JPS6012025B2 - Purification method of superoxide dismutase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to superoxide dismutase (hereinafter referred to as S
．． 0. (abbreviated as D)).

S. 〇． D was first discovered as an enzyme that decomposes superoxide (02-), an intermediate product of xanthine oxidase, in research on xanthine oxidase by King, Friedovich, and McCord et al.

After that, S. 0. D has attracted attention from the viewpoint of the body's oxygen toxicity defense mechanism, and is currently used as an anti-inflammatory agent to treat rheumatoid arthritis, osteoarthritis, side effects from radiation, and certain urinary disorders. Conventionally, S
．． 0. Purification methods for D include heat treatment, ammonium sulfate salting out, DEAE-cellulose [Jour of
BiologcaI ChemiSby, 244 (22
)6o49 (1949). ], DEAE-Sephadex, Sephadex G-100 Uom skin l of B
iochemist, 851397 (1979). ]
, CM-Cellulose, CM-Sephadex, Cephadex G-75 [Joumalof Biologca
l chemistry 248(10)3582(
1973). ]、Hydroxyapatite [Jouma
lofBiologicalchemisthi,252
(18) 6421 (1977). ] etc. are known.

However, these methods require desalination during the treatment process, take a long time to adsorb to the adsorbent,
In addition, it is difficult to say that it is an industrially advantageous method because of problems such as adsorption of the adsorbent.

The present inventors completed the present invention as a result of intensive research to solve these problems. That is, the present invention provides crude S
．． 0. The D solution was brought into contact with a cross-linked agarose gel bonded with phenyl groups or octyl groups, and the adsorbed S. 0. D
S. 0. This relates to the D purification method.

The crosslinked agarose gel with phenyl or octyl groups used in the present invention is manufactured by Pharmacia Fine Co., Ltd.
Those commercially available from Chemical Company under the trade names of "Phenyl Sepharose CL-Go" and "Octyl Sepharose CL-No" can be used.

Sepharose CL-Hime, which is used as a matrix to produce these Fenyl Sepharose CL-B or Octyl Sepharose CL-Hime, is agarose that is crosslinked with 2,3 dibromo propanol and further reduced. It was subjected to alkaline hydrolysis and desulfation under certain conditions.

The phenyl group or octyl group is Sepharose CL-
The glycity ethers corresponding to the capes were reacted and bonded to the respective matrices. Crude S.I. of the starting material used in the present invention. 0. D can be used as appropriate because it is distributed over a very wide range in the biological world, and the origin is not particularly limited, but usually,
Preferably, it is prepurified from blood or placenta of humans, cows, chickens, rabbits, monkeys, rats, etc. by known methods.

Particularly preferred are those prepurified from human or bovine blood red blood cells or placenta by any of the following methods. That is, fresh human or bovine blood is collected in the presence of blood coagulation inhibitors such as sodium citrate, centrifuged in a cooled state, the blood in the upper layer is removed, and the precipitated red blood cells are diluted with physiological saline for 2 to 3 hours. Wash three times and collect. This one is further
It is preferable to use it as a starting material in the present invention after processing it by any of the following methods. That is, either: ■ After dissolving the dish with ultrasonic treatment or saponin treatment, use a cellulose purified with DEAE cellulose, or ■ After adding water and hemolyzing, treat with the Tsuchihashi method [Cchibashi method; 63 (1923)], add (ethanol + chloroform + water), remove hemoglobin by centrifugation, add K2HP04, centrifuge again, and add 0.7 volume of supernatant to the obtained supernatant. The crude S. 0.0
used as In the present invention, phenylcephalose CL one side B or octylcephalose CL-4B
Crude S. 0. D is 1.3 to 1 in advance
．． Add salts at a concentration of 9M and adjust the pH to 5-9, preferably 6 with 0.2M potassium phosphate or dibasic potassium phosphate.
．． Adjust to 5 to 7.5 before use. The salts used here include P〇Ashi-, S〇Ko-, CH3C as anions.
〇〇-, C so-, Br-, N03-, C 04-,
1-, SCN-, iso-enteric ions, NH4+, Rb+
, K+, Na ten, Cs+, Li+, M ten, Ca2 ten,
Typical examples include appropriate combinations of Ba20, etc., but ammonium sulfate, potassium phosphate, sodium phosphate, ammonium phosphate, ammonium chloride, potassium chloride, sodium chloride, etc. are usually suitable.

The adsorbent gel was prepared by S. 0. D2300~460 mountain units/
It is appropriate to use it at a ratio of ', and the specific velocity of the flowing liquid (flow rate per hour/bed volume) in the adsorption step and washing step is preferably 1.5 or less.

S. 0. The twill weave of D is water with a pH of 7 to 9 or 1.3 to 1.8.
It is appropriate to carry out the reaction using the M ammonium sulfate-water concentration gradient method at a specific velocity of 0.5 or less. The used gel is 120qo after washing with water.
The purification method of the present invention can be reused by repeating treatment for 15 minutes or washing with 0.1M caustic soda and water and washing with 0.1M sodium acetate two to three times.The purification method of the present invention has the following features.

- Since the gel has good stability, it can be sterilized by autoclaving at 120°C and can be used repeatedly. ■ S. to gel. 0. Due to the high selective adsorption of D, the working time is short and S. 0. No decrease in D activity was observed.

■ Processing is easy because it can be adsorbed at high salt concentrations.

(2) Concentration is easy because water or an aqueous solvent is used for elution, and there is no need for a desalting step in post-treatment.

In the present invention, S. 0. To measure the activity of S.D., the reduction of oxidized cytochrome C by superoxide (02-) generated by the reaction between xanthine and xanthine oxidase is measured. 0. This was carried out based on the Pine cord and Friedovich method, which utilizes the inhibition caused by D.

The absorbance of each elution fraction was measured at 265h and 28h, and the absorbance ratio OD265/OD280>1 was used as a guideline. 0. I selected category D. [J. M. Matscord and Friedovich; Journal of Biological Chemistry, 244 (22) 6049 (196
9)], protein quantification was performed using the Lowry method [Lowry O.
Journal of Biological Chemistry, 193, 265 (1951)]. below,
Crude S. 0.
A preliminary purification method for preparing D is shown in a reference example.

Reference example 1. Fresh bovine blood 2. Collected in 1 volume of 3.8% sodium citrate, 2°C, 2500xG, 2 minutes,
Centrifuge.

After removing the supernatant fluid, the precipitated erythrocytes are washed three times with physiological saline, and 1 volume of water is added to each erythrocyte precipitate to cause hemolysis. According to the Shihashi method, this total amount was mixed with 0 ethyl alcohol.
．． Add 25 parts, 0.15 parts of chloroform, and 0.1 parts of water, mix well, stir at 5℃ for 2 minutes, and shake the dark brown mixture at 2500 x G for 2 minutes. After removing hemoglobin, etc., 0.0% dibasic potassium phosphate was added. Total/
Add at a ratio of '', mix thoroughly, and leave at room temperature for 15 minutes.
The pale brown upper layer separated into two layers was washed at 2500×G for 2 hours to obtain a pale yellow upper layer. Add 0.75 cold acetone to this.
The mixture was mixed and centrifuged at 2,500 x G for 20 minutes to obtain a white precipitate, which was dissolved in about 1/1 of the washed red blood cells with water and centrifuged at 10,000 x G for 20 minutes. A clear supernatant of 95' was obtained. This item is S. 0. Crude S.D. 3472 units/' (specific activity 417 units/9 proteins). 0.
D, and can be suitably used as a starting material for purification by the method of the present invention. Reference Example 2 1 kg of the outside of a delivered human placenta was washed thoroughly with water, placed in a polyethylene bag, and immediately frozen. The sample was extracted overnight at 5° C., and centrifuged at 2500×G for 20 minutes to remove solid matter.

Following Reference Example 1, a transparent sample 50' was obtained by the Dobashi method treatment, acetone treatment, cooling centrifugation, etc. This item is S.
0. D350 units/noto (specific activity: 750 units/female protein) crude S. 0. D and can be suitably used as a starting material for purification by the method of the present invention. Example 1 Crude S. 0. Add 17.1 g of ammonium sulfate to D, dissolve it, and adjust to pH 6.8' with 0.2 M potassium phosphate.
Correct this.

This solution was pre-equilibrated with a pH 6.8 buffer solution (1.4M ammonium sulfate + 0.09M phosphoric acid) using a force ram (diameter 2.0cm) filled with Phenyl Sepharose CL-80.
, height 25.5 mm) at a flow rate of 120 [/hour]. 0. Adsorb D. pH 6.8 buffer (
Washed with 80 1.4M ammonium sulfate and 0.09M potassium phosphate), and then washed with pH 9 water (0.1M caustic soda to pH 9).
Elute at a flow rate above 40/hour (adjusted to ). This elution pattern is shown in FIG. S. 0. D Highly active category (F
r14 to Frl9). 0. The specific activity of D is 7
200 units/9 protein, crude S. 0. The activity yield from D was 95%. Example 2 The same procedure as in Example 1 was carried out except that octyl Sepharose CL-block was used instead of phenyl-Sepharose CL-block, and S.C. 0. D was obtained with an active yield of 96%.

Example 3 Using human blood IZ stored at 5°C for 2 to 3 days in the presence of citrate dextrin, crude S.
0. D40 (2500 units/', specific activity 667 units/female protein) was obtained.

Next, this crude S. 0. D is ammonium sulfate7. Dissolve and pH 6
．． After adjusting the pH to 8, add a buffer solution of pH 6.8 (1.4
Column packed with phenylcephalose CL-Crab, 40', equilibrated with M ammonium sulfate + 0.09 M potassium phosphate) (
It is passed through the tower at a flow rate of 60/hour to a diameter of 2.0 mm and a height of 12.7 arc. Then, about 40% of the pH 6.8 buffer solution is added.
After washing with 100% of the same buffer, the eluate was washed with a concentration gradient of 200% of the same buffer and 200% of water at a flow rate of 20%/hour. 0. The specific activity of S.D. is 7400 units/female protein; 0. D
The activity yield was 97%. Example 4 The same procedure as in Example 3 was carried out except that octyl Sepharose CL- was used instead of phenyl Sepharose CL- to obtain a specific activity of 800 units/S. 0.0 was obtained with an active yield of 99%.

In addition, the crude S. 0. Regarding D, DEA
Regarding known methods using E cellulose and CM cellulose, and the methods of Examples 1 to 4 of the present invention, S. The results of comparing the purification effects of 0.0 are shown in Table 1. According to the method of the present invention, the yield,
It has excellent purity.

Table 1 Comparative Example of Purification Effects Using Various Adsorbents 5 The crude product obtained in Reference Example 5.0.0 and ammonium sulfate 9.0. After adjusting the solution in which the females were dissolved to pH 6.8, a column packed with 40 phenyl Sepharose CL-blocks (diameter 2.0 Celsius, height 12.7 Celsius) at a flow rate of 60/hour.

After washing with the same buffer solution at 40°C, it is eluted with water at pH 9 at a flow rate of 20°/hour. S. 0. The specific activity of D is 7500 units/9 protein, and the crude S.D. 0.
The activity yield from 0 was 96.5%. Example 6 The same procedure as in Example 5 was carried out except that Octyl Sepharose CL-Light was used instead of Fenyl Sepharose CL-5 to obtain a specific activity of 7650 units/S. 0. D was obtained with an active yield of 98%.

[Brief explanation of drawings]

FIG. 1 shows the elution pattern according to the present invention. The vertical axis is S. 0. D activity (units/unit) and absorbance at 268 h〆 and 28 h〃 are shown, and the horizontal axis shows the elution fraction (
[) of IFr=6 is shown. Flute 1 diagram

Claims (1)

[Claims] 1. A method for purifying superoxide dismutase, which comprises bringing a solution containing crude superoxide dismutase into contact with a cross-linked agarose gel to which phenyl groups or octyl groups are bonded, and eluating and collecting the adsorbed superoxide dismutase.