaline solution as a reaction raw material, which helps to improve the solubility of tyrosine and promote the reaction between tyrosine and a cross-linking agent. The present invention first reacts the tyrosine alkaline solution with the cross-linking agent to form a first reaction product containing a tyrosine-cross-linking agent composite product and a cross-linking agent that has not reacted with tyrosine. The first reaction product is then reacted with a hemoglobin monomer, so that the cross-linking agent that has not reacted with tyrosine can polymerize the hemoglobin monomer to form polymerized hemoglobin, and the tyrosine-cross-linking agent composite product can be modified on the surface of the polymerized hemoglobin.
[0019] According to the preparation method of the present invention, preferably, the pH of the tyrosine alkaline solution is 7-10, and the concentration of the tyrosine alkaline solution is 2-100 mmol / L; the reaction time of the tyrosine alkaline solution and the cross-linking agent is 20-180 min; the hemoglobin monomer is used in the form of a solution, and the concentration of the hemoglobin monomer solution is 10-60 mg / mL.
[0020] The pH of the tyrosine alkaline solution is 7-10, preferably 8-10.
[0021] The concentration of tyrosine in the tyrosine alkaline solution is 2 to 100 mmol / L, preferably 20 to 50 mmol / L.
[0022] The cross-linking agent can be used in the form of a solution. The concentration of the cross-linking agent solution can be 10 to 800 mmol / L, preferably 100 to 500 mmol / L.
[0023] The reaction time of the tyrosine alkaline solution and the cross-linking agent can be 20 to 180 minutes, preferably 60 to 120 minutes. The reaction can be carried out on a rotary shaker.
[0024] The concentration of the hemoglobin monomer solution may be 10 to 60 mg / mL, preferably 20 to 50 mg / mL.
[0025] According to the preparation method of the present invention, preferably, the reaction product obtained by reacting the first reaction product with the hemoglobin monomer is reacted with a reducing agent to obtain a reduced product; and optionally, a step of removing small molecular substances in the reduced product.
[0026] The reducing agent can be selected from one or more of dimethylaminoborane, triethylaminoborane and sodium borohydride.
[0027] The molar ratio of the crosslinking agent to the reducing agent may be 1:(7-60); preferably 1:(10-20).
[0028] Small molecules in the reduction product can be removed by dialysis or ultrafiltration.
[0029] The counteracting solution can be selected from one or more of physiological saline, Ringer's solution, and lactated Ringer's solution.
[0030] The present invention provides a polymerized hemoglobin, on which tyrosine is modified. The polymerized hemoglobin of the present invention has good antioxidant properties. The present invention also provides a method for preparing the polymerized hemoglobin. The method can modify tyrosine on the surface of the polymerized hemoglobin, thereby improving the antioxidant properties of the polymerized hemoglobin. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is the molecular weight distribution diagram of the polymerized hemoglobin of Example 1.
[0032] Figure 2 This is the molecular weight distribution diagram of the polymerized hemoglobin of Example 2.
[0033] Figure 3 This is the molecular weight distribution diagram of the polymerized hemoglobin of Example 3.
[0034] Figure 4 The UV spectra of the polymerized hemoglobin of Comparative Example 1 after reacting with hydrogen peroxide for different time periods.
[0035] Figure 5 The UV spectra of the polymerized hemoglobin of Example 1 after reacting with hydrogen peroxide for different time periods.
[0036] Figure 6 The UV spectra of the polymerized hemoglobin of Comparative Example 2 after reacting with hydrogen peroxide for different time periods.
[0037] Figure 7 The UV spectra of the polymerized hemoglobin of Example 2 after reacting with hydrogen peroxide for different time periods.
[0038] Figure 8 The UV spectra of the polymerized hemoglobin of Comparative Example 3 after reacting with hydrogen peroxide for different time periods.
[0039] Fig. 9 The UV spectra of the polymerized hemoglobin of Example 3 after reacting with hydrogen peroxide for different time periods. DETAILED DESCRIPTION
[0040] Example 1
[0041] (1) Adding a 100 mmol / L glutaraldehyde solution to 200 mL of a 20 mmol / L tyrosine solution having a pH of 8, until the molar ratio of tyrosine to glutaraldehyde is 1:1, to obtain a first mixed solution. The first mixed solution is reacted on a rotary shaker for 60 minutes to form a first reaction product.
[0042] Human oxygenated hemoglobin at a concentration of 50 mg / mL was added to the first reaction product until the molar ratio of human oxygenated hemoglobin to glutaraldehyde was 1: 13. After the reaction between the first reaction product and human oxygenated hemoglobin was completed, a second reaction product was obtained.
[0043] (2) Adding a 10 wt% dimethylaminoborane solution to the second reaction product for reduction reaction to obtain a reduction product. The molar ratio of glutaraldehyde to dimethylaminoborane is 1:10. The reduction product is placed in a dialysis bag, and the counter solution is physiological saline. The solution is changed once every 4 hours, and a total of four times, to obtain tyrosine-modified polymerized hemoglobin.
[0044] Comparative Example 1
[0045] Except step (1) as shown below, the rest is the same as in Example 1:
[0046] A 50 mg / mL human oxyhemoglobin solution was added to a 100 mmol / L glutaraldehyde solution until the molar ratio of human oxyhemoglobin to glutaraldehyde was 1: 13. After the reaction of glutaraldehyde and human oxyhemoglobin was completed, a second reaction product was obtained.
[0047] Example 2
[0048] (1) Adding a 500 mmol / L succinaldehyde solution to 200 mL of a 50 mmol / L tyrosine solution having a pH of 9, until the molar ratio of tyrosine to succinaldehyde is 20:1, to obtain a first mixed solution. The first mixed solution is reacted on a rotary shaker for 90 minutes to form a first reaction product.
[0049] A bovine deoxyhemoglobin solution with a concentration of 40 mg / mL was added to the first reaction product until the molar ratio of bovine deoxyhemoglobin to succinaldehyde was 1:39. After the reaction of the first reaction product and the bovine deoxyhemoglobin was completed, a second reaction product was obtained.
[0050] (2) Sodium borohydride is added to the second reaction product for reduction reaction to obtain a reduction product. The molar ratio of succinaldehyde to sodium borohydride is 1:10. The reduction product is passed through an ultrafiltration fiber pile with a pore size of 100 kDa to remove small molecules, and the counter solution is Ringer's solution to obtain tyrosine-modified polymerized hemoglobin.
[0051] Comparative Example 2
[0052] Except step (1) as shown below, the rest is the same as in Example 2:
[0053] (1) Adding a 40 mg / mL bovine deoxyhemoglobin solution to a 500 mmol / L succinaldehyde solution until the molar ratio of bovine deoxyhemoglobin to succinaldehyde is 1:39. After the reaction of succinaldehyde and bovine deoxyhemoglobin is completed, a second reaction product is obtained.
[0054] Example 3
[0055] (1) Adding a 180 mmol / L malondialdehyde solution to 200 mL of a 100 mmol / L tyrosine solution having a pH of 10, until the molar ratio of tyrosine to malondialdehyde is 5:1, to obtain a first mixed solution. The first mixed solution is reacted on a rotary shaker for 120 min to form a first reaction product.
[0056] A hemoglobin monomer solution with a concentration of 20 mg / mL is added to the first reaction product until the molar ratio of the hemoglobin monomer to malondialdehyde is 1:52. In the hemoglobin monomer solution, the hemoglobin monomer is composed of porcine oxyhemoglobin and porcine deoxyhemoglobin in a mass ratio of 1:1. After the reaction of the first reaction product and the hemoglobin monomer is completed, a second reaction product is obtained.
[0057] (2) Adding a 10 wt% dimethylaminoborane solution to the second reaction product for reduction reaction to obtain a reduction product. The molar ratio of malondialdehyde to dimethylaminoborane is 1:10. The reduction product is placed in a dialysis bag, and the counter solution is lactated Ringer's solution. The solution is changed once every 4 hours, and a total of four times, to obtain tyrosine-modified polymerized hemoglobin.
[0058] Comparative Example 3
[0059] Except step (1) as shown below, the rest is the same as in Example 3:
[0060] (1) Adding a hemoglobin monomer solution with a concentration of 20 mg / mL to a malondialdehyde solution with a concentration of 180 mmol / L, until the molar ratio of the hemoglobin monomer to the malondialdehyde is 1:52. In the hemoglobin monomer solution, the hemoglobin monomer is composed of porcine oxyhemoglobin and porcine deoxyhemoglobin in a mass ratio of 1:1. After the reaction of the malondialdehyde solution and the hemoglobin monomer is completed, a second reaction product is obtained.
[0061] Experimental Example 1
[0062] 1. Molecular weight distribution of polymerized hemoglobin: PB with a concentration of 20 mM and a pH of 7.4 was used as the mobile phase of the fully automatic nucleic acid protein purification instrument for determination. The sample determination concentration was 1 μM and the volume was 35 μL. The molecular weight distribution of polymerized hemoglobin in Examples 1-3 is shown in FIG. Figure 1-3 As shown. Figure 1-3 The content of hemoglobin polymer in the aggregated hemoglobin to the total hemoglobin is calculated as shown in Table 1.
[0063] Table 1
[0064]
[0065] 2. Using an ultraviolet spectrophotometer, the absorbance of the polymerized hemoglobin of the embodiment and the comparative example was measured under the condition that the wavelength of ultraviolet visible light was 280nm. The sample concentration was 1mM. The average absorbance of each polymerized hemoglobin (n=3) is shown in Table 2.
[0066] Table 2
[0067]
[0068] It can be seen from Table 1 that the content of hemoglobin polymer in the polymerized hemoglobin of the present invention is relatively high, and more than 78% of the hemoglobin monomers participate in the polymerization.
[0069] The molecular weight of the polymer product is proportional to the polymerization effect. The better the tyrosine modification effect, the larger the molecular weight of the polymer product. As shown in Table 2, the absorbance of the examples is higher than that of the comparative examples, indicating that the present invention can modify tyrosine on hemoglobin.
[0070] Experimental Example 2
[0071] Antioxidant performance: The corresponding polymerized hemoglobin of the embodiment and the comparative example was diluted to the same concentration as the test raw material; embodiment 1 corresponds to comparative example 1, embodiment 2 corresponds to comparative example 2, and embodiment 3 corresponds to comparative example 3. Hydrogen peroxide solution and ultrapure water were added to the test raw material to obtain a test sample. The concentration of total hemoglobin in the test sample was 1 μM, and the concentration of hydrogen peroxide was 100 μM. The spectra of the test sample at 0 min and 30 min of reaction were tested using an ultraviolet spectrophotometer. The obtained spectra are shown in FIG. Figures 4 to 9 shown.
[0072] Depend on Figures 4 to 9It can be seen that after the polymerized hemoglobin of the comparative example reacted with hydrogen peroxide for 30 minutes, its characteristic absorption peaks at 542nm and 577nm were significantly reduced, and the characteristic absorption peak at 630nm was significantly increased, which indicates that the ferrous hemoglobin was highly oxidized by hydrogen peroxide. After the polymerized hemoglobin of the embodiment reacted with hydrogen peroxide for 30 minutes, its characteristic absorption peaks at 542nm and 577nm were significantly reduced to a lesser extent than the corresponding comparative example, and the degree of increase of the characteristic absorption peak at 630nm was also significantly less than the corresponding comparative example. This shows that the polymerized hemoglobin of the present invention has good antioxidant activity after tyrosine modification.
[0073] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.
Claims
1. A tyrosine-modified polymerized hemoglobin, characterized in that: The polymerized hemoglobin is modified with tyrosine; the hemoglobin monomers forming the polymerized hemoglobin are selected from one or more of hemoglobin or hemoglobin derivatives.
2. The polymerized hemoglobin according to claim 1, characterized in that Tyrosine is modified on polymerized hemoglobin by a cross-linking agent; the cross-linking agent reacts with tyrosine and -NH2 contained in polymerized hemoglobin.
3. The polymerized hemoglobin according to claim 2, characterized in that The hemoglobin derivative is selected from one or more of deoxyhemoglobin, oxyhemoglobin, carboxyhemoglobin, and sulfhemoglobin; The cross-linking agent is selected from one or more of polyaldehydes, open-ring raffinose, and diaspirin.
4. The polymerized hemoglobin according to claim 3, characterized in that The polyaldehyde is an alkyl aldehyde or aromatic aldehyde containing 2 to 8 carbon atoms.
5. The polymerized hemoglobin according to claim 4, characterized in that The polyaldehyde is selected from one or more of glyoxal, malondialdehyde, succinaldehyde, glutaraldehyde, adipaldehyde and phthalaldehyde.
6. The polymerized hemoglobin according to any one of claims 1 to 5, characterized in that The polymerized hemoglobin is polymerized from raw materials including hemoglobin monomers, a cross-linking agent and tyrosine; The molar ratio of the hemoglobin monomer to the cross-linking agent is 1:(8-100), and the molar ratio of the tyrosine to the cross-linking agent is (0.7-25):
1.
7. The method for preparing polymerized hemoglobin according to any one of claims 1 to 6, characterized in that: The steps include: The tyrosine alkaline solution is reacted with a cross-linking agent to form a first reaction product; and the first reaction product is reacted with a hemoglobin monomer.
8. The preparation method according to claim 7, characterized in that: The pH of the tyrosine alkaline solution is 7-10, and the concentration of the tyrosine alkaline solution is 2-100 mmol / L; the reaction time of the tyrosine alkaline solution and the cross-linking agent is 20-180 minutes; the hemoglobin monomer is used in the form of a solution, and the concentration of the hemoglobin monomer solution is 10-60 mg / mL.
9. The preparation method according to claim 7, characterized in that: The following steps are also included: The step of reacting the reaction product obtained by reacting the first reaction product with the hemoglobin monomer with a reducing agent to obtain a reduced product; and optionally, removing small molecular substances in the reduced product.
10. The preparation method according to claim 9, characterized in that: The reducing agent is selected from one or more of dimethylaminoborane, triethylaminoborane, and sodium borohydride, and the molar ratio of the crosslinking agent to the reducing agent is 1:(7-60); dialysis or ultrafiltration is used to remove small molecules in the reduction product, and the counter solution used is selected from one or more of physiological saline, Ringer's solution, and lactated Ringer's solution.