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

A technology of hemoglobin and chemical modification, applied in the field of biomedicine, can solve the problems of ineffective expansion of blood volume and low colloid osmotic pressure, and achieve the effects of enhancing oxygen carrying/releasing activity, reducing affinity and improving tetramer stability.

Active Publication Date: 2020-08-07
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, because the intermolecular cross-linking mediated by glutaraldehyde reduces the number of colloidal particles of HbA, its colloid osmotic pressure is also lower than that of blood, and thus cannot effectively expand blood volume

Method used

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

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Experimental program
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Effect test

Embodiment 1

[0063] Separation and purification of adult hemoglobin (HbA):

[0064] Centrifuge adult whole blood and discard the supernatant. Suspend with PBS buffer (pH=7.4), centrifuge and discard the supernatant. Repeat washing twice. Red blood cells were lysed with 3 volumes of water. Centrifuge the erythrocyte lysate, take the supernatant, and filter it with a 0.22 μm filter membrane. The filtrate was separated and purified by Q Sepharose Fast Flow anion exchange chromatography medium, and the elution peak corresponding to adult hemoglobin (HbA) was collected, concentrated, dialyzed into PBS buffer (pH=7.4), and stored at -80°C for future use.

Embodiment 2

[0066] Preparation of glyoxylated hemoglobin:

[0067] Glyoxylic acid covalently modifies the N-terminal Val-1(α) of HbA, thereby introducing a carboxymethyl group, which will destroy the salt bridge that participates in the formation of the stable R-state structure and reduce the stability of the relaxed state (R-state) of HbA , so that the HbA quaternary structure shifts from the R state to the tense state (T state), reducing the affinity of HbA for oxygen.

[0068] Deoxygenated HbA was prepared by continuously bubbling nitrogen gas into the 0.5 mM HbA solution. Then 1.0 mM glyoxylic acid and 10 mM sodium cyanoborohydride (NaCNBH 3 ) solution, so that the mol ratio of HbA, glyoxylic acid and sodium cyanoborohydride is 1:2:20. Continuing to feed nitrogen, the reaction was carried out at 4° C. for 3.5 h under anaerobic conditions to obtain glyoxylic acid-modified HbA (expressed as Glx-Hb). Finally, 20 mM glycine solution was added to terminate the reaction, and unreacted gl...

Embodiment 3

[0070] Preparation of DBBF intramolecularly cross-linked hemoglobin:

[0071] When bis(3,5-dibromosalicylic acid) fumarate (DBBF) modified deoxy HbA, an intramolecular cross-linking reaction occurred between two Lys-99(α).

[0072] Deoxygenated HbA was prepared by continuously bubbling nitrogen gas into the 0.5 mM HbA solution. Subsequently, an equal volume of 4 mM inositol hexaphosphate (IHP) solution was added, nitrogen gas was continuously introduced, and the reaction was carried out at 25° C. for 3 h under anaerobic conditions. Subsequently, 0.5 mM DBBF solution was added so that the molar ratio of HbA to DBBF was 1:1, and reacted at 25° C. for 4 h to obtain intramolecularly cross-linked HbA (expressed as αα-Hb) of DBBF. Finally, 20 mM glycine solution was added to terminate the cross-linking reaction, and unreacted DBBF and glycine were removed by dialysis. The schematic diagram of its preparation is as Figure 13 shown.

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Abstract

The invention relates to a hemoglobin oxygen carrier based on double chemical modification as well as a preparation method and application thereof. In the hemoglobin oxygen carrier based on double chemical modification, two Val-1 (alpha) residues are subjected to carboxymethylation, and two Lys-99 (alpha) residues are subjected to intramolecular crosslinking. Two Lys-99 (alpha) residues are subjected to intramolecular crosslinking, and the formed fumarimide bridge can change the electrostatic interaction of human hemoglobin (HbA) molecules, stabilize the tension state (T state) of HbA and reduce the oxygen affinity of HbA; val-1 (alpha) is formed at the tail end of a center hole alpha alpha alpha- of HbA and can participate in formation of salt bridges contributing to stabilization of a relaxation state (R state) structure, carboxylation modification can damage the salt bridges, the R state stability of HbA is reduced, a quaternary structure of HbA is transferred from an R state to a Tstate, and the affinity of HbA to oxygen is reduced. Two modification means are organically combined, so that the stability of the HbA tetramer is improved, the electrostatic interaction is enhanced,the combination state of an oxygen combination region and oxygen is improved, and the hemoglobin oxygen carrier has very strong oxygen carrying / releasing activity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a hemoglobin oxygen carrier and its preparation method and application, in particular to a hemoglobin oxygen carrier based on double chemical modification and its preparation method and application. Background technique [0002] In recent years, with the rapid growth of clinical blood demand in my country and the implementation of the voluntary blood donation system, the contradiction between blood supply and demand has become increasingly prominent. The traditional blood transfusion method plays an important role in medical rescue, but also has various shortcomings, mainly reflected in: (1) Due to the existence of the window period, blood transfusion has the risk of pathogen transmission, such as HIV, hepatitis B virus and other pathogens may exist in the blood ; (2) There is a shortage of blood sources in my country, and clinical blood banks have insufficient rese...

Claims

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Application Information

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IPC IPC(8): C07K14/805C07K1/107C07K1/113C07K1/34C07K1/16A61K38/42A61P7/08
CPCC07K14/805A61P7/08
Inventor 胡涛颜文颖于卫立申莉娟
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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