CN111499732B - 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 double chemical modification, a preparation method and application thereof, wherein two Val-1(alpha) residues in the hemoglobin oxygen carrier based on double chemical modification are carboxymethylated, and two Lys-99(alpha) residues are intramolecular crosslinked. Wherein two Lys-99(alpha) residues are subjected to intramolecular cross-linking, and a formed fumarimide bridge can change into electrostatic interaction of human hemoglobin (HbA) molecules, stabilize the tense state (T state) of the HbA and reduce the oxygen affinity of the HbA; val-1(α) is located α α -terminal to the central cavity of HbA and can participate in the formation of salt bridges that help stabilize the relaxed state (R state) structure, and the carboxylation modification can disrupt these salt bridges and decrease the R state stability of HbA, thereby shifting the quaternary structure of HbA from the R state to the T state and decreasing the affinity of HbA for oxygen. The invention organically combines two modification means, improves the stability of HbA tetramer, enhances electrostatic interaction, improves the combination state of an oxygen combination area and oxygen, and has strong oxygen carrying/releasing activity.

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 (Hb) is a tetrameric protein composed of four subunits (α α β β), and has an oxygen-carrying function. Because the tetramer of the free hemoglobin molecule has poor stability, the dimer formed by depolymerization is easy to be filtered by glomerulus to cause nephrotoxicity; failure to efficiently supply oxygen to tissues due to loss of regulation by 2, 3-diphosphoglycerate (2, 3-DPG); the circulating half-life period in the organism is shortened because the protein is easily degraded by hydrolytic enzyme in the organism; the loss of the oxidoreductase system leads to the problem that inactive methemoglobin and oxygen free radicals are easily produced by autooxidation. The hemoglobin oxygen carrier is based on human or animal hemoglobin, and has its antigenicity and toxicity eliminated and its circulation half life prolonged so as to replace erythrocyte safely and effectively to exert oxygen carrying/releasing activity. The hemoglobin oxygen carrier is an ideal blood substitute, can be stored for a long time, does not need cross matching, can avoid diseases transmitted by menstrual blood, and is one of the hot spots of domestic and foreign researches at present.

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 blood volume cannot be effectively expanded.

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 Lys-99(α) residues are intramolecularly crosslinked.

The hemoglobin related by the invention is hemoglobin A (HbA), which accounts for 98% of hemoglobin of adult human, is tetrameric protein consisting of four subunits (alpha beta), and has an oxygen carrying function; wherein, two Val-1(alpha) residues (valine at position 1 of alpha subunit) are positioned at alpha-terminal of HbA central cavity and can participate in forming salt bridge which is helpful for stabilizing relaxed state (R state) structure, and Lys-99(alpha) residue is lysine at position 99 of alpha subunit.

Two Lys-99(alpha) residues in the hemoglobin oxygen carrier related by the invention are subjected to intramolecular crosslinking, and a formed fumarimide bridge can change the electrostatic interaction of HbA molecules, stabilize the T state of HbA and reduce the oxygen affinity; wherein Val-1(alpha) is positioned at alpha-end of a central cavity of HbA and can participate in forming salt bridges which are helpful for stabilizing a relaxed state (R state) structure, and carboxylation modification can damage the salt bridges and reduce the R state stability of HbA, so that the quaternary structure of HbA is transferred from the R state to a stressed state (T state), the affinity of HbA for oxygen is reduced, and the P of HbA is enhanced₅₀. The invention organically combines two modification means, can improve the tetramer stability of HbA, enhance electrostatic interaction and improve the combination state of an oxygen combination area and oxygen, and the dual-modified HbA (shown by Glx-alpha-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 Lys-99(alpha) residues of adult hemoglobin (HbA) by bis (3, 5-dibromo-salicylic acid) fumarate (DBBF) and site-specific modification of glyoxylic acid into a Val-1(alpha) residue of HbA, and the two modification operations can be optionally selected in sequence. The method is simple and easy to operate.

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 present invention uses polyethylene glycol-succinimidyl ester to perform intermolecular polymerization of the hemoglobin oxygen carrier (Glx- α α -Hb) based on the double chemical modification. The 2kDa-40kDa PEG can effectively lengthen the distance between HbA molecules and reduce the intermolecular space shielding effect, and the space shielding effect of the PEG on the HbA is reduced because a plurality of HbAs are combined with 1 PEG. This modification can largely maintain the high oxygen carrying/releasing activity of Glx- α -Hb. In addition, since PEG can bind to a plurality of water molecules, the colloid osmotic pressure of the polymerized hemoglobin can be enhanced to some extent.

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 invention relates to hemoglobin based on dual chemical modificationThe oxygen carrier introduces carboxymethyl through modification of Val-1(alpha) of HbA by glyoxylic acid, and breaks down salt bridges which participate in formation of structures contributing to stabilization of an R state, so that a quaternary structure of the HbA is transferred from the R state to a tense state (T state), the affinity of the HbA to oxygen is reduced, and the oxygen carrying/releasing activity of the HbA is further improved; two Lys-99(alpha) of HbA are subjected to intramolecular crosslinking by DBBF, so that the tetramer stability of the HbA can be improved, the electrostatic interaction of HbA molecules is changed, and the oxygen carrying/releasing activity of the HbA is enhanced; by utilizing the synergistic effect of the two modification methods, the two modification methods are organically combined, so that the stability of the tetramer of HbA can be improved, the electrostatic interaction is enhanced, the combination state of an oxygen combination area of the HbA and oxygen is improved, and the oxygen carrying/releasing activity of the HbA is enhanced. This dual modification of P of HbA (Glx- α α -Hb)₅₀The value reached 34.6 mmHg.

(2) The invention uses eight-arm PEG to perform intermolecular polymerization of Glx-alpha-Hb. P of Glx-alpha-Hb by the polymerization₅₀Value of little influence, P of the polymerization product PEG-Glx-alpha-Hb₅₀The value reaches 27.8mmHg, and the oxygen carrying/releasing activity of the Glx-alpha-Hb can be substantially maintained. P of PEG-Glx-alpha-Hb₅₀Values are much higher than glutaraldehyde cross-linked hemoglobin and pegylated hemoglobin.

(3) The eight-arm PEG performs intermolecular polymerization reaction on the Glx-alpha-Hb, can improve the colloid osmotic pressure of hemoglobin to a certain extent, and can play a role in carrying/releasing oxygen of an oxygen carrier and expanding blood volume during blood transfusion; in contrast, glutaraldehyde-crosslinked hemoglobin has a low colloid osmotic pressure and cannot function to expand blood volume.

Drawings

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

FIG. 2 is a schematic representation of the purified elution peaks of PEG-Hb, PEG-Glx-Hb, PEG- α -Hb, and PEG-Glx- α -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 in HbA, Glx-Hb, α α -Hb and Glx- α α -Hb and the thiol reactivity;

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

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

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

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

FIG. 11 is an oxygen balance curve for PEG-Hb, PEG-Glx-Hb, PEG- α α -Hb, and PEG-Glx- α α -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 (pH 7.4), 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 (pH 7.4), and freezing and storing at-80 ℃ for later use.

Example 2

Preparation of glyoxylated hemoglobin:

glyoxylic acid covalently modifies the N-terminal Val-1(alpha) of HbA, so that the introduction of a carboxymethyl group disrupts salt bridges involved in the formation of structures that contribute to the stabilization of the R state, reduces the stability of the relaxed state (R state) of HbA, shifts the quaternary structure of HbA from the R state to the stressed state (T state), and reduces 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-dibromosalicylic acid) fumarate (DBBF) modifies deoxy HbA, an intramolecular crosslinking reaction occurs between the two Lys-99 (. alpha.).

Deoxygenated HbA was prepared by continuously introducing nitrogen into a 0.5mM HbA solution. Subsequently, an equal volume of 4mM Inositol Hexaphosphate (IHP) solution was added, nitrogen was continued, and the reaction was carried out in the absence of oxygen at 25 ℃ for 3 h. Subsequently, a 0.5mM DBBF solution was added so that the molar ratio of HbA to DBBF was 1:1, and reacted at 25 ℃ for 4 hours to obtain DBBF intramolecularly crosslinked HbA (expressed by. alpha. -Hb). Finally, 20mM glycine solution was added to terminate the crosslinking reaction, and unreacted DBBF and glycine were removed by dialysis. The preparation scheme is shown in figure 13.

Example 4

Preparation of DBBF intramolecular cross-linked Glx-Hb:

deoxygenated Glx-Hb was prepared by continuously introducing nitrogen into a 0.5mM Glx-Hb solution. Subsequently, an equal volume of 4mM IHP solution was added and treated at 25 ℃ for 3 h. Subsequently, 0.5mM DBBF solution was added so that the molar ratio of Glx-Hb to DBBF was 1: 1. And continuously introducing nitrogen, and carrying out the reaction for 4h at 25 ℃ under the anaerobic condition to obtain Glx-Hb (expressed by Glx-alpha-Hb) with DBBF intramolecular cross-linking. Finally, 20mM glycine solution was added to terminate the crosslinking reaction, and unreacted DBBF and glycine were removed by dialysis. The preparation schematic diagram is shown in figure 14.

Example 5

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

Glx-Hb, α α α -Hb and Glx- α α -Hb were purified by Q Sepharose High Performance anion exchange chromatography (0.5 cm. times.5 cm). The Q Sepharose High Performance anion chromatography column was well equilibrated with 20mM Tris-acetate Buffer (pH 7.5, Buffer A). The reaction mixtures containing Glx-Hb, α α -Hb and Glx- α -Hb were loaded onto the chromatography columns, respectively, and after the columns were equilibrated with Buffer A, elution was carried out by gradient elution using 0-50% 20mM Tris-acetate Buffer (pH 7.5, Buffer B) containing 0.5M sodium chloride. The flow rate was 2.5 mL/min. As shown in fig. 1: the reaction mixture containing α α α -Hb eluted with 3 separate elution peaks, wherein peak 1, peak 2 and peak 3 correspond to HbA, α α -Hb and unreacted HbA bound to multiple DBBF molecules, respectively; after the reaction mixed liquor containing the Glx-Hb is eluted, 2 separated main elution peaks appear, wherein a peak 4 corresponds to the Glx-Hb; the reaction mixture containing Glx- α α -Hb eluted with 2 separate main elution peaks, wherein peak 6 corresponds to Glx- α -Hb. And respectively collecting elution peaks corresponding to the peak 2, the peak 4 and the peak 6, and concentrating for later use.

Example 6

Preparation of PEG modified hemoglobin:

the protein concentrations of HbA, α α -Hb, Glx-Hb, and Glx- α -Hb were each adjusted to 0.5mM and placed in PBS buffer (pH 7.4). An eight-arm polyethylene glycol-succinimidyl ester having a molecular weight of 10kDa was dissolved in PBS buffer (pH 7.4) to give a final concentration of 1.5 mM. Adding equal volume of eight-arm polyethylene glycol-succinimide ester into HbA, alpha-Hb, Glx-Hb and Glx-alpha-Hb solutions respectively, reacting for 24h at 4 ℃, and obtaining polymerization products of PEG-Hb, PEG-Glx-Hb, PEG-alpha-Hb and PEG-Glx-alpha-Hb respectively. Finally, 20mM glycine solution was added to stop the reaction.

Example 7

Separation and purification of PEG modified hemoglobin:

PEG-Hb, PEG-Glx-Hb, PEG- α α -Hb and PEG-Glx- α -Hb were separated and purified by Superdex 200 gel filtration column (1.6 cm. times.60 cm). The column was equilibrated and eluted with PBS buffer (pH 7.4) at a flow rate of 2.0mL/min, and the eluate was detected by visible light at 280 nm. As shown in fig. 2: eluting a reaction mixture containing PEG-Hb on a column to obtain 3 elution peaks, wherein a peak 1, a peak 2 and a peak 3 respectively correspond to the PEG-Hb, the HbA and unreacted eight-arm PEG, and the peak 1 is obviously earlier than the peak 2, which indicates that the molecular size of the HbA can be remarkably enhanced by the polymerization reaction mediated by the eight-arm PEG; similarly, the reaction mixture containing PEG-Glx-Hb, PEG- α α -Hb and PEG-Glx- α α -Hb was eluted separately from the column, and 3 elution peaks were obtained. Wherein, peak 4, peak 6 and peak 8 correspond to PEG- α -Hb, PEG-Glx-Hb and PEG-Glx- α -Hb, respectively. And collecting the eluent corresponding to the peaks, and concentrating for later use.

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, due to the fact that HbA (64.0kDa) is represented by alpha₁α₂β₁β₂The HbA tetrameric protein is completely depolymerized into a single subunit with the molecular weight of 16.0kDa in a boiling water bath, and a single electrophoresis strip shows that the HbA has high purity and reaches single-point electrophoresis purity; Glx-Hb (lane 6) exhibits an electrophoretic band similar to HbA; α α -Hb (lane 4) and Glx- α -Hb (lane 8) exhibit two electrophoretic bands, corresponding to molecular weights of 16kDa and 32kDa, since α α -Hb and Glx- α -Hb are composed of free β subunits (16kDa) and α α -dimers (32 kDa); PEG-Hb (lane 3) exhibits 4 electrophoretic bands with a larger molecular weight than the band of HbA, and the original electrophoretic band becomes weaker, which indicates that the whole HbA molecule is polymerized by the eight-arm PEG, but a few HbA subunits are involved in the polymerization of the eight-arm PEG; PEG-Glx-Hb (lane 7) exhibits an electrophoretic band similar to PEG-Hb; PEG- α -Hb (lane 5) exhibited 1 band compared to the band for α α -HbThe dispersive electrophoresis strip is presented, and the original two electrophoresis strips are weakened; PEG-Glx- α -Hb (lane 9) exhibits an electrophoretic band similar to PEG- α -Hb. Lane 1 is marker.

Example 9

Gel filtration analysis:

the samples were analyzed by analytical Superose 6 gel filtration column (1 cm. times.30 cm). As shown in FIG. 4, HbA, Glx-Hb, α α -Hb, and Glx- α α -Hb show single and symmetrical elution peaks after elution, and the elution peaks are at 18.1min, 17.8min, and 17.8min, respectively. The peak volumes of α α -Hb and Glx- α α -Hb are less than HbA and Glx-Hb, indicating that α α -intramolecular cross-linking can increase tetramer stability of HbA. In contrast, PEG-Hb, PEG- α α -Hb, PEG-Glx-Hb and PEG-Glx- α α -Hb eluted at peak position 7.8min, which is the external water volume of the gel filtration column. This indicates that the eight-arm PEG-mediated polymerization reaction can significantly enhance the molecular weight of HbA and its derivatives, resulting in apparent molecular weights of 4 samples exceeding the upper limit of detection of the gel filtration column.

Example 10

Identification of modification sites:

removing heme of hemoglobin by an acid acetone method to obtain globin. Trypsin can hydrolyze the carboxy side of lysine and arginine. Adding trypsin according to the mass ratio of 1:100 of trypsin to globin, mixing uniformly, incubating for 4h at 37 ℃, and freeze-drying in a freeze-dryer. Subsequently, the enzymatically cleaved peptide fragments were separated using reverse phase HPLC and the molecular weight of the peptide fragments was determined by electrospray ion trap mass spectrometry. The primary amino acid sequence of each resolved peptide fragment was identified with reference to hemoglobin. The reversed-phase HPLC column was a Proteonavi C4 column (4.6 mm. times.250 mm). Loading the enzymolysis peptide segment into a chromatographic column, and then carrying out gradient elution. The enzymatic peptide map of α α α -Hb or Glx-Hb is compared to HbA to determine the sites of α α -intramolecular cross-linking and carboxymethylation.

As shown in fig. 5: HbA, Glx-Hb, α α -Hb and Glx- α -Hb all exhibit some peptide profile after enzymatic cleavage. The peptide profile of Glx-Hb corresponds to alpha-T compared to HbA₁Peptide (alpha subunit 1 st peptide fragment) and alpha-T₁₊₂Peptide (alpha subunit) Basal 1 and 2 nd peptides) almost completely disappeared. Other peptide fragments, especially alpha-T₃The peptide (alpha subunit 3 rd peptide stretch) is hardly changed. Since Val-1 (. alpha.) (valine at position 1 of. alpha. subunit) is. alpha. -T₁Peptides and alpha-T₁₊₂Unique modifiable site of peptide, alpha-T₁Peptides and alpha-T₁₊₂Disappearance of the peptide indicates that Val-1 (. alpha.) is a specific modification site for glyceraldehyde. The peptide profile of α α -Hb compared to HbA corresponds to α -T₁₁Peptide (alpha subunit 11 th peptide fragment) and alpha-T₁₀₊₁₁The peptides (alpha subunit 10 th and 11 th peptides) were almost completely disappeared, while the other peptides were hardly changed. Since Lys-99(alpha) (alpha subunit lysine 11) is alpha-T₁₁Peptides and alpha-T₁₀₊₁₁Unique modifiable site of peptide, alpha-T₁₁Peptides and alpha-T₁₀₊₁₁The disappearance of the peptide indicates that Lys-99 (. alpha.) is the specific modification site for DBBF. The peptide profile of Glx- α α -Hb corresponds to α -T compared to HbA₁Peptide, alpha-T₁₊₂Peptide, alpha-T₁₁Peptides and alpha-T₁₀₊₁₁The peptide disappeared almost completely, while the other peptide fragments were hardly changed. Thus, Val-1(α) and Lys-99(α) of Glx- α α -Hb are specifically modified.

Example 11

Thiol reactivity of the sample:

the thiol reactivity of cysteine [ Cys-93 (beta) ] at position 93 of oxyhemoglobin can be used to assess the change in the hemoglobin α 1 β 2 interface. Since 4, 4' -dithiodipyridine (4-PDS) can react with-SH to form 4-thiopyridone, the substance has a maximum absorbance value at a wavelength of 324 nm. The quantity of the active sulfydryl and the reactivity of the sulfydryl in the Hb sample can be calculated by using the absorbance value as a function of time. As shown in fig. 6 and 7: the number of active thiols in all Hb samples was 2, which is the same as the theoretical number of active thiols of hemoglobin. However, FIG. 6 shows that HbA is slightly more thiol reactive than α α -Hb, but slightly less than Glx-Hb, indicating that α α α -intramolecular cross-linking stabilizes the α 1 β 2 interface, while carboxymethylation of Val-1(α) slightly disrupts the α 1 β 2 interface. Similarly, the thiol reactivity of PEG-Hb is shown in FIG. 7 to be slightly higher than PEG- α α -Hb but slightly lower than PEG-Glx-Hb, however, comparing FIG. 6 and FIG. 7 shows that the reactivity of PEG-Hb, PEG- α α -Hb and PEG-Glx-Hb are all slightly higher than the corresponding reactivity of HbA, α α -Hb and Glx-Hb, indicating that the eight arm PEG-mediated polymerization only slightly disrupts the α 1 β 2 interface of HbA.

Example 12

Circular dichroism spectrum analysis of the sample:

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

The light absorption value of the circular dichroism spectrum near 285nm can reflect the transition of the relaxed state (R state) of the hemoglobin to the tense state (T state). As shown in fig. 8: the absorption values of α α -Hb and Glx-Hb at 285nm are slightly lower than HbA, but slightly higher than Glx- α -Hb. In addition, Glx- α α -Hb exhibits 1 deeper negative valley at 285nm, indicating that Glx- α α -Hb is in a quaternary structure similar to the T state at the α 1 β 2 interface. As shown in fig. 9: similarly, PEG- α -Hb and PEG-Glx-Hb have slightly lower absorption values at 285nm than PEG-Hb, but slightly higher absorption values than PEG-Glx- α -Hb, indicating that PEG-Glx- α -Hb has a greater tendency 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 α α -Hb and Glx-Hb is higher in the Soret region than in HbA, and the maximum absorbance shifts from 417nm to 415nm, indicating that α α -intramolecular cross-linking and carboxymethylation of Val-1(α) perturb the microenvironment of hemoglobin. In contrast, the absorbance of Glx- α α -Hb is slightly higher than HbA, and the maximum absorbance is still at 417 nm. As shown in fig. 9: the absorbance of PEG-Glx- α -Hb in the Soret region was higher than that of Glx- α -Hb, indicating that the eight-arm PEG-mediated polymerization altered the interaction of the heme molecule with the surrounding microenvironment, where the heme molecule was more exposed.

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 curve₅₀A value for evaluating the oxygen affinity of the sample; the oxygen transport efficiency of the sample, i.e. the difference between the arterial oxygen partial pressure (40mmHg) and the venous oxygen partial pressure (110mmHg), was calculated. The oxygen transport efficiency can be used to evaluate the oxygen carrying/releasing activity of the sample; the Hill coefficient was calculated for evaluating the cooperativity of the four subunits of the sample. Adding a sample containing 6mg HbA into 4mL of HEMOX buffer solution, adding 20 mu L of bovine serum albumin and 10 mu L of defoaming agent, carrying out water bath at 37 ℃ for 5min, putting the sample into a sample pool, waiting for the temperature to rise to 37 ℃, setting parameters, and starting to measure.

As shown in fig. 10: the oxygen-carrying curve of HbA is saturated at physiological oxygen partial pressures (90-100 mmHg). In contrast, the oxygen-carrying curves for Glx-Hb and α α -Hb shift to the right and approach saturation at physiological oxygen partial pressures. The oxygen-carrying curve of Glx- α α -Hb shifts further to the right and assumes an unsaturated state under physiological oxygen partial pressure conditions. This indicates that these hemoglobin samples all have better oxygen carrying capacity. P of HbA, alpha-Hb, Glx-Hb and Glx-alpha-Hb₅₀The values were 14.8, 24.0, 25.6 and 34.6mmHg, respectively. This indicates that α α -intramolecular cross-linking and carboxymethylation of Val-1(α) can increase the P of HbA by way of a synergistic effect₅₀The value is obtained. As shown in fig. 11: p of PEG-Hb, PEG-alpha-bHb, PEG-Glx-bHb and PEG-Glx-alpha-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 influence of the value is large. The OTE values of HbA, α α -Hb, Glx-Hb and Glx- α -Hb were 9.1%, 25.5%, 25.7% and 33.1%, respectively. This indicates that α α -intramolecular cross-linking and carboxymethylation of Val-1(α) can alter the quaternary structural transition from the T state to the R state of HbA in a synergistic manner, significantly improving the ability of HbA to release oxygen. The values of oxygen transport efficiency for PEG-Hb, PEG- α -Hb, PEG-Glx-Hb and PEG-Glx- α -Hb were 8.3%, 23.2%, 21.9% and 30.5%, respectively. This indicates that PEG-Glx-alpha-Hb has strong propertiesOxygen releasing ability, while the polymerization reaction mediated by the eight-arm PEG can largely maintain the oxygen carrying/releasing function.

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

Example 14

Determination of the colloid osmotic pressure:

the osmolality of the sample was measured by a Wescor 4420 colloid osmometer (Wescor, USA) at a temperature of 25 ℃. Both HbA and PEG-Glx- α α -Hb protein concentrations were 40mg/mL and were dissolved in PBS buffer (pH 7.4). The oncotic pressure of HbA was determined to be 15.3mmHg, while the oncotic pressure of PEG-Glx- α -Hb was determined to be 19.6 mmHg.

Glutaraldehyde cross-linked HbA is then prepared, comprising the steps of: (1) HbA and glutaraldehyde were placed in PBS buffer (pH 7.4) at concentrations of 32mg/mL and 0.1% (v/v), respectively; (2) mixing 5mL of HbA solution with 5mL of glutaraldehyde solution, and reacting for 2 h; (3) adding 50mg/mL glycine aqueous solution (0.4mL) to terminate the crosslinking reaction; (4) the reaction mixture was placed in a dialysis bag with a molecular weight cut-off of 10kDa and dialyzed against PBS buffer (pH 7.4) to remove unreacted glutaraldehyde and glycine. The glutaraldehyde-crosslinked HbA solution was concentrated, and the concentration was adjusted to 40 mg/mL. The colloid osmotic pressure was determined to be 11.7 mmHg. This indicates that the colloid osmotic pressure of glutaraldehyde crosslinking to HbA is lower than that of HbA because glutaraldehyde crosslinking crosslinks HbA molecules, reducing the number of colloid particles of HbA, thereby lowering its colloid osmotic pressure; the colloid osmotic pressure of PEG-Glx-alpha-Hb is higher than that of HbA, because PEG can be combined with a large number of water molecules, so that the colloid osmotic pressure is enhanced, and the colloid osmotic pressure reduction caused by polymerization reaction is compensated to a certain extent.

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 modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the 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 (14)

1. The hemoglobin oxygen carrier based on the triple chemical modification is characterized in that two Val-1(alpha) residues of hemoglobin in the hemoglobin oxygen carrier based on the triple chemical modification are carboxymethylated, two Lys-99(alpha) residues are intramolecular cross-linked, and the hemoglobin oxygen carrier is modified by eight-arm polyethylene glycol-succinimide ester, wherein the eight-arm polyethylene glycol-succinimide ester is polyethylene glycol with 8 succinimide groups.

2. The method of claim 1, wherein the method comprises: carboxymethylation treatment is carried out on the hemoglobin by glyoxylic acid, and intramolecular cross-linking treatment is carried out on the hemoglobin by bis (3, 5-dibromo-salicylic acid) fumarate; the resulting product is then reacted with polyethylene glycol bearing 8 succinimide groups, terminating the reaction, to yield a hemoglobin-based oxygen carrier based on triple chemical modifications.

3. The method of claim 2 for preparing the hemoglobin-based oxygen carrier based on triple chemical modification, comprising the steps of:

(1) deoxidizing the hemoglobin to obtain deoxidized hemoglobin; mixing the deoxyhemoglobin with glyoxylic acid and sodium cyanoborohydride, reacting under the protection of protective gas, and terminating the reaction;

(2) mixing the product obtained in the step (1) with inositol hexaphosphate, and reacting under the protection of protective gas; then mixing the mixture with bis (3, 5-dibromo-salicylic acid) fumarate, reacting under the protection of protective gas, and stopping the reaction;

(3) and (3) reacting the product obtained in the step (2) with polyethylene glycol with 8 succinimide groups, and stopping the reaction to obtain the hemoglobin oxygen carrier based on triple chemical modification.

4. The method for preparing the hemoglobin-based oxygen carrier based on triple chemical modifications of claim 3, wherein the molar ratio of the deoxyhemoglobin, the glyoxylic acid and the sodium cyanoborohydride in the step (1) is 1 (1-4) to (10-20); the reaction temperature is 0-8 ℃, and the reaction time is 2-5 h; glycine is used for termination reaction; after the reaction is terminated, dialysis is carried out to remove impurities, and a product is separated and purified by using a Q Sepharose High Performance anion exchange chromatography medium.

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

6. The method of claim 1, wherein the method comprises: performing intramolecular cross-linking treatment on hemoglobin by using bis (3, 5-dibromo-salicylic acid) fumarate, and performing carboxymethylation treatment on the hemoglobin by using glyoxylic acid; the resulting product is then reacted with polyethylene glycol bearing 8 succinimide groups, terminating the reaction, to yield a hemoglobin-based oxygen carrier based on triple chemical modifications.

7. The method for preparing the hemoglobin-based oxygen carrier based on triple chemical modifications according to claim 6, 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.

8. The preparation method of the hemoglobin-based oxygen carrier based on triple chemical modification of claim 7, wherein the molar ratio of the deoxyhemoglobin to the inositol hexaphosphate in the step (2) is 1 (5-10), the reaction temperature is 20-30 ℃, and the reaction time is 1-5 h; the molar ratio of the product in the step (3) to the bis (3, 5-dibromo-salicylic acid) fumarate is 1 (0.5-2), the reaction temperature is 20-30 ℃, and the reaction time is 2-6 h; glycine was used for termination.

9. The method of claim 8, wherein the molar ratio of deoxyhemoglobin to inositol hexaphosphate in step (2) is 1:8.

10. The method for preparing the hemoglobin-based oxygen carrier based on triple chemical modification of claim 8, wherein the molar ratio of the product to bis (3, 5-dibromo-salicylic acid) fumarate in the step (3) is 1: 1.

11. The method of claim 2 or 6, wherein the polyethylene glycol has a molecular weight of 8-12 kDa; the molar ratio of the product to the polyethylene glycol with 8 succinimide groups is 1 (2-4); glycine is used for termination reaction; after the reaction is stopped, the product is separated and purified by Superdex 200 gel filtration chromatography medium.

12. The method of claim 11 wherein the polyethylene glycol has a molecular weight of 10 kDa.

13. The method of claim 11 wherein the molar ratio of the product to the product having 8 succinimide groups to polyethylene glycol is 1: 3.

14. Use of the hemoglobin oxygen carrier based on triple chemical modification of claim 1 for the preparation of a blood substitute.

Images