An oxygen carrying protein molecule and its preparation method and application

Abstract

This invention provides an oxygen-carrying protein molecule, its preparation method, and its applications, relating to the field of functional protein preparation technology. The oxygen-carrying protein molecule comprises at least one cysteine ​​moiety and at least one histidine moiety, wherein the cysteine ​​moiety comprises an acetyl or ethylmaleyl group, and the histidine moiety comprises a benzimidazole derivative group. This invention uses a benzimidazole derivative as an auxiliary molecule to participate in the modification process of hemoglobin by thiol-modified compounds, improving the selectivity of modification sites, reducing conformational changes, and precisely controlling the degree of modification, thereby effectively improving the oxygen-binding capacity (P50) of modified hemoglobin.

Description

Technical Field

[0001] This invention provides an oxygen-carrying protein molecule, its preparation method, and its application, relating to the field of functional protein preparation technology. Background Technology

[0002] Hemoglobin (Hb), a vital functional protein within red blood cells, plays crucial roles in maintaining life processes, including transporting oxygen, removing carbon dioxide, breaking down hydrogen peroxide, and transferring electrons. With the continuous development of medicine and biotechnology, the research and application of hemoglobin have become increasingly widespread, particularly in areas such as enhancing its oxygen-carrying capacity and developing novel blood substitutes, demonstrating immense potential and prospects. Hemoglobin consists of four subunits, each containing a heme group, which reversibly binds oxygen molecules. Hemoglobin exhibits an S-shaped oxygenation curve, a characteristic that allows it to efficiently absorb oxygen in the lungs and rapidly release it into tissues, thus meeting the body's oxygen demands. Furthermore, hemoglobin possesses allosteric properties; when one subunit binds to oxygen, it promotes the binding of oxygen to other subunits, a synergistic effect that significantly improves oxygen transport efficiency. In various disease states, such as anemia and shock, the body's oxygen demand increases dramatically, and traditional treatments often struggle to meet this demand quickly enough. Therefore, developing novel oxygen-carrying protein molecules, especially those that enhance the oxygen-binding capacity of hemoglobin through chemical modification, has become an important research direction. The significance of this technology lies in: improving treatment efficiency: In emergency treatments, such as shock resuscitation, hemoglobin with enhanced oxygen-binding capacity can rapidly replenish tissue oxygen supply, increasing the success rate of treatment. Reducing transfusion dependence: For patients requiring large transfusions, novel oxygen-carrying protein molecules can serve as blood substitutes, reducing blood shortages. Expanding application areas: Besides the medical field, this technology can also be applied to oxygen supply in special environments such as high-altitude operations, diving, and aerospace.

[0003] Currently, some progress has been made in the research of site-specific chemical modification of specific amino acids in hemoglobin molecules to enhance oxygen-binding capacity. Hemoglobin is a tetramer composed of two alpha subunits and two beta subunits. In the hemoglobin molecule, the active free cysteine ​​contains a sulfhydryl group, which can be easily modified site-specifically through controlled experimental conditions. The number of free cysteine ​​residues that can be modified site-specifically varies among different animals. (The paper by Acharya SA et al.) Biochem.J.Table 2 (2007;405:503-511) shows that, in the oxygen-bound state, human hemoglobin molecules have two active free sulfhydryl groups, dog, chicken, sheep, and mouse molecules each have four, while cat molecules have six. Chinese patent application No. 200710089647.6 from AGCO Inc. discloses that the cysteine ​​moiety of hemoglobin contains a sulfhydryl protecting group, reducing the ability of the cysteine ​​site to bind nitric oxide. The resulting hemoglobin oxygen-binding capacity P50 ranges from approximately 20 mmHg to 45 mmHg, and the reagent used to provide the sulfhydryl protecting group is iodoacetamide. In this patent application, the hemoglobin molecule undergoes additional intramolecular cross-linking; therefore, the oxygen-carrying protein differs in molecular structure and oxygen-binding capacity. (Paper: Garel M.C. et al.) Eur.J.Biochem. 1982;123:513-9 tested the effects of 21 thiol-modified compounds (including iodoacetamide and ethylmaleimide) on human hemoglobin. The data showed that the oxygen-binding capacity (P50) was not enhanced after modification with multiple compounds, suggesting that the different spatial structures of the modified proteins may be the reason for the different oxygen-binding capacities.

[0004] Modifying the active free cysteine ​​residues of hemoglobin with thiol-modifying compounds such as iodoacetamide or ethyl maleimide can enhance its oxygen-binding capacity. However, in some cases, this modification does not achieve the desired effect. The reasons for this may include: **Selectivity of modification sites:** Both iodoacetamide and ethyl maleimide can specifically modify cysteine ​​residues, but the selectivity and number of modification sites affect the conformational changes after modification. Inappropriate modification sites may disrupt the native conformation of hemoglobin, resulting in no significant increase in oxygen-binding capacity. **Spatial structure changes after modification:** Different modifying compounds may cause varying degrees of conformational changes in hemoglobin, which can affect its function. Studies by Garel MC et al. showed significant differences in oxygen-binding capacity after modification, possibly due to spatial structure changes caused by different compounds. **Control of modification degree:** Both excessive and insufficient modification can affect protein function. Excessive modification may lead to protein denaturation, while insufficient modification may not cause significant functional changes. Therefore, precise control of the modification reaction conditions is necessary to obtain the optimal modification effect. Summary of the Invention

[0005] To address the aforementioned problems, the present invention features a benzimidazole derivative designed as an auxiliary molecule to participate in the modification process of hemoglobin by acetyl or ethyl maleic groups. This improves the selectivity of modification sites, reduces conformational changes, and precisely controls the degree of modification, thereby effectively increasing the oxygen-carrying capacity of the modified hemoglobin. The specific scheme is as follows:

[0006] The present invention provides an oxygen-carrying protein molecule comprising at least one cysteine ​​moiety and at least one histidine moiety, wherein the cysteine ​​moiety comprises an acetyl group or an ethyl maleic group, and the histidine moiety comprises a benzimidazole derivative group.

[0007] Preferably, the protein molecule is derived from hemoglobin molecules.

[0008] Preferably, the molecular weight of the oxygen-carrying protein molecule is 64,000 to 66,000 Da.

[0009] Preferably, the oxygen-binding capacity P50 of the oxygen-carrying protein molecule ranges from 15.01 mmHg to 19.99 mmHg.

[0010] This invention provides a method for preparing the above-mentioned oxygen-carrying protein molecule, comprising the following steps:

[0011] S1. Preparation of benzimidazole derivatives: Add benzimidazole, a modifying agent and a catalyst to a solvent and react them. After the reaction is complete, purify to obtain benzimidazole derivatives.

[0012] S2. Prepare hemoglobin suspension and benzimidazole derivative solution, mix the two and incubate, and then purify to obtain benzimidazole derivative modified hemoglobin suspension.

[0013] S3. Iodoacetamide or ethyl maleimide was added to the obtained benzimidazole derivative-modified hemoglobin suspension, and the mixture was incubated and then purified to obtain oxygen-carrying protein molecules.

[0014] Preferably, the modifying agent in step S1 includes one or more of 3-chloropropanol, ethylene glycol, ethanolamine, hydroxylamine hydrochloride, and N-hydroxysuccinimide.

[0015] Preferably, the catalyst in step S1 includes NaOH or K2CO3.

[0016] Preferably, the solvent in step S1 includes one or more of dimethyl sulfoxide, acetonitrile, and ethanol.

[0017] Preferably, the molar ratio of benzimidazole to the modifying agent in step S1 is 1:(1-3).

[0018] Preferably, the molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0019] Preferably, the amount of solvent used in step S1 is 10-20 mL / 1g benzimidazole.

[0020] Preferably, the reaction temperature in step S1 is 25–80°C, and the reaction time is 2–24 h.

[0021] Preferably, the purification in step S1 is performed by column chromatography with silica gel as the solid phase and n-hexane / ethyl acetate as the mobile phase in a ratio of (5-1):1, or dichloromethane / methanol in a ratio of (10-1):1.

[0022] Preferably, in step S2, the hemoglobin suspension is in the form of PBS with a pH range of 7.0–7.4, and the hemoglobin content in the suspension is 0.5–0.8 mM; the benzimidazole derivative solution is in the form of DMSO or acetonitrile, and the concentration of the benzimidazole derivative in the solution is 1–2 mM; the amount of the benzimidazole derivative solution used is 2–4 times the volume of the hemoglobin suspension.

[0023] Preferably, the incubation in step S2 is carried out at a temperature of 37°C for 1 to 2 hours.

[0024] Preferably, the molar amount of iodoacetamide or ethyl maleimide added in step S3 is 5 to 10 times the molar amount of benzimidazole derivative-modified hemoglobin in the suspension.

[0025] Preferably, the incubation in step S3 is carried out at a temperature of 37°C for a time of 30–60 min.

[0026] Preferably, the purification in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 12–24 hours; wherein the dialysis solution is PBS buffer with a pH of 7.0–7.4, and the molecular weight cutoff of the dialysis bag is 7000–8000 Da; the dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0027] Preferably, the method for preparing hemoglobin in step S2 includes:

[0028] 1) Remove plasma components by methods such as centrifugation or microfiltration, and then wash cell components with physiological solutions;

[0029] 2) Add water or hypotonic solution to the cell components and control the reaction time to ensure selective lysis of red blood cells;

[0030] 3) Add hypertonic solution to restore the overall isotonic environment;

[0031] 4) Remove red blood cell membrane fragments and unbroken cells by centrifugation or ultrafiltration, and collect the supernatant or ultrafiltration filtrate rich in hemoglobin.

[0032] 5) Remove impurities by methods such as ultrafiltration or column chromatography to further improve the purity of hemoglobin.

[0033] This invention provides the application of the above-mentioned oxygen-carrying protein molecules in the preparation of compositions with in vivo and in vitro oxygen supply functions.

[0034] Preferably, the application includes the oxygen-carrying protein molecule forming a composition with in vivo and in vitro oxygen supply function together with other pharmaceutically acceptable carriers or pharmaceutically acceptable components.

[0035] The beneficial effects of this invention are:

[0036] This invention designs a benzimidazole derivative as an auxiliary molecule to participate in the modification process of hemoglobin by acetyl or ethyl maleic groups, thereby improving the selectivity of modification sites, reducing conformational changes, and precisely controlling the degree of modification, thus effectively improving the oxygen-binding capacity P50 of the modified hemoglobin. The oxygen-binding capacity P50 of the obtained oxygen-carrying protein molecule ranges from 15.01 mmHg to 19.99 mmHg. Attached Figure Description

[0037] Figure 1 The oxygen dissociation curve for Example 1;

[0038] Figure 2 The oxygen dissociation curve for Example 3;

[0039] Figure 3 The oxygen dissociation curve is shown in Comparative Example 1.

[0040] Figure 4 This is a comparison of the oxygen dissociation curves of the samples from Examples 1 and 3 with those of the standard bovine hemoglobin (bHb) sample;

[0041] Figure 5 The characteristic absorption spectrum of the sample prepared in Example 1;

[0042] Figure 6 The chromatogram of the sample was prepared for Example 1. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels (Sigma Company, analytical grade reagents) unless otherwise specified.

[0046] Preparation example: Preparation of hemoglobin:

[0047] 1) Bovine whole blood was obtained from the slaughterhouse, and sodium citrate was added as an anticoagulant (final concentration 0.38%). The plasma components were removed by centrifugation (2000g, 10min, Beckman JXN-26 centrifuge). Then, the cell components were washed with physiological solution (0.9% sodium chloride solution, Shanghai Lingfeng, AR500G).

[0048] 2) Add water to the cell components and control the reaction time to ensure selective rupture of red blood cells;

[0049] 3) Add hypertonic sodium chloride solution (5%, Shanghai Lingfeng, AR500G) to restore the overall isotonic environment;

[0050] 4) Centrifuge again to remove red blood cell membrane fragments and unbroken cells, and collect the supernatant rich in hemoglobin;

[0051] 5) Impurities are removed by dialysis (molecular weight cutoff of 7000-8000 Da), further improving the purity of hemoglobin.

[0052] Example 1: Preparation of oxygen-carrying protein molecules:

[0053] S1. Preparation of benzimidazole derivatives: Add benzimidazole, a modifying agent and a catalyst to a solvent and react them. After the reaction is complete, purify to obtain benzimidazole derivatives.

[0054] S2. Prepare hemoglobin suspension and benzimidazole derivative solution, mix the two and incubate, and then purify to obtain benzimidazole derivative modified hemoglobin suspension.

[0055] S3. Iodoacetamide was added to the obtained benzimidazole derivative-modified hemoglobin suspension, and after incubation, the oxygen-carrying protein molecule was obtained through purification.

[0056] The modifying agent in step S1 includes 3-chloropropanol.

[0057] The catalyst in step S1 includes NaOH.

[0058] The solvent in step S1 includes acetonitrile.

[0059] The molar ratio of benzimidazole to the modifying agent in step S1 is 1:1.5.

[0060] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0061] The amount of solvent used in step S1 is 10 mL / 1 g benzimidazole.

[0062] The reaction temperature in step S1 is 40°C and the reaction time is 12 hours.

[0063] The purification described in step S1 is performed using column chromatography with silica gel as the solid phase and dichloromethane / methanol as the mobile phase, in a ratio range of (10 to 1):1.

[0064] The hemoglobin suspension in step S2 is in PBS as solvent, with a pH range of 7.0–7.4, and the hemoglobin content in the suspension is 0.5 mM; the benzimidazole derivative solution is in DMSO or acetonitrile as solvent, and the concentration of the benzimidazole derivative in the solution is 1 mM; the volume of the benzimidazole derivative solution is twice the volume of the hemoglobin suspension.

[0065] The incubation in step S2 is carried out at a temperature of 37°C for 1 hour.

[0066] The molar amount of iodoacetamide added in step S3 is 5 times the molar amount of benzimidazole derivative-modified hemoglobin in the suspension.

[0067] The incubation process described in step S3 is carried out at a temperature of 37°C for 30 minutes.

[0068] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 12 hours; wherein the dialysis solution is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000 Da; the dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0069] Example 2: Preparation of oxygen-carrying protein molecules:

[0070] S1. Preparation of benzimidazole derivatives: Add benzimidazole, a modifying agent and a catalyst to a solvent and react them. After the reaction is complete, purify to obtain benzimidazole derivatives.

[0071] S2. Prepare hemoglobin suspension and benzimidazole derivative solution, mix the two and incubate, and then purify to obtain benzimidazole derivative modified hemoglobin suspension.

[0072] S3. Iodoacetamide was added to the obtained benzimidazole derivative-modified hemoglobin suspension, and after incubation, the oxygen-carrying protein molecule was obtained through purification.

[0073] The modifying agent in step S1 includes ethylene glycol.

[0074] The catalyst in step S1 includes NaOH.

[0075] The solvent in step S1 includes dimethyl sulfoxide.

[0076] The molar ratio of benzimidazole to the modifying agent in step S1 is 1:1.5.

[0077] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0078] The amount of solvent used in step S1 is 20 mL / 1 g benzimidazole.

[0079] The reaction temperature in step S1 is 70°C and the reaction time is 15 hours.

[0080] The purification described in step S1 is performed using column chromatography with silica gel as the solid phase and dichloromethane / methanol as the mobile phase, in a ratio range of (10 to 1):1.

[0081] The hemoglobin suspension in step S2 is in PBS as solvent, with a pH range of 7.0–7.4, and the hemoglobin content in the suspension is 0.8 mM; the benzimidazole derivative solution is in DMSO or acetonitrile as solvent, and the concentration of the benzimidazole derivative in the solution is 2 mM; the volume of the benzimidazole derivative solution is 4 times the volume of the hemoglobin suspension.

[0082] The incubation process described in step S2 is carried out at a temperature of 37°C for 2 hours.

[0083] The molar amount of iodoacetamide added in step S3 is 10 times the molar amount of benzimidazole derivative-modified hemoglobin in the suspension.

[0084] The incubation process described in step S3 is carried out at a temperature of 37°C for 60 minutes.

[0085] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 24 hours; wherein the dialysis solution is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000 Da; the dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0086] Example 3: Preparation of oxygen-carrying protein molecules:

[0087] S1. Preparation of benzimidazole derivatives: Add benzimidazole, a modifying agent and a catalyst to a solvent and react them. After the reaction is complete, purify to obtain benzimidazole derivatives.

[0088] S2. Prepare hemoglobin suspension and benzimidazole derivative solution, mix the two and incubate, and then purify to obtain benzimidazole derivative modified hemoglobin suspension.

[0089] S3. Ethyl maleimide was added to the hemoglobin suspension modified with the obtained benzimidazole derivative, and the mixture was incubated and then purified to obtain oxygen-carrying protein molecules.

[0090] The modifying agent in step S1 includes ethylene glycol.

[0091] The catalyst in step S1 includes NaOH.

[0092] The solvent in step S1 includes dimethyl sulfoxide.

[0093] The molar ratio of benzimidazole to the modifying agent in step S1 is 1:1.5.

[0094] The molar ratio of the modifying agent to the catalyst in step S1 is 1:1.

[0095] The amount of solvent used in step S1 is 20 mL / 1 g benzimidazole.

[0096] The reaction temperature in step S1 is 70°C and the reaction time is 15 hours.

[0097] The purification described in step S1 is performed using column chromatography with silica gel as the solid phase and dichloromethane / methanol as the mobile phase, in a ratio range of (10 to 1):1.

[0098] The hemoglobin suspension in step S2 is in PBS as solvent, with a pH range of 7.0–7.4, and the hemoglobin content in the suspension is 0.8 mM; the benzimidazole derivative solution is in DMSO or acetonitrile as solvent, and the concentration of the benzimidazole derivative in the solution is 2 mM; the volume of the benzimidazole derivative solution is 4 times the volume of the hemoglobin suspension.

[0099] The incubation process described in step S2 is carried out at a temperature of 37°C for 2 hours.

[0100] The molar amount of ethyl maleimide added in step S3 is 10 times the molar amount of benzimidazole derivative-modified hemoglobin in the suspension.

[0101] The incubation process described in step S3 is carried out at a temperature of 37°C for 60 minutes.

[0102] The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 24 hours; wherein the dialysis solution is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000 Da; the dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0103] Preparation of modified hemoglobin molecules in Comparative Example 1 (the difference from Example 3 is that no benzimidazole derivative modification was performed):

[0104] A hemoglobin suspension was prepared, ethyl maleimide was added, and the mixture was incubated and then purified to obtain oxygen-carrying protein molecules.

[0105] The molar amount of ethyl maleimide added is 10 times the molar amount of hemoglobin in the suspension.

[0106] The incubation was carried out at a temperature of 37°C for 60 minutes.

[0107] The purification process involves transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 24 hours. The dialysis solution is PBS buffer with a pH of 7.0–7.4, and the molecular weight cutoff of the dialysis bag is 7000–8000 Da. The dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0108] Preparation of modified hemoglobin molecules in Comparative Example 2 (the difference from Example 2 is that no benzimidazole derivative modification was performed):

[0109] A hemoglobin suspension was prepared, iodoacetamide was added, and the mixture was incubated and then purified to obtain oxygen-carrying protein molecules.

[0110] The amount of iodoacetamide added is 10 times the amount of hemoglobin in the suspension.

[0111] The incubation was carried out at a temperature of 37°C for 60 minutes.

[0112] The purification process involves transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 24 hours. The dialysis solution is PBS buffer with a pH of 7.0–7.4, and the molecular weight cutoff of the dialysis bag is 7000–8000 Da. The dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

[0113] Characterization and performance testing:

[0114] Oxygen binding capacity test (P50): The oxygen dissociation curve of the modified hemoglobin solution was measured using an oxygen dissociation curve analyzer. The P50 value was calculated, which is the partial pressure of oxygen at which hemoglobin reaches 50% oxygen saturation.

[0115] The characteristic absorption spectrum and chromatogram of the sample in Example 1 are shown below. Figure 5 , Figure 6 Its oxygen dissociation curve is shown in Figure 1The P50 values ​​for the samples in Example 1, 2, and 3 were all below 20.0 mmHg, indicating enhanced oxygen-binding capacity. The P50 value for the sample in Example 2 was 18.19 mmHg, for Example 3 it was 18.05 mmHg, and for Example 3 it was 18.54 mmHg. The modified hemoglobin exhibited enhanced oxygen-binding capacity. The P50 value for the sample in Comparative Example 1 was 27.29 mmHg, and for Comparative Example 2 it was 28.62 mmHg. The P50 values ​​for the comparative examples were comparable to those of unmodified hemoglobin in previous studies (26.0–30.0 mmHg). For the blank sample (standard bHb), the P50 value was 27.47 mmHg, which was also comparable to those of unmodified hemoglobin in previous studies (26.0–30.0 mmHg).

[0116] Through the above experiments and analyses, we obtained a technical solution that utilizes benzimidazole derivatives to participate in the modification of hemoglobin with acetyl or ethyl maleic groups, thereby effectively improving the oxygen-binding capacity of hemoglobin molecules. The binding of benzimidazole derivatives to hemoglobin may cause local conformational changes on the protein surface, making the overall structure of hemoglobin more open or flexible. In this open or flexible state, further modification with IAA or NEM may more effectively expose and modify key residues near the oxygen-binding site, leading to a significant change in oxygen affinity. The synergistic effect of the two modifications may significantly alter the quaternary structure of hemoglobin or the microenvironment of the oxygen-binding site, thereby greatly enhancing oxygen-binding capacity and significantly reducing the P50 value. Furthermore, we verified a scheme where our prepared benzimidazole derivative was replaced with benzimidazole, but experimental results showed that benzimidazole could not form a stable binding with hemoglobin.

[0117] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0118] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method of producing an oxygen carrying protein molecule, characterized by: Includes the following steps: S1. Preparation of benzimidazole derivatives: Add benzimidazole, a modifying agent and a catalyst to a solvent and react them. After the reaction is complete, purify to obtain benzimidazole derivatives. S2. Prepare hemoglobin suspension and benzimidazole derivative solution, mix the two and incubate, and then purify to obtain benzimidazole derivative modified hemoglobin suspension. S3. Iodoacetamide or ethyl maleimide was added to the obtained benzimidazole derivative-modified hemoglobin suspension, and the mixture was incubated and then purified to obtain oxygen-carrying protein molecules. The modifying reagent in step S1 is one or two of 3-chloropropanol and ethylene glycol; the catalyst is NaOH; the solvent is one or two of dimethyl sulfoxide and acetonitrile; the molar ratio of benzimidazole to the modifying reagent in step S1 is 1:(1~3); the molar ratio of the modifying reagent to the catalyst in step S1 is 1:1; the amount of solvent used in step S1 is 10~20mL / 1g benzimidazole; the reaction temperature in step S1 is 25~80℃, and the time is 2~24h; the purification in step S1 is carried out by column chromatography, with silica gel as the solid phase and dichloromethane / methanol as the mobile phase, in a ratio range of (10~1):

1.

2. The method of claim 1, wherein the oxygen carrying protein molecule is selected from the group consisting of hemoglobin, myoglobin, and hemocyanin. The hemoglobin suspension in step S2 is in PBS as solvent, with a pH range of 7.0-7.4, and the hemoglobin content in the suspension is 0.5-0.8 mM; the benzimidazole derivative solution is in DMSO or acetonitrile as solvent, and the concentration of the benzimidazole derivative in the solution is 1-2 mM; the volume of the benzimidazole derivative solution is 2-4 times the volume of the hemoglobin suspension.

3. The method for preparing oxygen-carrying protein molecules according to claim 1, characterized in that: The incubation process described in step S2 is carried out at a temperature of 37°C for 1 to 2 hours.

4. The method for preparing oxygen-carrying protein molecules according to claim 1, characterized in that: The molar amount of iodoacetamide or ethyl maleimide added in step S3 is 5 to 10 times the molar amount of benzimidazole derivative-modified hemoglobin in the suspension.

5. The method for preparing oxygen-carrying protein molecules according to claim 1, characterized in that: The incubation process described in step S3 is carried out at a temperature of 37°C for 30 to 60 minutes.

6. The method for preparing oxygen-carrying protein molecules according to claim 1, characterized in that: The purification described in step S2 or step S3 includes transferring the modified hemoglobin solution into a dialysis bag, placing the dialysis bag in a large amount of dialysis solution, and dialyzing at 4°C for 12 to 24 hours; wherein the dialysis solution is PBS buffer with a pH of 7.0 to 7.4, and the molecular weight cutoff of the dialysis bag is 7000 to 8000 Da; the dialysis solution is changed multiple times during the process to ensure complete removal of small molecules and solvents.

Images