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Method for preparing hemoglobin free of carbonic anhydrase

A technology of hemoglobin and carbonic anhydrase, which is applied to the preparation method of peptides, hemoglobin/myoglobin, chemical instruments and methods, etc., can solve the problems of difficult to achieve separation effect and separation, and achieve convenient reuse and shorten Operating time, good refolding effect

Active Publication Date: 2019-08-16
REDPHARM BEIJING BIOPHARMACEUTICAL INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The isoelectric point and dimer molecular weight of hemoglobin are very close, so it is difficult to separate them by general methods such as molecular sieve and ion exchange. At the same time, due to the interference of high-concentration hemoglobin, it is also difficult to use simple affinity chromatography to purify them. ideal separation effect

Method used

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  • Method for preparing hemoglobin free of carbonic anhydrase
  • Method for preparing hemoglobin free of carbonic anhydrase
  • Method for preparing hemoglobin free of carbonic anhydrase

Examples

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example 1

[0033] 1. Preparation of high-purity hemoglobin

[0034] Take fresh anticoagulated bovine blood, wash the blood three times with 0.9% NaCl, and dissolve the red blood cells with cold water. Centrifuge at 4°C for 30 minutes to remove cell debris and undissolved cells, and the supernatant is the hemoglobin solution to be purified; add filler Na to the hemoglobin solution to be purified 2 SO 4 , add 2.85g filler Na to 100ml of pure hemoglobin solution 2 SO 4 , to obtain the hemoglobin sample to be purified; before loading, equilibrate with 0.2M Na 2 SO 4 / 25mM Tris-HCl equilibrium affinity chromatography medium, the medium used for affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until it is flush with the medium level, and the flow-through solution is obtained. It is purified hemoglobin.

[0035] 2. Preparation of high-purity carbonic anhydrase

[0036] The first eluent of affinity chromatography was 30...

Embodiment 2

[0039] 1. Preparation of high-purity hemoglobin

[0040] Take fresh anticoagulated bovine blood, wash the blood three times with 0.9% NaCl, and dissolve the red blood cells with cold water. Centrifuge at 18,000g for 30 minutes at 4°C to remove cell debris and undissolved cells, and the supernatant is the hemoglobin solution to be purified; add filler Na to the hemoglobin solution to be purified 2 SO 4 , add 1.40g filler Na to 100ml of pure hemoglobin solution 2 SO 4 , to obtain the hemoglobin sample to be purified; before loading the sample, use the equilibrium solution 0.1M Na 2 SO 4 / 25mM Tris-HCl equilibrium affinity chromatography medium, the medium used for affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until it is flush with the medium level, and the flow-through solution is obtained. It is purified hemoglobin.

[0041] 2. Preparation of high-purity carbonic anhydrase

[0042] The first eluent...

Embodiment 3

[0045] 1. Preparation of high-purity hemoglobin

[0046] Take fresh anticoagulated bovine blood, wash the blood three times with 0.9% NaCl, and dissolve the red blood cells with cold water. Centrifuge at 18,000g for 30 minutes at 4°C to remove cell debris and undissolved cells, and the supernatant is the hemoglobin solution to be purified; add filler Na to the hemoglobin solution to be purified 2 SO 4 , add 4.30g filler Na to 100ml of pure hemoglobin solution 2 SO 4 , to obtain the hemoglobin sample to be purified; before loading, equilibrate with 0.3M Na 2 SO 4 / 50mM Tris-HCl equilibrium affinity chromatography medium, the medium used for affinity chromatography is CM Sephadex-Sulfanilamide, and then the prepared hemoglobin sample to be purified is loaded until it is flush with the medium level to obtain a flow-through solution, It is purified hemoglobin.

[0047] 2. Preparation of high-purity carbonic anhydrase

[0048] The first eluent of affinity chromatography was ...

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Abstract

The invention discloses a method for preparing hemoglobin free of carbonic anhydrase. The method includes steps: a, washing fresh anti-coagulation bovine blood with 0.9% NaCl, dissolving red blood cells with water, and centrifuging to remove cell debris and undissolved cells to obtain supernatant to-be-purified hemoglobin solution; b, adding filler Na2SO4 into the to-be-purified hemoglobin solution obtained in the step a to obtain a to-be-purified hemoglobin solution sample; c, before sample loading, equilibrating affinity chromatography medium with equilibrium liquid 0.1-0.3M Na2SO4 / 25-50mM Tris-HCl, loading the hemoglobin solution sample obtained in the step b to obtain liquid namely purified hemoglobin. By adoption of the method, carbonic anhydrase in the hemoglobin solution can be removed, high recovery rate can be obtained, high-purity carbonic anhydrase and high-purity hemoglobin are obtained at the same time, and double benefits are achieved.

Description

technical field [0001] The invention relates to a preparation method of hemoglobin, in particular to a preparation method of carbonic anhydrase-free hemoglobin. Background technique [0002] As the rigid demand for medical clinical blood increases at a rate of 10-15%, while the number of blood donations increases relatively slowly, the gap between supply and demand widens year by year. In addition, in the face of emergencies such as wars and natural disasters, the importance of blood protection is also highlighted. Due to the particularity of blood itself, such as short storage period, harsh transportation conditions, blood type matching, safety risks, etc., blood substitutes have irreplaceable advantages as medicines. [0003] Blood substitutes refer to artificial preparations that have the functions of formed and non-formed components of blood, and can be divided into red blood cell substitutes, platelet substitutes, and plasma substitutes. In view of the special efficac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/805C07K1/22
CPCC07K14/805
Inventor 游可为史国营闫晓玲孙新宇陈浩源
Owner REDPHARM BEIJING BIOPHARMACEUTICAL INST CO LTD
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