CN111499732A - Hemoglobin oxygen carrier based on double chemical modification and preparation method and application thereof - Google Patents

Abstract

The invention relates to a hemoglobin oxygen carrier based on dual chemical modification, and a preparation method and application thereof, wherein two Val-1(α) residues in the hemoglobin oxygen carrier based on dual chemical modification are carboxymethylated, and two L ys-99(α) residues are intramolecularly crosslinked, wherein two L ys-99(α) residues are intramolecularly crosslinked, a formed fumarimide bridge can change the electrostatic interaction of a human hemoglobin (HbA) molecule, stabilize the tense state (T state) of the HbA and reduce the oxygen affinity, and the Val-1(α) is positioned at the end of a central cavity αα -end of the HbA and can participate in the formation of salt bridges which are helpful for stabilizing the structure in the relaxed state (R state), and the carboxyl modification can destroy the salt bridges, reduce the R state stability of the HbA, so that the quaternary structure of the HbA is transferred from the R state to the T state and reduce the affinity of the HbA for oxygen.

Description

Hemoglobin oxygen carrier based on double chemical modification and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a hemoglobin oxygen carrier and a preparation method and application thereof, in particular to a hemoglobin oxygen carrier based on double chemical modification and a preparation method and application thereof.

Background

In recent years, with the rapid growth of the clinical blood demand and the implementation of a free blood donation system, the contradiction between blood supply and demand becomes more prominent. The traditional blood transfusion mode plays an important role in medical care, and has various defects, which are mainly reflected in that: (1) due to the existence of the window period, the risk of pathogen transmission exists in blood transfusion, such as HIV, hepatitis B virus and other pathogens possibly existing in blood; (2) the shortage of blood sources in China and the shortage of clinical blood bank reserves can cause that especially rare blood type patients lose lives due to the lack of blood of the same type; (3) the natural red blood cells have short storage time, can be stored in a blood bank at the temperature of 4 ℃ for only 5 to 6 weeks, and have harsh transportation conditions; (4) the cross matching is needed before the transfusion into the human body, which is not beneficial to the rescue in emergency. This makes the blood source that is in short supply unable to satisfy the needs of clinical treatment and emergency rescue, has also restricted the application of natural blood in the big blood loss first aid.

Hemoglobin (Hemoglobin, Hb) is tetrameric protein consisting of four subunits (ααββ) and has an oxygen carrying function, wherein the tetramer of free Hemoglobin molecules is poor in stability, a depolymerized dimer is easily filtered by glomeruli to cause renal toxicity, 2, 3-diphosphoglycerate (2,3-DPG) is not effectively supplied with oxygen to tissues due to loss of regulation, the circulating half-life period of the Hemoglobin in a body is shortened due to easy degradation by hydrolase in the body, and the Hemoglobin oxygen carrier is easy to generate inactive methemoglobin and oxygen free radicals through autoxidation due to loss of an oxidoreductase system.

Hemoglobin oxygen carriers are mainly prepared by methods such as intramolecular cross-linking, intermolecular polymerization and high polymer modification of hemoglobin. For example, glutaraldehyde polymerized bovine hemoglobin (HBOC-201) is used for the treatment of patients with acute blood loss; dextran is the main component of plasma volume expander, and its low cost, security are high, and the ability of expanding blood volume is strong, and dextran Hb has the dual function of expanding blood volume and oxygen suppliment, is applicable to acute big blood loss patient's treatment.

P₅₀The oxygen partial pressure when the oxygen saturation of the hemoglobin reaches 50 percent is a parameter reflecting the difficulty of the combination of Hb and oxygen, and the P of the hemoglobin can be improved by performing site-specific modification on the hemoglobin by adopting a small molecular chemical modification method₅₀Reducing the oxygen affinity and improving the oxygen carrying/releasing activity of the hemoglobin.

Hemoglobin oxygen carriers currently being developed generally have a higher oxygen affinity, i.e., a lower P₅₀In value, the bound oxygen is not efficiently released to hypoxic tissue. For example, PEGylation of P in Hb₅₀Is only 4-6mmHg, far below the P of the blood₅₀(-28 mmHg), due to the average binding of about 6 PEGs with a molecular weight of 5kDa per HbA molecule. The hydration layer formed by these PEG molecules shields some of the oxygen carrying/evolving active sites of HbA. P of glutaraldehyde polymerized bovine hemoglobin₅₀The value is also low, generally less than 10mmHg, because the polymerization of the bovine hemoglobin takes glutaraldehyde with low molecular weight as a connecting bridge, a plurality of bovine hemoglobin molecules are relatively close to each other, and then a large space shielding effect is generated, so that part of oxygen carrying/releasing active sites are shielded. In addition, since glutaraldehyde-mediated intermolecular crosslinking reduces the number of colloidal particles of HbA, its colloid osmotic pressure is also lower than that of blood, and thus it is not effective in expanding blood volumeAmount of the compound (A).

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a hemoglobin oxygen carrier and a preparation method and application thereof, in particular to a hemoglobin oxygen carrier based on double chemical modification and a preparation method and application thereof, comprising the hemoglobin oxygen carrier based on double chemical modification and a preparation method thereof, the hemoglobin oxygen carrier based on triple chemical modification and a preparation method thereof, and the application of the two products in the preparation of blood substitutes.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a dual chemical modification based hemoglobin oxygen carrier wherein two Val-1(α) residues are carboxymethylated and two L ys-99(α) residues are intramolecularly crosslinked.

The hemoglobin of the invention is hemoglobin A (HbA), which accounts for 98 percent of hemoglobin of adult, is tetrameric protein consisting of four subunits (ααββ), has oxygen carrying function, wherein two Val-1(α) residues (α subunit valine at position 1) are positioned at the αα -end of a central cavity of the HbA, can participate in forming a salt bridge which is helpful for stabilizing a relaxed state (R state) structure, and L ys-99(α) residues are α subunit lysine at position 99.

Two L ys-99(α) residues in the hemoglobin oxygen carrier are subjected to intramolecular crosslinking to form a fumeimide bridge, wherein the fumeimide bridge can change the electrostatic interaction of HbA molecules, stabilize the T state of HbA and reduce the oxygen affinity of HbA, the Val-1(α) is positioned at the αα -end of a central cavity of the HbA and can participate in the formation of a salt bridge which is helpful for stabilizing a relaxed state (R state) structure, and carboxylation modification can break the salt bridges to reduce the R state stability of the HbA, so that the quaternary structure of the HbA is transferred from the R state to a stressed state (T state), the affinity of the HbA to oxygen is reduced, and the P of the HbA is enhanced₅₀The present invention organically combines two modification means to improve the tetramer stability of HbA, enhance the electrostatic interaction, and improve the binding state of oxygen binding region and oxygen, and such double-modified HbA (hereinafter, referred to as "Glx- αα -Hb")) Has strong oxygen carrying/releasing activity.

In a second aspect, the present invention provides a method for preparing a hemoglobin oxygen carrier based on dual chemical modification as described above, the method comprising: carboxymethylation treatment is carried out on hemoglobin by glyoxylic acid, intramolecular cross-linking treatment is carried out on the hemoglobin by bis (3, 5-dibromo-salicylic acid) fumarate, and the hemoglobin oxygen carrier based on double chemical modification is obtained;

alternatively, the preparation method comprises: and (3) carrying out intramolecular cross-linking treatment on the hemoglobin by using bis (3, 5-dibromo-salicylic acid) fumarate, and then carrying out carboxymethylation treatment on the hemoglobin by using glyoxylic acid to obtain the hemoglobin oxygen carrier based on double chemical modification.

The hemoglobin oxygen carrier based on double chemical modification is prepared by intramolecular crosslinking of two L ys-99(α) residues of adult hemoglobin (HbA) by using bis (3, 5-dibromo-salicylic acid) fumarate (DBBF) and site-specific modification of glyoxylic acid into a Val-1(α) residue of HbA, and the two modification operations can optionally select the sequence.

Preferably, the carboxymethylation of hemoglobin by glyoxylic acid specifically comprises the following steps:

(1) deoxidizing the hemoglobin to obtain deoxidized hemoglobin;

(2) and (2) mixing the deoxyhemoglobin obtained in the step (1) with glyoxylic acid and sodium cyanoborohydride, reacting under the protection of protective gas, and terminating the reaction.

Preferably, the molar ratio of the deoxyhemoglobin, the glyoxylic acid and the sodium cyanoborohydride in the step (2) is 1 (1-5) to (5-100), wherein 1-5 can be selected from 1, 2,3, 4 or 5, 5-100 can be selected from 5, 10, 20, 40, 50, 60, 70, 80, 90 or 100, and the like, and 1:2:20 is preferred.

Preferably, the reaction temperature in step (2) is 0-8 ℃, such as 0 ℃, 2 ℃,3 ℃,4 ℃,5 ℃, 6 ℃ or 8 ℃, and the like, and the time is 2-5h, such as 2h, 3h, 4h or 5h, and other specific values in the above range can be selected, and are not repeated herein.

Preferably, glycine is used for the termination reaction in step (2).

Preferably, dialysis is carried out on the product after the termination reaction in the step (2) to remove impurities.

Preferably, the product is separated and purified by anion exchange chromatography medium after the termination reaction in step (2), wherein the medium is Q Sepharose High Performance anion exchange chromatography medium.

Preferably, the intramolecular cross-linking treatment of hemoglobin by bis (3, 5-dibromo-salicylic acid) fumarate specifically comprises the following steps:

(1) deoxidizing the hemoglobin to obtain deoxidized hemoglobin;

(2) mixing the deoxyhemoglobin obtained in the step (1) with inositol hexaphosphate, and reacting under the protection of protective gas;

(3) and (3) mixing the product obtained in the step (2) with bis (3, 5-dibromo-salicylic acid) fumarate, reacting under the protection of protective gas, and stopping the reaction.

Preferably, the molar ratio of the deoxycarboxymethylated hemoglobin to inositol hexaphosphate in step (2) is 1: 5-10, such as 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc., preferably 1:8.

Preferably, the reaction temperature in step (2) is 20-30 ℃, for example 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃ and the like, and the time is 1-5h, for example 1h, 2h, 3h, 4h or 5h and the like, and other specific values in the above range can be selected, and are not described again.

Preferably, the molar ratio of the deoxycarboxymethylated hemoglobin to the bis (3, 5-dibromo-salicylic acid) fumarate in the step (3) is 1 (0.5-5), such as 1:0.5, 1:1, 1:2, 1:3, 1:4 or 1:5, preferably 1:1, and other specific values within the above range can be selected, and are not repeated herein.

Preferably, the reaction temperature in step (3) is 20-30 ℃, for example 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃ or 30 ℃ and the like, and the time is 2-6h, for example 2h, 3h, 4h, 5h or 6h and the like, and other specific values in the above range can be selected, and are not described again.

Preferably, glycine is used for the termination reaction in step (3).

Preferably, dialysis is carried out on the product after the termination reaction in the step (3) to remove impurities.

Preferably, after the termination reaction in step (3), the product is separated and purified by using an anion exchange chromatography medium, wherein the medium is a Q Sepharose High Performance anion exchange chromatography medium.

In a third aspect, the invention provides a hemoglobin oxygen carrier based on triple chemical modification, which is obtained by modifying the hemoglobin oxygen carrier based on double chemical modification as described above through polyethylene glycol-succinimidyl ester.

In a fourth aspect, the present invention provides a method for preparing a hemoglobin oxygen carrier based on triple chemical modification as described above, the method comprising: and mixing the hemoglobin oxygen carrier based on the double chemical modification with polyethylene glycol-succinimidyl ester for reaction, and terminating the reaction to obtain the hemoglobin oxygen carrier based on the triple chemical modification.

Preferably, the polyethylene glycol-succinimide ester is a polyethylene glycol with 8 succinimide groups, i.e. an eight-arm polyethylene glycol-succinimide ester.

Preferably, the polyethylene glycol-succinimidyl ester has a number average molecular weight of 2kDa to 40kDa, such as 2kDa, 8kDa, 10kDa, 12kDa, 15kDa, 20kDa, 30kDa or 40kDa, etc., preferably 8kDa to 12kDa, and other specific values within the above range can be selected, and are not described in detail herein.

Preferably, the molar ratio of the hemoglobin oxygen carrier based on double chemical modification to the polyethylene glycol-succinimidyl ester is 1 (2-4), such as 1:2, 1:3 or 1:4, and other specific values within the above range can be selected, and are not described in detail herein.

Preferably, glycine is used for the termination reaction.

Preferably, after the termination reaction, the product is separated and purified by using a gel filtration chromatography medium, and the chromatography medium is a Superdex 200 gel filtration chromatography medium.

The PEG with the molecular weight of 2kDa to 40kDa can more effectively lengthen the distance between HbA molecules and reduce the space shielding effect among molecules, and the space shielding effect of the PEG on the HbA is reduced because a plurality of HbAs are combined with 1 PEG.

In a fifth aspect, the present invention provides the use of a hemoglobin oxygen carrier based on a dual chemical modification as described above or a hemoglobin oxygen carrier based on a triple chemical modification as described above for the preparation of a blood substitute.

Compared with the prior art, the invention has the following beneficial effects:

(1) the hemoglobin oxygen carrier based on double chemical modification introduces carboxymethyl by modifying Val-1(α) of HbA through glyoxylic acid, breaks salt bridges which participate in forming structures which are helpful for stabilizing R state, enables the quaternary structure of HbA to transfer from R state to tense state (T state), reduces the affinity of HbA to oxygen, further improves the oxygen carrying/releasing activity of HbA, utilizes DBBF to carry out intramolecular crosslinking on two L ys-99(α) of HbA, can improve the tetramer stability of HbA, simultaneously changes the electrostatic interaction of HbA molecules, enhances the oxygen carrying/releasing activity of HbA, utilizes the synergistic effect of the two modification methods, organically combines the two modification methods, can improve the tetramer stability of HbA, enhances the electrostatic interaction, improves the combination state of the oxygen combination region of HbA and oxygen, further enhances the oxygen carrying/releasing activity of HbA, and the P of the double modified HbA (Glx- αα -Hb) is used for improving the oxygen carrying/releasing activity of HbA₅₀The value reached 34.6 mmHg.

(2) The invention uses eight-arm PEG to perform intermolecular polymerization reaction on Glx- αα -Hb, and the polymerization reaction is performed on P of Glx- αα -Hb₅₀Value of P of the polymerization product PEG-Glx- αα -Hb with little effect₅₀The value reached 27.8mmHg and,p of PEG-Glx- αα -Hb substantially maintains oxygen carrying/releasing activity of Glx- αα -Hb₅₀Values are much higher than glutaraldehyde cross-linked hemoglobin and pegylated hemoglobin.

(3) The eight-arm PEG performs intermolecular polymerization reaction on the Glx- αα -Hb, can improve the colloid osmotic pressure of hemoglobin to a certain extent, can play a role in carrying/releasing oxygen of an oxygen carrier and can play a role in expanding blood volume during blood transfusion, and in contrast, the colloid osmotic pressure of glutaraldehyde cross-linked hemoglobin is lower, and cannot play a role in expanding blood volume.

Drawings

FIG. 1 is a schematic of the purified elution peaks of Glx-Hb, αα -Hb, and Glx- αα -Hb;

FIG. 2 is a schematic representation of the purified elution peaks of PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb, and PEG-Glx- αα -Hb;

FIG. 3 is a diagram showing the result of SDS-PAGE electrophoretic analysis;

FIG. 4 is a graph showing the results of gel filtration analysis;

FIG. 5 is a schematic representation of the peak elution of the enzymatically hydrolyzed peptide fragment for each sample;

FIG. 6 is a graph showing the number of active thiols and thiol reactivity in HbA, Glx-Hb, αα -Hb and Glx- αα -Hb;

FIG. 7 is a graph showing the number of active thiols and thiol reactivity in PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb and PEG-Glx- αα -Hb;

FIG. 8 is a graph of the results of circular dichroism spectroscopy analysis of HbA, Glx-Hb, αα -Hb, and Glx- αα -Hb;

FIG. 9 is a graph of results of circular dichroism spectroscopy analysis of PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb and PEG-Glx- αα -Hb;

FIG. 10 is an oxygen balance curve for HbA, Glx-Hb, αα -Hb, and Glx- αα -Hb;

FIG. 11 is an oxygen equilibrium curve for PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb, and PEG-Glx- αα -Hb;

FIG. 12 is a schematic diagram of the preparation of glyoxylated hemoglobin of example 2;

FIG. 13 is a schematic diagram of the preparation of DBBF intramolecular cross-linked hemoglobin in example 3;

FIG. 14 is a schematic of the preparation of DBBF intramolecular cross-linked Glx-Hb in example 4.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

Separation and purification of adult hemoglobin (HbA):

adult whole blood was centrifuged and the supernatant was discarded. The suspension was suspended in PBS buffer (pH7.4), and the supernatant was discarded by centrifugation. The cleaning was repeated twice. Erythrocytes were lysed with 3 volumes of water. The erythrocyte lysate was centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter. Separating and purifying the filtrate by Q Sepharose Fast Flow anion exchange chromatography medium, collecting the corresponding elution peak of adult hemoglobin (HbA), concentrating, dialyzing into PBS buffer (pH7.4), and freezing and storing at-80 ℃ for later use.

Example 2

Preparation of glyoxylated hemoglobin:

glyoxylic acid covalently modifies the N-terminal Val-1 of HbA (α) such that the introduction of a carboxymethyl group disrupts the salt bridges involved in the formation of structures that contribute to the stabilization of the R state, decreasing the stability of the relaxed state (R state) of HbA, shifting the quaternary structure of HbA from the R state to the stressed state (T state), decreasing the affinity of HbA for oxygen.

Deoxygenated HbA was prepared by continuously introducing nitrogen into a 0.5mM HbA solution. Followed by the addition of 1.0mM glyoxylic acid and 10mM sodium cyanoborohydride (NaCNBH)₃) The solution was such that the molar ratio of HbA, glyoxylic acid to sodium cyanoborohydride was 1:2: 20. Nitrogen was further introduced, and the reaction was carried out at 4 ℃ for 3.5 hours under an anaerobic condition to obtain glyoxylate-modified HbA (indicated by Glx-Hb). Finally, 20mM glycine solution was added to terminate the reaction, and unreacted glyoxylic acid and glycine were removed by dialysis. The preparation schematic diagram is shown in figure 12.

Example 3

Preparation of DBBF intramolecular cross-linked hemoglobin:

when bis (3, 5-dibromo-salicylic acid) fumarate (DBBF) modifies deoxy HbA, an intramolecular crosslinking reaction occurs between two L ys-99(α).

A deoxygenated HbA was prepared by continuously introducing nitrogen into a 0.5mM HbA solution, then adding an equal volume of 4mM Inositol Hexaphosphate (IHP) solution, continuing to introduce nitrogen, and the reaction was performed at 25 ℃ for 3h under an anaerobic condition, then adding a 0.5mM DBBF solution so that the molar ratio of HbA to DBBF was 1:1, and reacting at 25 ℃ for 4h to obtain a DBBF intramolecular cross-linked HbA (represented by αα -Hb). finally, adding a 20mM glycine solution to terminate the cross-linking reaction, and dialyzing to remove unreacted DBBF and glycine, the preparation scheme thereof is shown in FIG. 13.

Example 4

Preparation of DBBF intramolecular cross-linked Glx-Hb:

continuous nitrogen gas introduction into 0.5mM Glx-Hb solution to prepare deoxygenated Glx-Hb. then an equal volume of 4mM MIHP solution was added and treated at 25 ℃ for 3h, then 0.5mM DBBF solution was added such that the molar ratio of Glx-Hb to DBBF was 1:1 the nitrogen gas introduction was continued and the reaction was carried out at 25 ℃ for 4h in the absence of oxygen to obtain DBBF intramolecular cross-linked Glx-Hb (denoted by Glx- αα -Hb). finally, 20mM glycine solution was added to terminate the cross-linking reaction and dialyse to remove unreacted DBBF and glycine.A schematic of its preparation is shown in FIG. 14.

Example 5

Separation and purification of Glx-Hb, αα -Hb and Glx- αα -Hb:

purifying Glx-Hb, αα -Hb and Glx- αα -Hb. by using a Q Sepharose High Performance anion exchange column (0.5cm × cm) and a Q Sepharose High Performance anion column by using a 20mM Tris-acetate Buffer (pH 7.5, Buffer A) to fully balance the Glx-Hb, αα -Hb and Glx- αα -Hb, respectively loading the reaction mixture containing the Glx-Hb, the αα -Hb and the Glx- αα -Hb on the column, and after fully balancing the column by using Buffer A, carrying out gradient elution by using a 20mM Tris-acetate Buffer (pH 7.5, Buffer B) containing 0.5M sodium chloride in an amount of 0.5M L/min, as shown in figure 1, 3 separate elution peaks appear after the reaction containing αα -Hb appears, wherein the peaks 1, 2 and 3 correspond to DBF 5, 5-Hb molecules bound, 4835-Hb and 5-Hb, respectively, and the main peak 1, 2-6-Hb is collected after elution, and the main peak is concentrated, wherein the main peak is eluted, and the main peak 1, the auxiliary peak is eluted.

Example 6

Preparation of PEG modified hemoglobin:

the protein concentrations of HbA, αα -Hb, Glx-Hb and Glx- αα -Hb are all adjusted to 0.5mM and placed in PBS buffer (pH 7.4). Octadine-succinimidyl ester with the molecular weight of 10kDa is dissolved in PBS buffer (pH7.4) to make the final concentration of 1.5 mM., and equal volumes of octandaine polyethylene glycol-succinimidyl ester are respectively added into the solutions of HbA, αα -Hb, Glx-Hb and Glx- αα -Hb and reacted for 24h at 4 ℃, and finally 20mM glycine solution is added to stop the reaction, wherein the obtained polymerization products are respectively PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb and PEG-Glx- αα -Hb..

Example 7

Separation and purification of PEG modified hemoglobin:

PEG-Hb, PEG-Glx-Hb, PEG- αα -Hb and PEG-Glx- αα -Hb are separated and purified by a Superdex 200 gel filtration column (1.6cm × cm), the chromatographic column is balanced and eluted by PBS buffer solution (pH7.4), the flow rate is 2.0m L/min, and the eluent is detected by visible light at 280nm, as shown in FIG. 2, the reaction mixture containing PEG-Hb is eluted on the column to obtain 3 elution peaks, wherein the peak 1, the peak 2 and the peak 3 respectively correspond to PEG-Hb, HbA and unreacted eight-arm PEG, the peak 1 is significantly earlier than the peak 2, which shows that the polymerization reaction mediated by the eight-arm PEG can significantly enhance the molecular size of HbA, and similarly, the reaction mixture containing PEG-Glx-Hb, PEG- αα -Hb and PEG-Glx- αα -Hb is respectively eluted on the column to obtain 3 elution peaks, wherein the peak 4, the peak 6 and the peak 8 correspond to PEG-Glx- αα, PEG-Glx-678678, and the eluate is respectively collected after PEG-Glx-Hb and PEG-865-Hb are concentrated.

Example 8

SDS-PAGE electrophoretic analysis:

the concentrated peak eluate was added to an equal volume of running buffer, placed in a boiling water bath for 5min, stained with 15% electrophoresis gel, Coomassie Brilliant blue, and the purity of the sample was analyzed by SDS-PAGE electrophoresis.

As shown in FIG. 3, HbA (lane 2) shows 1 single electrophoretic band, corresponding to a molecular weight of about 16kDa, since HbA (64.0kDa) is represented by α₁α₂β₁β₂The four subunits are composed, HbA tetrameric protein is completely depolymerized into a single subunit with the molecular weight of 16.0kDa in a boiling water bath, a single electrophoresis band shows that the purity of HbA is high and single-point electrophoresis purity is achieved, Glx-Hb (lane 6) shows an electrophoresis band similar to HbA, αα -Hb (lane 4) and Glx- αα -Hb (lane 8) show two electrophoresis bands with the corresponding molecular weights of 16kDa and 32kDa, because αα -Hb and Glx- αα -Hb are composed of free β subunit (16kDa) and αα -dimer (32kDa), compared with the HbA band, PEG-Hb (lane 3) shows a electrophoresis band with 4 larger molecular weights, while the original electrophoresis band is weakened, which shows that the whole HbA molecule is polymerized by eight-arm PEG, a small number of subunits are involved in polymerization reaction of eight-arm PEG, compared with the HBA band, PEG-Glx-HbA tetrameric protein (lane 7) shows a diffusion electrophoresis band similar to PEG- αα, compared with the original PEG-5631-9-Hb shows a diffusion electrophoresis band, and a diffusion band of HBa single-1-369-9-Hb shows a single electrophoresis band.

Example 9

Gel filtration analysis:

as shown in FIG. 4, HbA, Glx-Hb, αα -Hb and Glx- αα -Hb show single and symmetrical elution peaks after elution, and the elution peak positions are respectively 18.1min, 17.8min and 17.8min, the peak volumes of αα -Hb and Glx- αα -Hb are smaller than those of HbA and Glx-Hb, indicating that αα -intramolecular cross-linking can increase tetramer stability of HbA. in contrast, the elution peak positions of PEG-Hb, PEG- αα -Hb, PEG-Glx-Hb and PEG-Glx- αα -Hb are 7.8min and are the external water volume of the gel filtration column.

Example 10

Identification of modification sites:

adding trypsin according to the mass ratio of 1:100 of the trypsin to the globin, uniformly mixing, incubating for 4 hours at 37 ℃, and freeze-drying in a freeze-drying machine, then separating enzymatic hydrolysis peptide segments by using reverse-phase HP L C, determining the molecular weight of the peptide segments by electrospray ion trap mass spectrum, identifying each analyzed peptide segment according to a primary amino acid sequence of the hemoglobin, selecting a Proteonavi C4 column (4.6mm × 250mm) as a chromatographic column of the reverse-phase HP L C, loading the enzymatic hydrolysis peptide segments into the chromatographic column, carrying out gradient elution, comparing an enzymatic hydrolysis peptide graph of αα -Hb or Glx-Hb with HbA, and determining αα -intramolecular cross-linking and carboxymethylation sites.

As shown in FIG. 5, HbA, Glx-Hb, αα -Hb, and Glx- αα -Hb all exhibited some peptide spectra after enzymatic cleavage, compared to HbA, the peptide spectrum of Glx-Hb corresponds to α -T₁Peptides (α subunit 1 st peptide stretch) and α -T₁₊₂Peptides (α subunit 1 st and 2 nd peptides) almost completely disappeared other peptides, especially α -T₃The peptide (α subunit 3 rd peptide stretch) was hardly changed, since Val-1(α) (α subunit 1 st valine) was α -T₁Peptides and α -T₁₊₂Unique sites of modification of the peptide, α -T₁Peptides and α -T₁₊₂Disappearance of the peptide indicates that Val-1(α) is a specific modification site for glyceraldehyde compared to HbA, αα -Hb corresponds to α -T in the peptide profile₁₁Peptides (α subunit 11 th peptide stretch) and α -T₁₀₊₁₁Peptides (α subunit 10 th and 11 th peptides) almost completely disappeared, while the other peptides were hardly changed, since L ys-99(α) (α subunit 11 th lysine) was α -T₁₁Peptides and α -T₁₀₊₁₁Unique sites of modification of the peptide, α -T₁₁Peptides and α -T₁₀₊₁₁The disappearance of the peptide showed L ys-99(α) to be a specific modification site for DBBF compared to HbA, the peptide profile of Glx- αα -Hb corresponds to α -T₁Peptide α -T₁₊₂Peptide α -T₁₁Peptides and α -T₁₀₊₁₁The peptide almost completely disappeared, while the other peptide fragments almost completely disappearedTherefore, Val-1(α) and L ys-99(α) of Glx- αα -Hb were specifically modified.

Example 11

Thiol reactivity of the sample:

the amount of active thiols and thiol reactivity in Hb samples were calculated as shown in FIGS. 6 and 7, where the amount of active thiols in all Hb samples was 2, as compared to the theoretical active thiol number of hemoglobin, however, FIG. 6 shows that HbA has a slightly higher thiol reactivity than α -Hb, but slightly lower thiol reactivity than Glx-Hb, which indicates that α -intramolecular cross-linking stabilizes α β interfaces, and Val-1(α) carboxymethylation destroys slightly α 1 β interfaces, similarly, FIG. 7 shows that PEG-thiol reactivity of PEG-thiol is slightly higher than that of α -Hb, but slightly lower thiol reactivity than that of Glx-Hb, and thus PEG-1- α -Lys 3-Lys-9-Hb, and PEG-1- β -Lys-are slightly higher than that of PEG-Lys- α -Lys-9-Hb, but only PEG-5-9-Hb-4-Hb-is slightly higher than that of Hb-Hb, and the PEG-Hb-4-Hb-3-Hb-4-Hb-3-Hb-has a maximum absorbance at 324nm wavelength.

Example 12

Circular dichroism spectrum analysis of the sample:

the L peak (around 260 nm) of the circular dichroism spectrum is very sensitive to the interaction of hemoglobin with surrounding globulins and is influenced by the interaction of ligands (such as oxygen molecules). As shown in FIG. 8, the L peak absorption values of αα -Hb and Glx-Hb are slightly lower than HbA but slightly higher than Glx- αα -Hb, which indicates that both αα -intramolecular cross-linking and carboxymethylation of Val-1(α) alter the interaction of oxygen with hemoglobin.As shown in FIG. 9, the L peak absorption values of PEG-Hb, PEG- αα -Hb, PEG-Glx-Hb and PEG-Glx- αα -Hb are comparable.

The light absorption values of αα -Hb and Glx-Hb at 285nm are slightly lower than HbA but slightly higher than Glx- αα -Hb.. furthermore, Glx- αα -Hb exhibits 1 deeper negative valley at 285nm, indicating that Glx- αα -Hb is in a quaternary structure similar to the T state at α 1 β 2 interface, as shown in FIG. 9. similarly, PEG- αα -Hb and PEG-Glx-Hb have slightly lower absorption values at 285nm than PEG-Hb but slightly higher than PEG-Glx- αα -Hb, indicating that PEG-Glx- αα -Hb is more prone to transition from the R state to the T state, thereby releasing more oxygen from Hb to hypoxic tissue.

As shown in FIG. 8, the absorbance of αα -Hb and Glx-Hb is higher in the Soret region than HbA, and the maximum absorbance shifts from 417nm to 415nm, indicating that αα -intramolecular cross-linking and carboxymethylation of Val-1(α) perturbs the hemoglobin microenvironment.

Example 13

Measurement of oxygen carrying/releasing Activity:

the sample was measured by a Hemox blood oxygen analyzer to obtain an oxygen balance curve. P can be directly obtained from the oxygen equilibrium curve₅₀The measurement was started by adding a sample containing 6mg HbA to 4m L HEMOX buffer, adding 20 μ L bovine serum albumin and 10 μ L antifoam, bathing in a water bath at 37 ℃ for 5min, placing the sample in a cell, waiting for the temperature to rise back to 37 ℃, and setting parameters.

As shown in FIG. 10, the oxygen-carrying curve of HbA is at saturation at physiological oxygen partial pressure conditions (90-100 mmHg.) in contrast, the oxygen-carrying curves of Glx-Hb and αα -Hb shift to the right and approach saturation at physiological oxygen partial pressure conditions the oxygen-carrying curve of Glx- αα -Hb shifts further to the rightThe hemoglobin samples all have better oxygen carrying capacity, P of HbA, αα -Hb, Glx-Hb and Glx- αα -Hb₅₀The values were 14.8, 24.0, 25.6 and 34.6mmHg, respectively, which indicates that αα -intramolecular cross-linking and carboxymethylation of Val-1(α) can increase the P of HbA by way of a synergistic effect₅₀Values shown in FIG. 11 for P of PEG-Hb, PEG- αα -bHb, PEG-Glx-bHb, and PEG-Glx- αα -Hb₅₀The values were 10.8, 18.1, 20.1 and 27.8mmHg, respectively, and thus, the eight-arm PEG-mediated polymerization reaction slightly reduced the P of HbA and its derivatives₅₀The value is obtained.

Oxygen transport efficiency by P₅₀The OTE values of HbA, αα -Hb, Glx-Hb and Glx- αα -Hb are 9.1%, 25.5%, 25.7% and 33.1%, respectively, which indicates that αα -intramolecular cross-linking and carboxymethylation of Val-1(α) can significantly improve the oxygen-releasing ability of HbA by changing the four-stage structural transition from the T state to the R state of HbA in a synergistic manner, and the oxygen transport efficiency values of PEG-Hb, PEG- αα -Hb, PEG-Glx-Hb and PEG-Glx- αα -Hb are 8.3%, 23.2%, 21.9% and 30.5%, respectively, which indicates that PEG-Glx- αα -Hb has a strong oxygen-releasing ability, while the eight-arm PEG-mediated polymerization reaction can largely maintain its oxygen-carrying/releasing function.

The Hill coefficient of HbA is 2.7, which indicates that four subunits have better synergistic effect, αα -Hb, the Hill coefficients of Glx-Hb and Glx- αα -Hb are 2.0, 2.1 and 2.0 respectively, which indicates that αα -intramolecular cross-linking and carboxymethylation of Val-1(α) can influence the subunit synergistic effect of HbA to some extent, and the Hill coefficients of PEG-Hb, PEG- αα -bHb, PEG-Glx-bHb and PEG-Glx- αα -Hb are 1.8, 1.6, 1.7 and 1.4 respectively, which indicates that eight-arm PEG-mediated polymerization reaction further reduces the subunit synergistic effect of HbA.

Example 14

Determination of the colloid osmotic pressure:

the osmolality of the sample was determined by a Wescor 4420 osmolarity Meter (Wescor, USA) at 25 deg.C, with HbA and PEG-Glx- αα -Hb both at a protein concentration of 40mg/m L, dissolved in PBS buffer (pH 7.4). As determined, the osmolality of HbA was 15.3mmHg and the osmolality of PEG-Glx- αα -Hb was 19.6 mmHg.

Then, glutaraldehyde-crosslinked HbA is prepared by (1) placing HbA and glutaraldehyde in PBS buffer (pH7.4) at concentrations of 32mg/m L and 0.1% (v/v), respectively, (2) mixing 5m L HbA solution with 5m L glutaraldehyde solution and reacting for 2h, (3) adding 50mg/m L glycine aqueous solution (0.4m L) to terminate the crosslinking reaction, (4) placing the reaction mixture in a dialysis bag having a molecular weight cut-off of 10kDa, and dialyzing with PBS buffer (pH7.4) to remove unreacted glutaraldehyde and glycine, concentrating the glutaraldehyde-crosslinked HbA solution and adjusting its concentration to 40mg/m L, which is determined to have an osmolarity of 11.7. this indicates that glutaraldehyde crosslinking to HbA has an osmolarity lower than HbA because glutaraldehyde crosslinking causes intermolecular crosslinking, reduces the number of colloidal particles of HbA, thereby reducing its osmolarity, and the osmolarity of PEG- αα HbA is higher than the osmolarity of the PEG-HbA polymerization reaction, thereby compensating for the increased osmolarity of the colloid-HbA.

The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (10)

1. A dual chemical modification based hemoglobin oxygen carrier, wherein two Val-1(α) residues are carboxymethylated and two L ys-99(α) residues are intramolecularly crosslinked.

2. The method of claim 1, wherein the method comprises: carboxymethylation treatment is carried out on hemoglobin by glyoxylic acid, intramolecular cross-linking treatment is carried out on the hemoglobin by bis (3, 5-dibromo-salicylic acid) fumarate, and the hemoglobin oxygen carrier based on double chemical modification is obtained;

alternatively, the preparation method comprises: and (3) carrying out intramolecular cross-linking treatment on the hemoglobin by using bis (3, 5-dibromo-salicylic acid) fumarate, and then carrying out carboxymethylation treatment on the hemoglobin by using glyoxylic acid to obtain the hemoglobin oxygen carrier based on double chemical modification.

3. The method for preparing the hemoglobin-based oxygen carrier based on dual chemical modification of claim 2, wherein the carboxymethylation of hemoglobin by glyoxylic acid comprises the following steps:

(1) deoxidizing the hemoglobin to obtain deoxidized hemoglobin;

(2) and (2) mixing the deoxyhemoglobin obtained in the step (1) with glyoxylic acid and sodium cyanoborohydride, reacting under the protection of protective gas, and terminating the reaction.

4. The method for preparing the dual chemical modification based hemoglobin oxygen carrier of claim 3, wherein the molar ratio of the deoxyhemoglobin, the glyoxylic acid and the sodium cyanoborohydride in the step (2) is 1 (1-5) to (5-100), preferably 1:2: 20;

preferably, the reaction temperature of the step (2) is 0-8 ℃, and the reaction time is 2-5 h;

preferably, glycine is used for the termination reaction in step (2);

preferably, after the termination reaction in the step (2), performing dialysis to remove impurities from the product;

preferably, the product is separated and purified by anion exchange chromatography medium after the termination reaction in step (2), wherein the medium is Q Sepharose High Performance anion exchange chromatography medium.

5. The method for preparing the hemoglobin-based oxygen carrier based on dual chemical modification of claim 2, wherein the intramolecular cross-linking treatment of hemoglobin with bis (3, 5-dibromosalicylic acid) fumarate comprises the following steps:

(1) deoxidizing the hemoglobin to obtain deoxidized hemoglobin;

(2) mixing the deoxyhemoglobin obtained in the step (1) with inositol hexaphosphate, and reacting under the protection of protective gas;

(3) and (3) mixing the product obtained in the step (2) with bis (3, 5-dibromo-salicylic acid) fumarate, reacting under the protection of protective gas, and stopping the reaction.

6. The method for preparing the hemoglobin-based oxygen carrier based on dual chemical modification of claim 5, wherein the molar ratio of the deoxycarboxymethylated hemoglobin to inositol hexaphosphate in the step (2) is 1 (5-10), preferably 1: 8;

preferably, the reaction temperature of the step (2) is 20-30 ℃, and the time is 1-5 h;

preferably, the molar ratio of the deoxycarboxymethylated hemoglobin to the bis (3, 5-dibromo-salicylic acid) fumarate in the step (3) is 1 (0.5-5), preferably 1: 1;

preferably, the reaction temperature of the step (3) is 20-30 ℃, and the time is 2-6 h;

preferably, glycine is used for the termination reaction in step (3);

preferably, after the termination reaction in the step (3), performing dialysis to remove impurities from the product;

preferably, after the termination reaction in step (3), the product is separated and purified by using an anion exchange chromatography medium, wherein the medium is a Q Sepharose High Performance anion exchange chromatography medium.

7. A hemoglobin oxygen carrier based on triple chemical modification, which is obtained by modifying the hemoglobin oxygen carrier based on double chemical modification according to claim 1 with polyethylene glycol-succinimidyl ester.

8. The method of claim 7, wherein the method comprises: and mixing the hemoglobin oxygen carrier based on the double chemical modification with polyethylene glycol-succinimidyl ester for reaction, and terminating the reaction to obtain the hemoglobin oxygen carrier based on the triple chemical modification.

9. The method of claim 8, wherein the polyethylene glycol-succinimide ester is polyethylene glycol with 8 succinimide groups;

preferably, the polyethylene glycol-succinimidyl ester has a number average molecular weight of 2kDa to 40kDa, preferably 8kDa to 12 kDa;

preferably, the molar ratio of the hemoglobin oxygen carrier based on double chemical modification to the polyethylene glycol-succinimidyl ester is 1 (2-4);

preferably, the termination reaction uses glycine;

preferably, after the termination reaction, the product is separated and purified by using a gel filtration chromatography medium, and the chromatography medium is a Superdex 200 gel filtration chromatography medium.

10. Use of the dual chemical modification based hemoglobin oxygen carrier of claim 1 or the triple chemical modification based hemoglobin oxygen carrier of claim 7 in the preparation of a blood substitute.

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