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Method for extracting superoxide dismutase from erythrocyte

A technology of superoxide and red blood cells, applied in the biological field, can solve the problems of reduced SOD yield, strong toxicity of chloroform, high cost, etc., and achieve the effects of complete thermal denaturation, increased ionic strength, and low cost

Inactive Publication Date: 2008-03-12
抚顺丹阳生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this process is that the process is complicated, the cost is high, and chloroform has strong toxicity
In addition, simply use chloroform ethanol to extract hemoglobin, because SOD is adsorbed on the hemoglobin, resulting in a decrease in the yield of SOD

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Add 10L of 10% sodium citrate solution to 200L of fresh pig blood for anticoagulation, and separate 90L of red blood cells and 120L of plasma with a tube centrifuge. Plasma is spray-dried to become plasma protein powder. Red blood cells were pumped into the reaction tank, and 180 L of distilled water was added and stirred for 30 minutes. Add 10.2Kg of potassium chloride and 300g of copper chloride dihydrate under continuous stirring, heat to 65°C and keep it warm for 60 minutes, then quickly cool it in water to below 30°C, filter with a plate and frame filter press to obtain a total of 190L of light blue SOD crude extract , unit activity 210U / ml. Add 190L of 4°C pre-cooled acetone under stirring, and continue stirring for 15 minutes. Centrifuge in a tube centrifuge, add 76 L of pre-cooled acetone to the supernatant under stirring, continue stirring for 15 minutes and then centrifuge to obtain a light blue-green precipitate. The precipitate was dissolved with 1 L of p...

Embodiment 2

[0028] 14L of fresh pig blood was anticoagulated with 140ml of 10% sodium citrate solution, and separated by a tube centrifuge to obtain 6L of red blood cells and 8.14L of plasma. Place the red blood cells in a reaction tank, add 10 L of distilled water, and stir at room temperature for 30 min. Add 80g of sodium chloride, 112g of potassium chloride and 2.5g of copper sulfate pentahydrate under continuous stirring, heat to 68°C for one hour, and press filter to obtain 12L of blue-green clear solution with a unit activity of 230U / ml. While stirring, 8.4 L of 4°C precooled acetone was added, and the stirring was continued for 15 minutes. Centrifuge in a tube centrifuge, add 9.6 L of pre-cooled acetone to the supernatant under stirring, continue stirring for 15 minutes and then centrifuge to obtain a light blue-green precipitate. Dissolve the precipitate with about 100ml of 2.5mM phosphate buffer solution with a pH of 7.6, desalt the solution on a sephadex G25 column chromatograp...

Embodiment 3

[0032] Take 10L of fresh pig blood, add 300ml of 10% sodium citrate solution for anticoagulation, centrifuge to obtain 4.5L of red blood cells, add 20.5L of distilled water, and stir at room temperature for 30min. Add 4.3Kg of sodium chloride and 62.5g of copper sulfate pentahydrate under continuous stirring, heat to 83°C for 20 minutes, and press filter to obtain 21L of blue-green clear solution with a unit activity of 60U / ml. Add acetone to the filtrate for fractional precipitation, add 16.8 L of pre-cooled acetone while stirring, continue to stir for 15 minutes, and centrifuge to obtain the supernatant; continue to add 14.7 L of pre-cooled acetone to the supernatant, continue to stir for 15 minutes, and centrifuge to obtain the precipitate. The precipitate was dissolved with about 100ml of pH 8.0 concentration 2.5mM phosphate buffer solution, and the solution was dialyzed to the same buffer solution for desalting, a total of 5 times, each time for 5 hours.

[0033]The desal...

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PUM

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Abstract

The invention discloses a method for extracting super oxide mutase from red blood cells, which comprises the following procedures: carrying out anti-coagulation treatment for fresh animal blood, collecting in a centrifugal way red blood cells, haemolyting, thermal modifying, settling by stages through acetone, freezing dry to get crude SOD, separating the crude SOD by ion exchanging separation and / or molecular sieve separation to get refined SOD. The invention has completely eliminates chloroform alcohol, just uses thermal modification and stage-by-stage settlement through acetone to separate crude SOD with high activity. The method can maintain to the max. extent the original SOD activity, and the yield rate is higher than prior chloroform alcohol method. The invention is easy to operate and is applicable for industrialized production.

Description

technical field [0001] The invention relates to a method for extracting superoxide dismutase from red blood cells, belonging to the field of biotechnology. Background technique [0002] Superoxide dismutase (SOD) is a metalloenzyme ubiquitous in organisms that can scavenge superoxide anion free radicals. It has many functions such as anti-inflammation, anti-radiation, and anti-tumor. It has been widely used in medicine, food, health food and cosmetics. [0003] The current methods for extracting superoxide dismutase from red blood cells are mainly blood collection, anticoagulation, washing with normal saline, low-temperature hemolysis, extraction of hemoglobin with chloroform and ethanol, extraction with potassium phosphate, heat denaturation, acetone precipitation, and ion exchange chromatography , molecular sieve column chromatography. The disadvantage of this process is that the process is complicated, the cost is high, and chloroform has strong toxicity. In addition, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08
Inventor 郦剑勇龚兴国曾冬云叶跃国
Owner 抚顺丹阳生物科技股份有限公司
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