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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, complicated process, etc., and achieve the effects of complete thermal denaturation, increased ionic strength, and simple operation

Inactive Publication Date: 2009-09-09
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. The precipitate was dissolved with about 100ml of pH 7.6 concentration 2.5mM phosphate buffer solution, and the solution was subjected to sephadex G25 c...

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 desa...

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PUM

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Abstract

The method disclosed by the invention for extracting superoxide dismutase from red blood cells comprises the following steps: after anticoagulant treatment of fresh animal blood, collecting red blood cells by centrifugation, undergoing hemolysis, thermal denaturation, fractional precipitation with acetone, and freeze-drying to obtain crude SOD. The crude product was subjected to ion-exchange chromatography and / or molecular sieve chromatography to obtain SOD fine product. The present invention completely abandons chloroform and ethanol, and only adopts thermal denaturation and acetone fractional precipitation to easily separate crude SOD with relatively high activity. The method for extracting superoxide dismutase from red blood cells of the present invention can keep the original SOD activity to the greatest extent, and the yield is higher than the traditional chloroform-ethanol method. The invention has simple operation and is suitable 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 Patents(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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