Process for purifying hematoglobin

A technology of hemoglobin and buffer solution, applied in the field of direct purification of hemoglobin, can solve the problems of membrane fouling concentration, increased cost and pollution, and is only suitable for laboratory or small-scale preparation.

Inactive Publication Date: 2004-04-28
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has membrane fouling and concentration polarization problems, and the membrane separation process takes a long time, which increases the cost and the chance of contamination
At present, most of the separation and purification technologies of hemoglobin are only suitable for laboratory or small-scale preparation, so it is necessary to develop new methods for efficient large-scale preparation of hemoglobin

Method used

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  • Process for purifying hematoglobin
  • Process for purifying hematoglobin
  • Process for purifying hematoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] (1) Sample treatment: 1ml of stacked bovine erythrocytes washed with 0.9% NaCl, according to erythrocytes: steamed

[0035] Distilled water = 1:29 (volume ratio) mixed in the ratio, put in the refrigerator at 4 ° C with a magnetic

[0036] The force stirrer stirred slowly for 30 minutes, at this time, the red blood cells swelled rapidly and ruptured, and the intracellular

[0037] The hemoglobin was released into the solution to obtain the red blood cell lysate, and the solution was tested by Benesch et al.

[0038] (Benesch, R.E., Benesch.R. and Yung, Suzanna..Equations

[0039] for the spectrophotometric analysis of hemoglobin mixture,

[0040] Analytical Biochemistry.1973, 55:245-248) established multi-wavelength method

[0041] Detection, Hb concentration is 8.0mg / ml -1 .

[0042] (2) Bed balance: Use 250ml balance buffer 40mmol / L -1 PBS

[0043] (K 2 HPO 4 / NaH 2 PO 4 , pH6.0) Bottom-up equilibrium expanded bed medium, the selecte...

Embodiment 2

[0061] (1) Sample treatment: 1ml of packed human erythrocytes washed with 0.9% NaCl, according to erythrocytes:

[0062] Distilled water = 1:29 (volume ratio) mixed, put in the refrigerator at 4°C

[0063] Stir slowly with a magnetic stirrer for 30 minutes, at this time the red blood cells swell rapidly and rupture.

[0064] Intracellular hemoglobin is released into solution.

[0065] (2) Bed balance: use 3 times column volume of equilibration buffer (10mmol / L -1 Barbital Sodium-

[0066] hydrochloric acid, pH6.9) to balance the expanded bed medium from bottom to top, and the selected expanded bed medium is

[0067] Spherosil, feed line flow rate 2cm / min -1 , so that it forms a stable expanded bed.

[0068] (3) Feed adsorption: take 8.0mg / ml -1 The erythrocyte lysate 10ml that step (1) obtains,

[0069] Adjust the pH to 5.0 with the same flow rate of 2cm / min -1 Bottom-up feed adsorption to expansion

[0070] bed.

[0071] (4) Rinse: then rinse wi...

Embodiment 3

[0086] (1) Sample treatment: 1ml of stacked pig erythrocytes washed with 0.9% NaCl, according to erythrocytes:

[0087] Distilled water = 1:29 (volume ratio) mixed, put in the refrigerator at 4°C

[0088] Stir slowly with a magnetic stirrer for 30 minutes, at this time the red blood cells swell rapidly and rupture.

[0089] Intracellular hemoglobin is released into solution.

[0090] (2) Bed balance: use 3 times column volume of equilibration buffer (80mmol / L -1 Acetic acid - acetic acid

[0091] Sodium buffer, pH 5.0) to balance the expanded bed medium from bottom to top, the selected expanded bed medium

[0092] The quality is Streamline SP, the feed flow rate is 5cm / min -1 , so that it forms a stable expansion

[0093] bed.

[0094] (3) Feed adsorption: take 8.0mg / ml -1 The erythrocyte lysate 5ml that step (1) obtains,

[0095] Adjust the pH to 8.0 and feed from bottom to top at the same flow rate.

[0096] (4) Rinse: Then rinse with the same e...

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Abstract

The present invention provides the process of purifying hematoglobin. Expanding bed adsorption technology is adopted, and through material feeding from bottom to top and chromatographic medium expanding, cell fragment, hybrid protein and other impurities are made to pass through the bed layer and hematoglobin is adsorbed by the medium, so as to realize direct purification of hematoglobin from disintegrated human or animal blood cell liquid. Without centrifugation or filtering to eliminating segment, the product reach electrophoresis pure level of hematoglobin in short time, high yield and less loss in product activity. The present invention is suitable for industrial production of natural hemotoglobin.

Description

technical field [0001] The invention belongs to the field of protein separation and purification, and in particular relates to a method for directly purifying hemoglobin from red blood cell lysates of animals by adopting an expanded bed adsorption technique. Background technique [0002] Human blood substitutes are research hotspots and difficulties that are concerned by the international scientific community and business circles, and all major developed countries in the world have listed them as major cross-century projects. Especially in the past two decades, anxiety about the safety of the natural blood supply and the expected high return potential have stimulated great interest in the development of blood substitutes. The currently studied human blood substitutes can be roughly divided into three categories: perfluorocarbons, hemoglobin-based blood substitutes, and red blood cells through blood type conversion. Among them, because perfluorocarbons do not have the biolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K14/805
Inventor 苏志国路秀玲金业涛赵东旭
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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