Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof

A hemoglobin and antioxidant enzyme technology, applied in the field of modified polyhemoglobin and its preparation, can solve problems such as limiting the use of modified hemoglobin, and achieve the effects of wide applicability, low auto-oxidation rate, and strong antioxidant properties

Inactive Publication Date: 2009-02-04
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is these factors that limit the use of modified hemoglobin as an RBC substitute

Method used

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  • Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof
  • Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof
  • Antioxidant enzyme crosslinking polyhemoglobin and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of stroma-free hemoglobin

[0037] 1. Acquisition and preservation of expired red blood cells

[0038] The expired human blood with a shelf life of 3-4 weeks was stored in a refrigerator at 4°C+-2°C before further manipulation. All expired human blood has passed the source of disease detection before storage.

[0039] 2. Preparation of washed red blood cells

[0040] Divide the expired blood into several sterile centrifuge tubes and centrifuge at 3500rpm for 10 minutes. After taking out, the upper layer of plasma and middle layer of white blood cells were sucked out, and the lower layer of red blood cells was mixed with 0.9% normal saline and centrifuged at 3500 rpm for 10 minutes. The supernatant was discarded, and the red blood cells in the lower layer were washed with normal saline again, and this was repeated three times to obtain washed red blood cells.

[0041] 3. Lysis and centrifugation of washed red blood cells to remove matrix

[004...

Embodiment 2

[0045] Example 2 Preparation of Polyhemoglobin Crosslinked by Antioxidant Enzyme

[0046] 1. Antioxidant enzyme cross-linked hemoglobin

[0047] Two antioxidant enzymes were added to the purified hemoglobin solution at the ratio of 300000U catalase: 15000U superoxide dismutase: 1g hemoglobin. Add 1.5% glutaraldehyde at a molar ratio of 12:1 (glutaraldehyde:hemoglobin), and monitor the molecular cross-linking degree by HPLC. When the desired degree of cross-linking was achieved, 2M lysine solution was added to the solution to terminate the cross-linking reaction.

[0048] 2. Purification of modified hemoglobin

[0049]The polyhemoglobin solution that terminated the reaction was subjected to hollow fiber ultrafiltration with a filtration limit of 300kd, balanced with phosphate buffer, and circulated for 8-10 volumes until the content of unmodified hemoglobin was less than 5%. The purified modified hemoglobin solution was concentrated to a final concentration of 7 g / dl, and su...

Embodiment 3

[0052] Example 3 Preparation of Polyhemoglobin Crosslinked by Antioxidant Enzyme

[0053] The ratio of catalase: superoxide dismutase: hemoglobin is 200000U: 10000U: 1g, and the rest of the steps are the same as in Example 2.

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Abstract

The invention relates to a blood substitute and discloses poly hemoglobin cross-linked with antioxidase, and the poly hemoglobin is formed by chemically cross-linking superoxide dismutase and catalase. The poly hemoglobin cross-linked with the antioxidase has lower autoxidation rate and stronger oxidation resistance, can resist exogenous oxidants, does not need cross matching and laboratory detection during the application and has extensive applicability.

Description

technical field [0001] The present invention relates to blood substitutes, in particular to modified polyhemoglobin and its preparation. Background technique [0002] The reversible oxygenation of red blood cells in the blood is mainly performed by hemoglobin. In mammals, hemoglobin is a class composed of about 6% heme and 94% globulin, with a molecular weight of 64458 dalton. The hemoglobin molecule is composed of two α subunits and two β subunits, each subunit (α or β) contains a heme and a globulin. [0003] With the increasing demand for clinical blood and the unfavorable factors existing in the existing blood supply, such as the potential risk of blood transfusion infection, the cumbersome matching of blood for emergency use, and the inability to store blood for a long time, people continue to invest in blood substitution. In the research and development of supplies. [0004] The so-called blood substitute is a blood product that can supply oxygen to tissues, and can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/805C07K1/34C07K1/18C07K1/107
Inventor 范华骅杨懿铭王永彬杨洁任亚娜谢如锋
Owner SHANGHAI BLOOD CENT
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