JPH03994B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides copper (Cu), zinc (Zn)-superoxide dismutase;
EC.1.15.1.1 (hereinafter abbreviated as SOD)), more specifically, Cu, Zn- purified to a considerable extent from animal tissues, red blood cells, or yeast by various methods. Efficiently removes impurities from SOD-containing fractions to achieve high purity.
This invention relates to a method for isolating and purifying Cu, Zn-SOD.

SOD is a superoxide anion radical (hereinafter referred to as "superoxide anion radical") produced by one-electron reduction of oxygen molecules.
(abbreviated as O ₂ −), i.e. O ₂ −
It is an enzyme that catalyzes +O ₂ − +2H ⁺ →O ₂ +H ₂ O ₂ and is known to exist widely in the living world.
Since 1969, many enzymes with this activity have been isolated. Has Cu and Zn in its active site
Cu, Zn-SOD is found in the cytoplasm of eukaryotes. Cu, Zn derived from various eukaryotes
-SOD shows high homology in its amino acid sequence (C. Petersen)
et al., Carlsberg Res.Commun, Volume 42, 391-395.
Page, 1977), with interrelated physicochemical properties (AEG
Cass) et al., Carlsberg Res.Commun., Vol. 43, 439-449.
Page, 1978) is known to be a dimeric molecule. Another type of SOD is one that contains iron or manganese in the active site and is found in prokaryotic and eukaryotic mitochondria. This type shows high homology in amino acid composition and N-terminal amino acid sequence.

However, Cu, Zn−SOD and Fe or Mn−
There is no significant homology between SODs.

As mentioned above, SOD quickly removes O ₂ - generated from the in-vivo reactions _of living organisms.
- itself and various reactive oxygen species derived from O ₂ - are thought to reduce cytotoxicity. In other words, O ₂ − is said to be involved in various inflammatory processes in tissues and to be a cause of rheumatoid arthritis, and it has been proposed that SOD be used to treat these, and it has been shown to have a therapeutic effect on inflammation and rheumatoid arthritis. This is because it has been confirmed by tests (H.
Marberger) et al.
Nephrol ((Intl.J.Urol.Nephrol.)) Volume 6, 61
~74 pages, 1974; M. Walravens ((M.
Walravens)), Jay Detzker ((J.
Dequeker), Current Therap.Res, Vol. 20, pp. 62-69,
(1976). Therefore, research and development of high-yield SOD recovery and purification methods have been carried out on an industrial scale.

Cu, Zn-SOD in red blood cells is derived from Orgotein (International Nonproprietary Name for Pharmaceutical Substances), which is purified from higher animal materials.
It belongs to the group of metal chelating proteins named Pharmaceutical Substauces); international non-proprietary name).
208, No. 8, p. 1399, May 26, 1969), which has been previously isolated in various degrees of purity and is equivalent to a protein with the following name: Namely, Hemocuprein (T.Mann), Dei Keilin (D.Keilin), Proc Roy Sok Series Bee Biol Sai ((Proc.
Roy.Soc.Ser.B.Biol.Sci) Volume 126, page 303,
1939), Erythrocuprein (H. Markowitz et al., J. Biol.Chem), Vol. 234, p. 40, 1959;・Kimmel ((J.R. Kimmel))
et al., J. Biol.Chem.
234, p. 46, 1959), Cerebrocuprein (H.
Porter), J.Folch, J.Neurochem, Volume 1,
260 pages, 1959) and Hepatocuperin (T.Mann),
D. Keilin, Proc.Roy.Soc.Ser.B.Biol.Sci., Volume 126, 303
Page, 1939; M.S. Mohammed ((MS
Mohamed), DM Greenberg, J. Gen. Physiol, Vol. 37, p. 433, 1953).

Of these, SOD, which has been well-clarified from protein and enzyme chemistry, is Cu, Zn− in bovine erythrocytes.
It is SOD, and its complete amino acid sequence and three-dimensional structure determined by X-ray crystallography are already known. In other words, it has a molecular weight of 32,000, is composed of equal subunits of 151 amino acids, and has a 3D structure with less α-helical structure and a cylindrical β-helix structure.
It is said to be resistant to degeneration because its structure occupies most of the structure. As a chelate metal, Cu and
Each contains 2g atoms of Zn, and the isoelectric point is
Shows pH around 4.9 to 5.5. It is a non-toxic, injectable protein that is difficult to recognize as a foreign substance by the immune system when administered into the body, and has pharmacological effects such as inflammation that occurs in autoimmune diseases. arthritis, chronic rheumatism,
Development is being promoted as a treatment and prevention agent for serious side effects of radiotherapy (edited by WHBannister (WHBannister) and JVBannister (JVBannister)).
Biological and Clinical Aspects of Superoxide and Superoxide Dismutase (Biological
and Clinical Aspects of Superoxide and
Superoxide Dismutase) 1980, Elsevier (Elsevier).

By the way, several methods are known for separating Cu and Zn-SOD. Cu, Zn-SOD, for example from cattle and other higher animals, is a water-soluble protein solution obtained, for example, from organs and tissues, especially the liver, by extracting minced tissue with cold buffer, or by hemolysis of red blood cells from blood. From Cu,
It is recovered by separating Zn-SOD.
In addition, Cu, Zn-SOD from yeast can be recovered by separating Cu, Zn-SOD from a water-soluble protein solution obtained by autolysis by adding an organic solvent that is immiscible with water, such as ether. There is. Separation may be carried out by fractional precipitation, for example with organic solvents or ammonium sulfate, optionally by heating in the presence of divalent metals, together with denaturation of the thermolabile protein;
DEAE group or carboxymethyl group (hereinafter referred to as
It is carried out by chromatography using an ion exchange resin having a CM group (abbreviated as CM group). Generally, low molecular weight substances are removed by purification using dialysis and gel filtration methods.

For example, in Special Publication No. 53-31206, Cu, Zn,
Heating red blood cell lysate in the presence of divalent metal salts such as Co, Mn, and Mg to precipitate and remove hemoglobin and carbonic anhydrase, Cu, Zn-SOD
Discloses a method for isolating. In addition, the special public
-39832, erythrocyte lysate was treated with an organic chlorine compound to remove hemoglobin, Cu, Zn-SOD
A method for isolating is disclosed. Also, JMMcCord, I.
I. Frido vich (J.Biol.Chem.) Volume 244, 6049~
6055, 1969) also used an organochlorine compound to precipitate and remove hemoglobin.
Cu, Zn-SOD is isolated and purified. In addition, in JP-A-49-50195, after heating the raw blood, the supernatant was treated with a protein hydrolase, and then subjected to column chromatography, gel filtration, etc. to remove Cu,
A method for purifying Zn-SOD is presented. Furthermore, in Japanese Patent Publication No. 53-22137, hemolysis of red blood cells was directly treated with a weakly basic ion exchange resin, Cu,
A method for isolating Zn-SOD by adsorbing it onto a gel is presented. However, the Cu, Zn-SOD fractions obtained by these methods do not provide protein chemically pure Cu, Zn-SOD, and in order to obtain pure Cu, Zn-SOD, further steps are required. There is a problem in that a purification step by means of purification, such as ion exchange chromatography, must be added.

Cu, Zn−SOD purified to a considerable degree
Several methods have already been demonstrated for purification by ion exchange chromatography to obtain pure Cu, Zn-SOD from fractions. JMMcCord, I.
I.Fridovich, J.Biol.Chem (Vol. 244, 6049-6055)
After adsorbing Cu, Zn-SOD onto a column packed with DEAE-cellulose equilibrated with 2.5 mmole/potassium phosphate buffer (PH7.4), the concentration of potassium phosphate buffer was PH
They report a method to specifically elute and purify Cu and Zn-SOD using a concentration gradient elution method in which the concentration is maintained at 7.4 and increased to 0.2 mole/.

However, this paper also describes ``pure Cu,'' which is said to have been purified by ion-exchange chromatography.
The ultraviolet absorption spectrum of ``Zn-SOD'' has a wavelength of 280nm.
A large shoulder appears nearby, with absorbance A 260 at 260 nm, which is the absorption maximum, and absorbance A ₂₈₀ at ₂₈₀ nm.
The ratio of A ₂₆₀ /A ₂₈₀ is 1.29, and Cu, Zn−SOD
and accompanying minute proteins coexist,
This indicates the need for further purification.

Another problem with this method is that the reproducibility or stability of the operation is poor. That is, it is difficult to apply it to ordinary ion exchange resins.

Furthermore, in JP-A-46-548, JP-A-50-69215 and JP-A-56-19876, substantially pure Cu,
In the method of removing impure proteins (hereinafter referred to as accompanying proteins) contained in Zn-SOD, the ionic strength
A Cu, Zn-SOD solution containing 10 mmole or less of impure protein dissolved in a buffer solution with a pH of 5.5 to 8 is passed onto a column of weakly basic ion exchange resin.
Ionic strength and pH increased to 100 mmole/
A method is disclosed for selectively eluting Cu,Zn-SOD from the column with a buffer having a pH of about 5.5 to 8. However, this method also requires specific gels and operating conditions, and similarly to the above, there are problems with reproducibility and stability.

Furthermore, JP-A-56-35983, JP-A-56-35984
Cu, Zn−
Cu from an aqueous solution containing both SOD and associated proteins.
To separate Zn-SOD, first perform cation exchange chromatography at pH 4.7 to 5.5.
A two-step method has been disclosed in which a Cu, Zn-SOD active fraction is obtained and then subjected to anion exchange chromatography, but this method has problems such as operability and stability.

As detailed above, the conventional method for purifying Cu, Zn-SOD to a uniform level can be summarized as follows:
Cu and Zn-SOD are adsorbed onto a weakly basic ion exchange resin using a phosphate buffer with a pH of 5.5 to 8 under an ionic strength of 10 mmole or less, and then eluted by increasing the ionic strength. In reality, these methods have extremely poor reproducibility, and the adsorption of Cu, Zn-SOD to the gel used
In addition to problems with PH adjustment, buffer type, resin regeneration, etc., these conventional methods also have problems with Cu, Zn−
There is a problem that it cannot be said that SOD is purified and purified to complete homogeneity.

As a result of intensive research to improve these conventional problems, the present inventor has devised a method that can efficiently, easily, and reproducibly isolate and purify impure proteins that accompany Cu, Zn-SOD. I came to invent it.
That is, in the present invention, Cu, Zn-
In the method for isolating SOD, in order to efficiently adsorb Cu, Zn-SOD using an anion exchange resin, a cationic buffer solution whose acid component is hydrochloric acid, preferably with a pH of 5.5 to 11, is used. The ionic strength of the buffer solution is maintained at a range of 7 to 10, more preferably 8 to 10, and the ionic strength of the buffer is maintained at a level that has a sufficient buffering effect, and the gel is recyclable, that is, 10 to 50 mmole/Ion exchange chromatography. It was discovered that good isolation of Cu and Zn-SOD can be obtained by carrying out this process.

In the method of the present invention, the PH, type, ion concentration, etc. of the buffer solution when adsorbing Cu, Zn-SOD to anion exchange gel are specified within the ranges described above, but this is due to its reproducibility, stability, and operability. The effects of the present invention will not be achieved if any of these ranges are exceeded.

That is, the isoelectric point of Cu, Zn-SOD is between PH4.9 and 5.5, and in order to effectively adsorb this Cu, Zn-SOD, from this point of view, the alkali side is preferable.
In addition, the buffer solution used at that time must maintain sufficient buffering capacity and be within a range that does not cause problems in activation, regeneration, etc. of the anion exchange gel used.

In addition, the anion exchange gel used in the present invention is preferably one that can sufficiently adsorb and elute Cu, Zn-SOD, and the anion exchange resin includes diethylaminoethyl (DEAE) group, triethylaminoethyl (TEAE) group, etc. cellulose gels substituted with DEAE or diethyl-(2-hydroxypropyl)-aminoethyl (QAE) groups; cross-linked dextran gels substituted with DEAE or QAE groups; cross-linked agarose gels substituted with DEAE groups; or a hydrophilic polyvinyl resin substituted with DEAE groups, preferably a cellulose gel substituted with QAE groups or a crosslinked dextran gel substituted with DEAE groups; N-tris-(hydroxymethyl)-methylacrylamide gel or hydrophilic polyvinyl resin is suitable. Furthermore, as a buffer solution, a cationic buffer solution whose acid component is hydrochloric acid is used,
Preferably imidazole/hydrochloric acid, 2:4:6-
Trimethylpyridine/hydrochloric acid, triethanolamine/hydrochloric acid, N-ethylmorpholine/hydrochloric acid, tris-(hydroxymethyl)-aminomethane/hydrochloric acid,
2-Amino-2-methyl-1:3-propanediol/hydrochloric acid, diethanolamine/hydrochloric acid, sodium tetraborate/hydrochloric acid, dimethylglycine sodium/hydrochloric acid, or ammonium chloride/ammonia.

Cu, Zn−SOD adsorbed on anion exchange resin
The buffer used for selective elution has an ionic strength of 0.2 mmole/or higher or a pH of 5 or lower. Furthermore, anion exchange chromatography is desirably carried out at 0 to 30°C.

This is because it is preferable to use monovalent ions as much as possible for the adsorption and elution of Cu, Zn-SOD to the anion exchange gel used or the regeneration and activation of the gel, and other buffers However, the excellent effects of the present invention in terms of operability, stability, and reproducibility cannot be obtained.

The buffer conditions during elution include Cu, Zn.
-The isoelectric point of SOD is between PH4.9 and 5.5, so it can be carried out under these conditions. Also, the operating temperature is 0.
If the temperature is below 30°C, samples and solutions will freeze, salts will precipitate, etc. If the temperature exceeds 30°C, the sample will rot, the gel will deteriorate, the adsorption to the gel will decrease, etc.

The Cu, Zn-SOD specimen obtained by the method of the present invention was found to be single by polyacrylamide gel electrophoresis, and was given a molecular weight of 16,000 by electrophoresis in the presence of sodium dodecyl sulfate. Further, ultracentrifugal analysis using the precipitation equilibrium method revealed that the molecular weight was 32,000, and atomic absorption analysis revealed that each molecule contained 2 g atoms each of Cu and Zn. The activity was 3500 units per mg of standard weight.

Next, the present invention will be further explained with reference to Examples, but the present invention is not limited to these Examples.

Example 1 ACD as an anticoagulant in fresh bovine blood 10
150 ml of the solution was added, and the packed red blood cells were separated by centrifugation. 10 ml of physiological saline was added to the packed red blood cells, stirred, and washed by centrifugation. Approximately 5 of the packed red blood cells obtained in this manner were hemolyzed by adding the same amount of distilled water to give a hemolysis of approximately 10, and the total SOD activity of the hemolysate was determined by the aforementioned JMMc Cord and I. Fridowicz. Fridovich) (J.Biol.
Chem) Vol. 244, pp. 6049-6055, 1969), it was 4.0×10 ⁶ units.

This JM pine cord (JM
McCord) and I.Fridovich
Following the method of (1) removal of hemoglobin by ethanol-chloroform treatment, (2) separation of ethanol and aqueous layers by K ₂ HPO ₄ , (3) removal of Cu from the ethanol layer by acetone fractionation,
The precipitate obtained by performing the precipitation operation of Zn-SOD has a pale green color, and is dissolved in a small amount of distilled water, and the insoluble matter is separated with paper. The resulting liquid is called "solution (A)." The total SOD activity at this time was 2.1×10 ⁶ units. The dialysate obtained by dialyzing "Solution (A)" against an ionic strength of 25 mmole/tris(hydroxymethyl)-aminomethane/hydrochloric acid buffer (PH8.2) is called "Solution (B)."
Ionic strength 25 mmole/Tris-(hydroxymethyl)-aminomethane/hydrochloric acid buffer (PH8.2)
The above "solution (B)" was flowed into a column filled with "QAE-Sephade x A-50" gel (manufactured by Pharmacia; trade name) equilibrated with Cu, Zn-
Adsorbed SOD. This gel chromatogram and the measurement results of the SOD activity absorbance at 280 nm and 680 nm are shown in FIG. Further, the ultraviolet absorption spectrum and electropherogram of "Solution (B)" are shown in FIG.

A light green band appeared at a thickness of about 2.5 cm from the top, and about 1 part of the same buffer solution was subsequently flowed into the column to wash away proteins that did not adsorb to the gel. Then ionic strength 25mmole/
The adsorbed Cu, Zn-SOD was eluted using a linear concentration gradient in which NaCl was varied from zero to 1 mole/tris-(hydroxymethyl)-aminomethane/hydrochloric acid buffer (PH8.2). The flow rate was approximately 10 ml/hr, and the operation was performed at room temperature. Immediately after starting to apply the NaCl concentration gradient, connect the eluate tube from the column to a fraction collector,
Fractionated in drops (4.6 ml). The absorbance of each fraction was measured at 280 nm and 680 nm.
At the same time, SOD activity was also recorded. SOD activity was eluted in fraction numbers 41-65. The results are shown in FIG. After recording the ultraviolet absorption spectrum (240 to 400 nm) of each fraction using a self-recording spectrophotometer, calculate the absorbance at 260 nm.
The ratio of A ₂₆₀ and absorbance at 28 nm A ₂₈₀ is A ₂₆₀ /
Fraction numbers 47 to 52 with A ₂₈₀ greater than 1.50 were collected. This pooled solution was called "solution (C)" and its ultraviolet absorption spectrum and electropherogram are shown in FIG. This was dialyzed against distilled water and then freeze-dried. The weight of the obtained specimen was 400 mg.
The total SOD activity was 1.4×10 ⁶ units, the specific activity was 3500 units per mg of dry weight, and there was a single band electrophoretically.

Example 2 "Solution (A)" obtained in Example 1 was dialyzed against diethanolamine/hydrochloric acid buffer (PH9.0) with an ionic strength of 20 mmole/solution to obtain "solution (B)'". "DEAE-Toyopearl 650M" (Toyo Soda Kogyo Co., Ltd.) equilibrated with the same buffer solution as above.
After washing thoroughly with the same buffer, elute stepwise with diethanolamine/hydrochloric acid buffer (PH9.0) with an ionic strength of 20 mmole/containing 0.2 mmole/NaCl. I went there. It was fractionated in the same manner as in Example 1,
Fractions with A ₂₆₀ /A ₂₈₀ >1.50 (number of fractions 39 to 46) were collected, dialyzed against distilled water, and then lyophilized to obtain 390 mg of Cu, Zn-SOD.
The total SOD activity was 1.37×10 ⁶ units, giving a single electrophoretic band.

Example 3 The buffer solution used in Example 1 was adjusted to ionic strength.
Change to 30 mmole/ammonium chloride/ammonia buffer (PH10.5) and use "DEAE-Tris Acrylic M" (manufactured by LKB) as an anion exchange gel.
Cu, Zn−
After adsorbing SOD, NaCl was added in the same buffer solution.
It was eluted with a linear concentration gradient varying from 0 to 1 mole/mole. From the pool of active fractions
405 mg of lyophilized sample was obtained. The total SOD activity is
It was 1.42×10 ⁶ units, and its electrophoretic pattern was single.

Example 4 5 Kg of fresh beef liver was mixed with 10 mmole/25 mmole/Tris-glycine buffer (PH7.5) containing Mn ²⁺ under water cooling. Next, add 250 ml of toluene and centrifuge to remove 10 ml of supernatant.
Collect 12.5 mmol of cold acetone to this supernatant to form a precipitate, and collect the precipitate by centrifugation to obtain the same 10 mmole/25 mmole containing Mn ²⁺ as above.
It was suspended in Tris-glycine buffer (PH7.5).

After stirring, the supernatant obtained by centrifugation was heated and then centrifuged to obtain a supernatant. This supernatant
Ammonium sulfate was added to give a concentration of 45%, the precipitate was removed, and ammonium sulfate was further added to give a concentration of 65%, and the precipitate was collected. This precipitate
30mmole/2-amino-2-methyl-1:3
- The dialysate obtained by dissolving in propanediol/hydrochloric acid buffer (PH9.0) and dialyzing against the same buffer is referred to as "Solution D". This "solution D" is a nearly pure Cu, Zn-SOD solution containing some accompanying proteins, and the total SOD activity contained in this solution is 1.75×
It was ¹⁰⁶ units. This “solution D” has an ionic strength of
30 mmole/2-amino-2-methyl-1:
The gel was equilibrated with 3-propanediol/hydrochloric acid (PH9.0), poured into a column packed with "Selectacel-QAE" (manufactured by Brown, trade name) at 4°C, and the gel was thoroughly washed with the same buffer solution. 0.2mole／
The same buffer solution containing NaCl was poured in the same manner as in Example 2, and the adsorbed Cu and Zn-SOD were eluted. SOD active fractions with A ₂₆₀ /A ₂₈₀ of 1.50 or more were pooled, thoroughly dialyzed against distilled water, and then freeze-dried to obtain 340 mg of a freeze-dried sample. The total SOD activity was 1.20 x 10 ⁶ units and was electrophoretically unitary.

Example 5 “Solution D” obtained in Example 4 was
10 mmole/imidazole/hydrochloric acid buffer (PH
"Servacel TEAE-23" (manufactured by Serva: trade name) equilibrated with the same buffer after dialysis at 7.0)
and "DEAE B10-GEL A" (manufactured by Bio-Rad:
(trade name) and eluted in the same manner as in Example 4 to obtain 345 mg of each electrophoretically single freeze-dried sample.

Example 6 Yeast cake was mixed with an equal volume of 5:3 ethanol-chloroform mixture by volume, stirred at 25°C, and the mixture was then centrifuged to obtain a supernatant liquid.
SOD activity is 1.42×10 ⁷ when starting from 10 kg of yeast
It was a unit. Add to this supernatant liquid at a rate of 300g/
After adding solid K ₂ HPO ₄ and mixing, the mixture was left to stand, and the upper layer was separated. This salting-out organic phase was centrifuged to remove insoluble matter, then 0.75 volume of cold acetone was added to the supernatant, the resulting acetone precipitate was collected by centrifugation, and a small amount (approximately 200 ml) of ionic strength 10 mmole Cu ，
Adsorb Zn-SOD and use the same buffer from 0 to
The active fraction was eluted with a linear concentration gradient of up to 1 mole/NaCl, and a lyophilized product was obtained from the active fraction in the same manner as in Example 1. The total SOD activity was 6.02 x 10 ⁶ units and electrophoretically unitary.

Example 7 The acetone precipitate obtained in Example 6 was dissolved in 5:5-diethylbarbiturate sodium hydrochloride buffer (PH9.5) with an ionic strength of 50 mmole/
The ion-exchange chromatography operation was changed to the same buffer at 30℃, and the ion-exchange resin was ``DEAE-''.
Toyopeorl 650M” (manufactured by Toyo Soda Kogyo Co., Ltd.; product name)
and “Cellex QAE” (manufactured by Bio-Rad: product name)
The active fractions were collected in the same manner as in Example 6 except that . Electrophoretically single Cu, Zn−SOD
178 mg of freeze-dried sample was obtained.

Example 8 "Solution (A)" obtained in Example 1 was dialyzed against 5:5-sodium diethylbarbiturate/hydrochloric acid buffer (PH5.8) with an ionic strength of 10 mmole/ “DEAE-Sepharose CL-6B” equilibrated with the same buffer
(manufactured by Pharmacia: trade name) and Cu, Zn−
After adsorbing SOD, the procedure was repeated in the same manner as in Example 1 to obtain 372 mg of a single freeze-dried specimen electrophoretically.

Example 9 In Example 2, DEAE-Toyopearl 650M
When selectively eluting SOD adsorbed to
Ionic strength containing 0.2mole/ NaCl
Except that a sodium acetate buffer (PH4.5) with an ionic strength of 20 mmole/was used instead of a diethanolamine/hydrochloric acid buffer (PH9.0) with an ionic strength of 20 mmole/.
I did the same thing. The obtained chromatogram was the same as that obtained in Example 2, and 390 mg of an electrophoretically homogeneous freeze-dried sample of Cu, Zn-SOD was obtained from the active fraction.

Comparative Example 1 In Example 1, the buffer solution was adjusted to ionic strength.
Change to 25 mmole/phosphate buffer (PH8.1),
"DEAE-Toyopearl650M" (Toyo Soda Kogyo Co., Ltd.)
(Product name) and “DEAE-Sephadex A-50”
(manufactured by Pharmacia, trade name) and "DEAE-Cellulose DE-32" (manufactured by Watman, trade name) were used in the same manner, except that Cu, Zn -SOD was not adsorbed and flowed out of the column.

Comparative Example 2 The same operation as in Example 1 was performed at an ionic strength of 5 mmole/
The test was carried out in a phosphate buffer solution (PH7.8). As a result, 95% of the total SOD activity leaked out of the column when sample dialysate was introduced or when washed with buffer solution. On the other hand, the remaining 5% activity was eluted by flowing 0.2 mole/phosphate buffer (PH7.8) through the column.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram in Example 1. −−○・−− is SOD activity (unit/ml)−・−
indicates A ₂₈₀ , −×− indicates A ₆₈₀ , and − indicates NaCl concentration (mole/). Figure 2 shows the ultraviolet absorption spectrum and electropherogram of "Solution (B)" in Example 1, and Figure 3 shows the "Solution (B)" in Example 1.
The ultraviolet absorption spectrum and electropherogram of (C) are shown.

Claims (1)

[Claims] 1. From an aqueous solution containing Cu, Zn-superoxide dismutase and accompanying proteins.
In a method for isolating and purifying Cu, Zn-superoxide dismutase by ion exchange chromatography using an anion exchange resin, PH
Cu, Zn-superoxide dismutase is adsorbed onto an anion exchange resin in a cationic buffer solution whose acid component is hydrochloric acid, with an ionic strength of 10 to 50 mmole/5.5 to 11, and then the adsorbed Cu , Zn-superoxide dismutase at an ionic strength of 0.2 mole/
A method characterized by elution using a buffer solution with a pH of 5 or higher, or a pH of 5 or lower. 2 Anion exchange resin has diethylaminoethyl (DEAE) group, triethylaminoethyl (TEAE) group
or diethyl-(2-hydroxypropyl)-aminoethyl (QEA) groups; cross-linked dextran substituted with DEAE or QAE groups; cross-linked agarose substituted with DEAE groups; -Tris-
(Hydroxymethyl)-methylacrylamide; or a hydrophilic polyvinyl alcohol resin substituted with a DEAE group. 3 Anion exchange chromatography at 0-30℃
The method according to claim 1 or 2, which is carried out in