Hemoglobin oxygen carrier based on propionaldehyde and polyethylene glycol modification as well as preparation method and application of hemoglobin oxygen carrier

Abstract

The invention relates to a propionaldehyde and polyethylene glycol modified hemoglobin oxygen carrier as well as a preparation method and application thereof. The preparation method of the hemoglobin oxygen carrier based on propionaldehyde and polyethylene glycol modification comprises the following steps: carrying out propylation on a val-1 (alpha) residue of hemoglobin by propionaldehyde to obtain val-1 (alpha) residue propylated hemoglobin; two Val-1 (beta) residues of val-1 (alpha) residue propylated hemoglobin are subjected to site-specific modification by polyethylene glycol aldehyde, and the hemoglobin oxygen carrier based on propionaldehyde and polyethylene glycol modification is obtained. The hemoglobin oxygen carrier prepared by the method has relatively high oxygen carrying / releasing activity and relatively high structural stability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a hemoglobin oxygen carrier and a preparation method and application thereof, and in particular to a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol and a preparation method and application thereof. Background Art

[0002] Hemoglobin is a tetrameric protein composed of four subunits (ααββ). It has the function of carrying oxygen and can be used as a temporary substitute for red blood cells. It is an ideal oxygen carrier. Since hemoglobin can cause tubular necrosis or other toxic effects after being filtered by the kidneys, hemoglobin must be appropriately chemically modified to meet the requirements of blood transfusion. Hemoglobin oxygen carrier can effectively carry oxygen and expand volume. It also has the advantages of sufficient source, non-toxicity, non-immunogenicity and long circulation time in the body. In recent years, scientific research institutions and companies at home and abroad have developed a series of chemically modified and genetically engineered hemoglobin as blood substitutes, mainly including intramolecular cross-linked hemoglobin, polymerized hemoglobin, genetically engineered hemoglobin and polyethylene glycol (PEG) modified hemoglobin.

[0003] Intramolecular cross-linked hemoglobin, such as the αα-cross-linked hemoglobin developed by Baxter, has been withdrawn from the market due to its severe vasoactivity in the human body, leading to increased blood pressure and other toxic effects. Polymerized hemoglobin, such as the polymerized bovine hemoglobin developed by Biopure, and the polymerized human hemoglobin developed by Northfield, has not been approved by the US FDA due to its side effects such as gastrointestinal cramps. Genetic recombination and transgenic plant and animal technology have been used in the research of blood substitutes. The cross-linked human hemoglobin mutant produced by genetic engineering has been successfully expressed in Escherichia coli and yeast, and the production of blood containing human hemoglobin using transgenic animals has also been reported. However, this type of technology faces some problems, such as whether the amount of hemoglobin expressed can meet large-scale commercial production, which remains to be studied.

[0004] After PEG modification of hemoglobin, the molecular weight of hemoglobin is increased, and a protective layer is formed around the hemoglobin molecule. Therefore, PEG modification can maintain the structural integrity of the oxygen binding site of hemoglobin, reduce the phagocytosis of the reticuloendothelial system, passivate the immunogenicity and antigenicity of hemoglobin, increase the circulation half-life of hemoglobin in the body, and reduce possible allergic reactions and immune reactions. In addition to being used for acute blood loss or hemorrhagic shock, the potential indications of PEG-modified hemoglobin include surgical blood preparation, anemia and sickle cell anemia, extracorporeal tissue perfusion, local ischemia, and adjuvant for tumor treatment. In addition to effectively eliminating vascular activity, PEG-modified hemoglobin can also effectively expand volume and carry oxygen. This shows that PEG-modified hemoglobin can be used as a potential oxygen carrier. However, because PEG molecules destroy the hydrogen bonds and hydrophobic interactions on the surface of hemoglobin molecules, PEG modification of hemoglobin will cause the hemoglobin tetramer (ααββ) to disaggregate into dimers (αβ), which greatly destroys the structural and functional properties of hemoglobin. For example, the dissociation of hemoglobin tetramers into dimers will lead to the loss of the quaternary structure of hemoglobin, affecting the transition of hemoglobin molecules from the relaxed state (R state) to the tense state (T state), thereby changing the binding and release function of hemoglobin with oxygen; the heme molecules of hemoglobin are more exposed to solvent molecules, and the Fe in hemoglobin is 2+ Easily oxidized to Fe 3+ , resulting in heme being unable to effectively bind to oxygen molecules, while generating oxygen free radicals (such as hydrogen peroxide), which further damage the molecular structure of hemoglobin; the binding force between heme molecules and globulin molecules is weakened, resulting in the loss of heme molecules. Therefore, we need to develop new chemical modification methods to improve the stability of PEG-modified hemoglobin tetramers, thereby improving the structural and functional properties of PEG-modified hemoglobin.

[0005] PEG-modified hemoglobin currently has the disadvantage of poor uniformity of the modified product. For example, MP4, a product developed by Sangart in the United States, is a PEG-modified hemoglobin; its molecular weight distribution is uneven, and the hemoglobin molecule is connected to one or more PEG chains, with an average of 6 PEG chains connected to each hemoglobin molecule; the modification site of PEG is also uncertain, distributed on Val-1 (α), Val-1 (β), Cys-93 (β) and some Lys residues. These shortcomings are not conducive to the quality control and biosafety evaluation of PEG-modified hemoglobin. In contrast, specific PEG-modified hemoglobin is conducive to approval as a new drug because of its determined modification site and uniform molecular weight distribution. Therefore, we also need to develop PEG-specific modification methods to achieve site-specific chemical modification of PEG.

[0006] Studies have shown that PEG modification at the Val-1(α) site of hemoglobin will cause the hemoglobin tetramer to dissociate into dimers, while propylation modification at the Val-1(α) site will improve the stability of the hemoglobin tetramer. On this basis, we propose to introduce a propyl group at the Val-1(α) site of hemoglobin, and further use polyethylene glycol aldehyde to perform specific PEG modification at the Val-1(β) site. The present invention organically combines the specific small molecule chemical modification of hemoglobin with the specific PEG modification, and is expected to improve the stability of the PEG-modified hemoglobin tetramer, and prepare PEG-modified hemoglobin with a defined modification site and uniform molecular weight, which has important guiding significance for the design of new hemoglobin oxygen carriers. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a hemoglobin oxygen carrier modified based on propionaldehyde and polyethylene glycol, and a preparation method and application thereof.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol, the preparation method comprising:

[0010] The Val-1(α) residue of hemoglobin is propylated with propionaldehyde to obtain Val-1(α) residue propylated hemoglobin (Pr-Hb);

[0011] The two Val-1(β) residues of hemoglobin, which were propylated with Val-1(α) residue by polyethylene glycol aldehyde, were site-specifically modified to obtain hemoglobin oxygen carrier based on propionaldehyde and polyethylene glycol modification (PEG-Pr-Hb).

[0012] The present invention creatively proposes to introduce a propyl group at the Val-1 (α) site of hemoglobin, and further uses polyethylene glycol aldehyde (PEG aldehyde) to perform specific PEG modification at the Val-1 (β) site. The present invention organically combines the specific small molecule chemical modification of hemoglobin with the specific PEG modification, improves the stability of the PEG-modified hemoglobin tetramer, and prepares PEG-modified hemoglobin (hemoglobin oxygen carrier modified based on propionaldehyde and polyethylene glycol) with a defined modification site and uniform molecular weight.

[0013] Preferably, the step of propylating the Val-1(α) residue of hemoglobin by propionaldehyde comprises:

[0014] (1) Adding phytate to hemoglobin to block the Val-1(β) site;

[0015] (2) Propionaldehyde and sodium cyanoborohydride are added, and unreacted propionaldehyde, sodium cyanoborohydride and phytic acid are removed by dialysis to obtain hemoglobin with propylation of the Val-1(α) residue.

[0016] Preferably, the molar ratio of hemoglobin, propionaldehyde and sodium cyanoborohydride is 1:(1-10):(10-100), preferably 1:4:40.

[0017] Among them, the specific point values ​​​​in 1-10 can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific point values ​​​​in 10-100 can be selected from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.

[0018] Preferably, the step of site-specifically modifying two Val-1(β) residues of hemoglobin propylated with Val-1(α) residue by polyethylene glycol aldehyde comprises:

[0019] (I) reacting hemoglobin with propylated Val-1(α) residue with polyethylene glycol aldehyde and sodium cyanoborohydride;

[0020] (ⅠⅠ) Add glycine solution to terminate the reaction, and dialyze to remove unreacted glycine and sodium cyanoborohydride to obtain a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol.

[0021] Preferably, the molar ratio of the Val-1(α) residue propylated hemoglobin, polyethylene glycol aldehyde and sodium cyanoborohydride is 1:(2-20):(20-200), preferably 1:10:100.

[0022] Among them, the specific point values ​​in 2-20 can be selected from 2, 5, 10, 15, 20, etc., and the specific point values ​​in 20-200 can be selected from 20, 50, 100, 150, 200, etc.

[0023] Preferably, the molecular weight of the polyethylene glycol aldehyde is 2 kDa-40 kDa, preferably 5 kDa.

[0024] Preferably, after obtaining the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol, the method further comprises: separating and purifying the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol by using a gel filtration chromatography medium.

[0025] Preferably, the gel filtration chromatography medium comprises Superdex 200 gel filtration chromatography medium.

[0026] In a second aspect, the present invention provides a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol prepared by the preparation method of the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol as described in the first aspect.

[0027] In a third aspect, the present invention provides a use of the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol as described in the second aspect in the preparation of a blood substitute.

[0028] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention develops a method for specific chemical modification of hemoglobin, which realizes the site-specific chemical modification of Val-1 (β) site by PEG and the site-specific chemical modification of Val-1 (α) site by propionaldehyde.

[0031] (2) The present invention organically combines PEG chemical modification and propionaldehyde chemical modification to improve the tetramer stability of hemoglobin.

[0032] (3) The present invention organically combines PEG chemical modification and propionaldehyde chemical modification, which can reduce the self-oxidation rate of hemoglobin and at the same time has higher oxygen carrying / releasing activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the separation and purification of hemoglobin;

[0034] Figure 2 This is the result of SDS-PAGE electrophoresis analysis of purified hemoglobin;

[0035] Figure 3 It is a schematic diagram of the separation and purification of hemoglobin (Pr-Hb) with propylation of Val-1(α) residue;

[0036] Figure 4 It is a schematic diagram of separation and purification of PEG-Pr-Hb;

[0037] Figure 5 It is the purity result diagram of PEG-Hb and PEG-PrB-Hb analyzed by SDS-PAGE electrophoresis;

[0038] Figure 6 It is the result figure of reverse phase-HPLC analysis of PEG-Hb and PEG-Pr-Hb;

[0039] Figure 7 is the gel filtration analysis graph of the sample;

[0040] Figure 8 This is the result of the trypsin cleavage map to identify the modification sites of PEG aldehyde in Hb and Pr-Hb;

[0041] Fig. 9 is the circular dichroism spectrum analysis diagram of the sample;

[0042] Fig.10 is the dissociation constant plot of the sample;

[0043] Fig.11 It is a test chart of the percentage of methemoglobin in the sample. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] Example 1

[0046] Separation and purification of hemoglobin:

[0047] Centrifuge the whole blood of an adult, discard the supernatant to obtain the blood cells, then wash twice with PBS buffer (pH 7.4), and take the precipitate after centrifugation. Add 3 times the volume of ultrapure water, stir with a glass rod, and let it stand for 2 hours to rupture the red blood cells. Then centrifuge to obtain the supernatant, filter with a 0.22μm filter membrane, and you can get the red blood cell rupture liquid. The rupture liquid is separated and purified using a Q Sepharose High Performance anion exchange chromatography column. Since hemoglobin accounts for more than 90% of the total protein in red blood cells, the last elution peak is the target protein, such as Figure 1 The target elution peak was collected, concentrated and dialyzed into PBS buffer (pH 7.4), and stored in a -80°C refrigerator for later use.

[0048] Example 2

[0049] Identification of Hemoglobin Separation and Purification:

[0050] The concentrated elution peak was identified by SDS-PAGE electrophoresis. Figure 2 As shown in the figure, the concentrated elution peak solution presents a single electrophoretic band, corresponding to a molecular weight of about 16.0 kDa. This is because hemoglobin (64.0 kDa) is composed of four subunits, α1α2β1β2, and the tetrameric protein is depolymerized into a single subunit with a molecular weight of 16.0 kDa in a boiling water bath. This further proves that the last elution peak is purified hemoglobin with a high purity, reaching single-point electrophoresis purity.

[0051] Example 3

[0052] Preparation, separation and purification of Val-1(α) residue propylated hemoglobin (Pr-Hb):

[0053] Inositol hexaphosphate (IHP) is an allosteric effector of hemoglobin, which can reversibly bind to Val-1(β), thereby preventing aldehyde modifiers from modifying the Val-1(β) site. Aldehyde-mediated modification reactions tend to modify the N-terminus of proteins under acidic conditions, so the modification reaction may be concentrated on Val-1(α). A 16 mg / ml hemoglobin solution (i.e., 0.25 mM) was mixed with 1.0 mM IHP dissolved in 50 mM BisTris-acetate buffer (pH 6.5) for 1 hour. The IHP-bound hemoglobin solution reacted with 1.5 mM propionaldehyde. In the presence of 7.5 mM NaCNBH3, the modification reaction was carried out in 50 mM BisTris-acetate buffer (pH 6.5) at 4°C overnight. Unreacted propionaldehyde and NaCNBH3 in the reaction solution were then dialyzed to remove them. Separation and purification were performed using a Mono S chromatography column. The results are shown in Figure 3 As shown in the figure, the reaction solution modified with propionaldehyde was eluted by pH gradient, and four elution peaks (a1, b1, c1 and d1) appeared. The elution position of peak d1 is the same as that of hemoglobin, and it can be considered as the unmodified hemoglobin in the reaction solution. By analogy, peak c1 may be the product of propionaldehyde modification on Val-1(α), while peaks a1 and b1 may be the products of propionaldehyde modification on Val-1(α) and other sites. Therefore, we collected peak c1 and concentrated it for the next step of identification experiment of modification sites.

[0054] Example 4

[0055] Preparation, separation and purification of hemoglobin oxygen carrier (PEG-PrB-Hb) modified with propionaldehyde and polyethylene glycol:

[0056] The protein concentrations of hemoglobin (Hb) and hemoglobin (Pr-Hb) propylated with Val-1 (α) residues were adjusted to 0.1 mM, respectively. Then 0.5 mM polyethylene glycol propionaldehyde (molecular weight 5 kDa) and 5.0 mM sodium cyanoborohydride were reacted with 0.1 mM Pr-Hb and Hb, respectively. The reaction buffer was 50 mM BisTris-acetate buffer with a pH of 6.5, a reaction temperature of 4°C, and the reaction was allowed to proceed overnight (12-14 hours). Subsequently, Superdex 200 gel filtration columns (1.6 cm × 70 cm) were used for separation and purification. The buffer used for the chromatography column was PBS buffer (pH 7.4), the flow rate was 2.0 mL / min, and the detection wavelength of the eluent was 280 nm. The results are as shown in Figure 4 As shown by Figure 4As shown in a, the modified mixture is mainly composed of Pr-Hb and PEG-modified Pr-Hb, and the elution peak corresponding to PEG-modified Pr-Hb is collected (as indicated by the arrow). After the collected elution peak is concentrated, the concentrate is further separated and purified by a Superdex 200 gel filtration column. Figure 4 b shows that after loading, a peak with poor symmetry appeared, corresponding to PEG-modified Pr-Hb. Collect the elution peak as indicated by the arrow. After concentrating the collected elution peak, continue to load the column and elute to obtain a peak with good symmetry ( Figure 4 c) Therefore, from Figure 4 The elution peak collected on c has a high purity and can be used for the next experiment. PEG-Hb modified with PEG aldehyde is also separated and purified by a similar method.

[0057] Example 5

[0058] Purity identification of hemoglobin oxygen carrier modified with propionaldehyde and polyethylene glycol (PEG-PrB-Hb):

[0059] The purity of PEG-Hb and PEG-PrB-Hb was analyzed by SDS-PAGE electrophoresis. Figure 5 As shown, Hb (lane 2) and Pr-Hb (lane 3) both show a single electrophoretic band, corresponding to a molecular weight of approximately 16 kDa. This indicates that under the electrophoretic conditions, the four subunits of Hb and Pr-Hb with a molecular weight of 64 kDa are completely depolymerized into a single subunit of 16 kDa. After PEG aldehyde modification of Hb and Pr-Hb, two subunits of Hb and Pr-Hb bind to PEG molecules, while the other two subunits do not bind to PEG molecules. Therefore, PEG-Hb (lane 4) and PEG-Pr-Hb (lane 5) both show two electrophoretic bands, corresponding to molecular weights of approximately 16 kDa and 25 kDa. No other miscellaneous bands were observed for PEG-modified proteins.

[0060] The modification of the four subunits of Hb and Pr-Hb by PEG aldehyde was analyzed using a reverse phase HPLC column (C4 column, 0.46 cm × 25 cm). Figure 6As shown in the figure, hemoglobin (Hb) and hemoglobin (Pr-Hb) propylated with Val-1 (α) residues exhibit three elution peaks on the reversed-phase HPLC column, corresponding to the heme, β-subunit and α-subunit of hemoglobin, respectively. However, the elution peak of the α-subunit (Pr-α) of Pr-Hb shifts to the right. At the same time, PEG aldehyde modification of Pr-Hb also exhibits three elution peaks. Compared with the β-subunit and α-subunit, two elution peaks shift to the right, corresponding to the propylated α-subunit (Pr-α) and the β-subunit bound to one PEG molecule (PEG-β), respectively. PEG aldehyde modification of Hb also exhibits three elution peaks, the latter two of which correspond to the α-subunit and PEG-β, respectively. Compared with the α-subunit, the elution peak corresponding to Pr-α also shifts to the right. The above indicates that Pr-α has one more propyl group and has stronger hydrophobicity. The results showed that PEG aldehyde specifically modified the β-subunits of Pr-Hb and Hb, while the α-subunits were not modified. Since the hemoglobin molecule has two β-subunits and two α-subunits, it is speculated that Pr-Hb and Hb are bound to two PEG molecules with a molecular weight of 5 kDa, respectively. Therefore, both PEG-Pr-Hb and PEG-Hb have strong molecular homogeneity.

[0061] Example 6

[0062] Gel filtration analysis of Hb, Pr-Hb, PEG-Hb, PEG-Pr-Hb samples:

[0063] The samples were analyzed by analytical Superdex 200 gel chromatography column (1 cm × 30 cm). Figure 7 As shown in the figure, the purified Hb, Pr-Hb, PEG-Hb and PEG-Pr-Hb all showed a single and symmetrical elution peak. In contrast, the elution peak position of Pr-Hb was comparable, and the elution peak position of the PEG-modified product was significantly advanced, indicating that propionaldehyde modification of hemoglobin did not change its hydration volume, while PEG modification could significantly enhance the molecular weight of Hb and its derivatives. In addition, the elution time of PEG-Pr-Hb was shorter than that of PEG-Hb, indicating that propionaldehyde modification of hemoglobin Val-1(α) could reduce the tetramer disaggregation effect caused by PEG modification.

[0064] Example 7

[0065] Identification of modification sites of PEG-modified hemoglobin:

[0066] Trypsin cleavage patterns are used to identify the modification sites of hemoglobin. The heme of hemoglobin in the sample is removed by the acid acetone method. After centrifugation to remove the supernatant, the precipitate is dissolved in 1 ml of 0.1% trifluoroacetic acid and lyophilized overnight. The lyophilized sample is dissolved in 0.1M ammonium bicarbonate solution, and a certain amount of trypsin is added. The mass ratio of trypsin to sample is 1:100. After mixing, it is placed at 37°C for 3 hours and then lyophilized overnight. The lyophilized sample is dissolved in 0.3% trifluoroacetic acid solution.

[0067] After hemoglobin was treated with trypsin, the enzyme fragments were analyzed by C18 reverse phase chromatography column (0.46cm×25cm). Figure 8 As shown in Figure 2, multiple enzyme fragments appeared after hemoglobin was treated with trypsin. Compared with Hb, α-T1, α-T 1+2 The β-T1 and α-T2 peptides disappeared completely, while other peptides (such as α-T3) remained almost unchanged. This indicates that Val-1(α) was specifically modified by propionaldehyde, and its hydrophobicity changed, which ultimately led to the changes in α-T1 and α-T3. 1+2 The peptide segment completely disappeared; Val-1(β) was specifically modified by PEG, resulting in the complete disappearance of the β-T1 peptide segment. The other peptide segments did not change, indicating that the amino acid residues at other sites were not modified. Therefore, the modification site of PEG aldehyde in Hb and Pr-Hb is on the Val-1(β) residue.

[0068] Example 8

[0069] Circular dichroism analysis of Hb, Pr-Hb, PEG-Hb, and PEG-Pr-Hb samples:

[0070] The L peak region of the circular dichroism spectrum (near 260 nanometers) is very sensitive to the interaction between heme and its surrounding globin and is affected by the interaction with ligands (such as oxygen molecules). Fig. 9 As shown, propylation did not change the L peak of hemoglobin (Hb). PEGylation of Val-1(β) also did not significantly reduce the L peak of Hb. In addition, the near-ultraviolet circular dichroism spectra of PEG-aldehyde-modified Pr-Hb (PEG-Pr-Hb) and Hb (PEG-Hb) almost overlap. At 420 nanometers, PEGylation of Val-1(β) can increase the maximum absorbance of Hb. This indicates that PEGylation of Val-1(β) can affect the microenvironment of hemoglobin. In contrast, propylation can reduce the light absorption of PEG-Hb at 420 nanometers. This indicates that propylation can slightly weaken the interference of PEG modification on the microenvironment structure of hemoglobin.

[0071] Example 9

[0072] Study on the tetramer stability of Hb, Pr-Hb, PEG-Hb, and PEG-Pr-Hb samples:

[0073] like Fig.10 As shown, propylation of Val-1(α) results in a dissociation constant (K d ) decreased by 9.6 times. This indicates that the propylation of Val-1(α) can effectively enhance the tetramer stability of hemoglobin. On the other hand, PEG modification caused the dissociation constant of Hb to increase by 6.6 times, indicating that the tetramer stability of hemoglobin decreased. The propylation of Val-1(α) caused the dissociation constant (K) of PEG-Hb to increase by 6.6 times. d ) decreased by 11.8 times. This indicates that the propylation of Val-1(α) can effectively enhance the tetramer stability of PEG-Hb.

[0074] Example 10

[0075] Study on the self-oxidation of Hb, Pr-Hb, PEG-Hb, and PEG-Pr-Hb samples:

[0076] Hemoglobin undergoes self-oxidation, which is manifested by the increase of methemoglobin and the generation of oxygen free radicals. Methemoglobin is hemoglobin that loses its oxygen-carrying capacity when the ferrous ions of heme are oxidized to ferric ions. It is one of the important indicators for evaluating the function of blood substitutes. The results of the determination of the methemoglobin content of Hb, Pr-Hb, PEG-Hb, and PEG-Pr-Hb samples over time are shown in Figure 2. Fig.11 As shown in the figure, after two hours, the content of methemoglobin is in descending order of PEG-Hb, PEG-Pr-Hb, Hb, and Pr-Hb, indicating that PEG modification accelerates the self-oxidation rate of hemoglobin. This is because PEG modification severely exposes the heme microenvironment of Hb, making heme more susceptible to attack by oxygen free radicals in the solution. 2+ Oxidized to Fe 3+ , generating methemoglobin without oxygen-carrying function. It also shows that propylation can reduce the interference of PEG modification on the microenvironment structure of heme.

[0077] Embodiment 11

[0078] Oxygen affinity study of Hb, Pr-Hb, PEG-Hb, and PEG-Pr-Hb samples:

[0079] The oxygen affinity of the sample is evaluated by P50, which is the oxygen partial pressure of the blood at 50% oxygen saturation, reflecting the oxygen carrying capacity of the blood. The sample is measured by a Hemox oximeter to obtain an oxygen balance curve, from which the P50 value can be obtained. The P50 values ​​of Hb, Pr-Hb, PEG-Hb and PEG-Pr-Hb are 14.0, 7.8, 5.3 and 4.6 mmHg, respectively, indicating that propionaldehyde and PEG modification of hemoglobin Val-1 (α) can reduce its P50 value and improve oxygen affinity, and the two have a synergistic effect.

[0080] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0081] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0082] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol, characterized in that: The preparation method comprises: The Val-1(α) residue of hemoglobin is propylated with propionaldehyde to obtain hemoglobin with propylated Val-1(α) residue; The two Val-1(β) residues of hemoglobin, in which the Val-1(α) residue was propylated by polyethylene glycol aldehyde, were site-specifically modified to obtain a hemoglobin oxygen carrier based on propionaldehyde and polyethylene glycol modification.

2. The preparation method according to claim 1, characterized in that: The step of propylating the Val-1(α) residue of hemoglobin by propionaldehyde comprises: (1) Adding phytate to hemoglobin to block the Val-1(β) site; (2) Propionaldehyde and sodium cyanoborohydride are added, and unreacted propionaldehyde, sodium cyanoborohydride and phytic acid are removed by dialysis to obtain hemoglobin with propylation of the Val-1(α) residue.

3. The preparation method according to claim 2, characterized in that: The molar ratio of hemoglobin, propionaldehyde and sodium cyanoborohydride is 1:(1-10):(10-100), preferably 1:4:

40.

4. The preparation method according to any one of claims 1 to 3, characterized in that The step of site-specifically modifying two Val-1(β) residues of hemoglobin propylated with Val-1(α) residue by polyethylene glycol aldehyde comprises: (I) reacting hemoglobin with propylated Val-1(α) residue with polyethylene glycol aldehyde and sodium cyanoborohydride; (ⅠⅠ) Add glycine solution to terminate the reaction, and dialyze to remove unreacted glycine and sodium cyanoborohydride to obtain a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the Val-1(α) residue propylated hemoglobin, polyethylene glycol aldehyde and sodium cyanoborohydride is 1:(2-20):(20-200), preferably 1:10:

100.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The molecular weight of the polyethylene glycol aldehyde is 2 kDa-40 kDa, preferably 5 kDa.

7. The preparation method according to any one of claims 1 to 6, characterized in that: After obtaining the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol, the method further comprises: using a gel filtration chromatography medium to separate and purify the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol.

8. The preparation method according to claim 7, characterized in that: The gel filtration chromatography medium comprises Superdex200 gel filtration chromatography medium.

9. A hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol prepared by the method for preparing a hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol as described in any one of claims 1 to 8.

10. Use of the hemoglobin oxygen carrier modified by propionaldehyde and polyethylene glycol as claimed in claim 9 in the preparation of blood substitutes.

Images