Method for preparing narrow-distribution low average molecular weight polymerized hemoglobin

A technology of average molecular weight and hemoglobin, applied in the direction of hemoglobin/myoglobin, specific peptides, etc., can solve the problems of low utilization rate of anion exchange medium, increased colloidal osmotic pressure, excessive modification of hemoglobin, etc. The effect of reducing colloid osmotic pressure

Inactive Publication Date: 2008-06-18
天津协和生物科技发展有限公司
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Problems solved by technology

However, unmodified polymerized hemoglobin cannot be used directly as a blood substitute, mainly because (1) unmodified hemoglobin will lose the regulation of 2,3-bisphosphoglycerate, which will increase the oxygen affinity of hemoglobin and cannot Effective supply of oxygen to tissues
(2) Unpolymerized hemoglobin will increase the colloid

Method used

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  • Method for preparing narrow-distribution low average molecular weight polymerized hemoglobin
  • Method for preparing narrow-distribution low average molecular weight polymerized hemoglobin

Examples

Experimental program
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Effect test

Embodiment

[0026] Embodiment: take 5 g of PLP-modified human hemoglobin solution (pH=6.3) with a concentration of 75 mg / ml and pass nitrogen at 20° C. for 2 hours to make the hemoglobin in a deoxygenated state; add 2.8 ml of 2% glutaraldehyde to react for 3 hours for pretreatment Polymerization; fill Φ1.6×30cm CM-52 cation exchange column, equilibrate with deoxygenated 10mM PBS (pH=6.3) buffer solution (see figure 1 ); the hemoglobin solution is pumped into the chromatographic column to circulate, and the final volume of the solution in the reservoir is 40ml; after 1 hour, add 4.4ml glutaraldehyde in the reservoir for 12 times every 0.5 hour; continue the reaction 2 hours. Finally, it was eluted with 10mM PBS buffer (pH7.4); after the collection was concentrated by ultrafiltration, it was reduced with sodium borohydride; it was concentrated by ultrafiltration; M NaCl (pH7.2), the flow rate is 0.50ml / min, and the eluate is detected under 280nm ultraviolet light; 3 elution peaks can be de...

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Abstract

A preparation method of narrow-distribution polymerized hemoglobin with low average molecular weight is characterized in that the method includes the following steps: {1} the hemoglobin is added into the gel retardation spectrum for balance through a pump and then distributively added with double-function crosslinker for polyreaction; {2} buffer solution is used to elute and collect the polymer produced in the step {1}, amide acid is used in the collected solution for stopped reaction and then hyperfiltration and concentration are carried out; {3} the obtained polymerized hemoglobin is treated by reduction by sodium borohydride and then is separated and purified through hyperfiltration and chromatography. The advantages of the present invention are that 1. the polymerized hemoglobin prepared by the present invention is narrow-distribution polymerized hemoglobin with low average molecular weight less than 200kD; 2. the present invention overcomes the poor homogeneity of the polymer in the traditional glutaraldehyde preparation method; 3. the polymerized hemoglobin is easily separated from the unpolymerized single hemoglobin and the reduced number of protein colloid decreases the osmotic pressure without any obvious change in viscosity.

Description

(1) Technical field: [0001] The invention relates to a preparation method of polymerized hemoglobin, in particular to a preparation method of narrowly distributed low average molecular weight polymerized hemoglobin. (two) background technology: [0002] Although the traditional blood transfusion method has played an important role in medical rescue, there are also various problems, such as blood shortage, virus transmission, immune response, and the disposal of expired blood. In various clinical medical treatment and first aid, the quality and quantity requirements for blood transfusion are becoming more and more stringent, so the research and development and use of blood substitutes have generally been paid attention to. [0003] Hemoglobin consists of four subunits (α 1 alpha 2 beta 1 beta 2 ), a protein in red blood cells responsible for oxygen transport. Because of its unique oxygen binding characteristics, normal physiological metabolic pathways, etc., hemoglobin-b...

Claims

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Application Information

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IPC IPC(8): C07K14/805C07K1/14C07K1/10
Inventor 王翔杨成民黄磊李燊张鸿辉梁伟光李洪英许丽丽
Owner 天津协和生物科技发展有限公司
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