JPH0134033B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for purifying copper-zinc superoxide dismutase (hereinafter abbreviated as Cu-Zn-SOD). SOD is an enzyme that decomposes superoxide ion (O ^- ₂ ), which is one of the active oxygen molecular species that exists in living tissues and blood of bacteria, mammals, and humans, and exhibits oxygen toxicity in living organisms. Due to the difference in the chelating metal, Fe−SOD, Mn−
It is classified into three types: SOD and Cu・Zn−SOD.
SOD is an effective therapeutic agent for inflammation that is thought to occur from tissue damage caused by superoxide ions generated from oxygen molecules in the body, such as osteoarthritis and rheumatoid arthritis, as well as damage caused by radiation. Attention has been paid. Methods for purifying and isolating SOD from living tissues and blood include ammonium sulfate salting-out method and heat treatment method (Japanese Patent Application Laid-open No. 1983-1999).
12993), Organochlorine Compound Treatment Method (Journal of
Biological Chemistry (JBC) 244 6051 (1969))
,
Ion exchange resin treatment method (Special Publication No. 53-22137), etc.
Various methods are known. However, these methods have poor purification efficiency, making it difficult to obtain products with high specific activity in high yields, and require complicated pretreatments such as desalting and removal of contaminant proteins, making industrial purification difficult. It is not necessarily a good method as a law. Therefore, as a result of various studies on simple purification methods for SOD, the present inventors found that
When the crude extract of Cu・Zn−SOD is brought into contact with a nonpolar high porous polymer resin, SOD is selectively and efficiently adsorbed to the nonpolar high porous polymer resin, and the Cu・Zn−
Only SOD was selectively eluted, Fe−SOD and Mn
- It was found that SOD was not eluted. The present invention has been completed based on the above findings. Therefore, in the present invention, after contacting a non-polar high porous polymer resin with a solution containing impure Cu/Zn-SOD, the resin is washed, and then the Cu/Zn-SOD adsorbed on the resin is eluted. This invention relates to a method for purifying Cu/Zn-SOD. The non-polar highly porous polymer resin used in the present invention is not particularly limited as long as it has Cu/Zn-SOD adsorption ability, but examples of resin base materials include styrene and divinylbenzene, or A polystyrene-based resin copolymerized by adding a small amount of other components such as 2-vinylpyridine is desirable, such as Amberlite.
XAD-2, XAD-4, XAD-9 (trademark, manufactured by Rohm and Haas), Diaion HP-
10, HP-20, HP-21, HP-30, HP-40, HP
-50 (trademark, manufactured by Mitsubishi Chemical Industries, Ltd.), Diuolite S-861, S-862, ES-863, ES-865, ES
−866 (trademark, manufactured by Diamond Shamlok),
Examples include Revachit OC-1031 (trademark, manufactured by Bayer AG). More preferably higher than 150Å and 1500Å
It is a polystyrene-based non-polar highly porous polymer with the following most frequent pore diameters and pore volumes of 0.4 to 1.1 ml/g, including Amberlite XAD-2, Diaion HP-10, HP-20, HP-21, HP-30,
HP-40, Diuolite S-861, ES-863, ES
-865 and Rebachit OC-1031 are superior in terms of yield and purification efficiency. Impure materials used as raw materials in the present invention
The solution containing Cu・Zn−SOD is Cu・Zn−SOD
There are no particular restrictions as long as the solution contains
During the purification of the crude extract of Cu・Zn−SOD
Examples include solutions containing Cu.Zn-SOD, but crude extracts are preferred. Materials for obtaining the Cu/Zn-SOD-extracted solution or the solution containing Cu/Zn-SOD that can be used in the present invention may include any material containing Cu/Zn-SOD, including yeast, plants, Includes those of animal and bacterial origin. Of particular importance among these are those of yeast and mammalian origin. In the case of mammals, it may be either blood or tissue, and the species does not matter, such as humans, cows, horses, pigs, and sheep. Furthermore, any tissue containing Cu/Zn-SOD, such as liver, kidney, placenta, etc., can be used. In the case of blood, blood may be used as it is, but it is more preferable to use red blood cells after washing and removing blood plasma. In order to hemolyze red blood cells, for example, 1 to
A method such as adding 4 times the volume of water is used. In the case of tissue, any number of times is fine, but 1 to 10 times the volume,
Preferably, 2.5 to 5 times the volume of water or 0.1 to 0.3M inorganic neutral salt aqueous solution is added, and the tissue cells are crushed using an ordinary cell crusher to obtain a water-soluble protein containing Cu/Zn-SOD. A crude extract can be obtained by extracting. Water may be used as a cleaning agent to remove impurities other than Cu/Zn-SOD adsorbed on the resin, but any salt that is acidic and has a buffering effect may be used, and salts that provide ionic strength may be used. The type is not important. For example, salts of acetic acid, citric acid, succinic acid, phthalic acid, cacodylic acid, maleic acid, etc. are used. The ionic strength and PH of the cleaning agent should be 1M or less.
4 to 6 is preferable, and more preferably an acidic buffer with an ionic strength of 0.2M or less and a pH of 4.5 to 5.5. Examples of eluents that selectively elute Cu.Zn-SOD include water-soluble organic solvents and salt solutions.
Examples of water-soluble organic solvents include lower alcohols such as methanol, ethanol, propyl alcohol,
Butanol and the like can be used, and acetone and the like can be used as the ketone water-soluble organic solvent, but among these, methanol and acetone are particularly preferred. As a salt solution, neutral salt itself or PH6
It may be a buffer that maintains a buffering capacity of ~11, and the type of salt that provides ionic strength is not necessarily important. For example, neutral salts such as common salt and potassium chloride, cacodylic acid, maleic acid, phosphoric acid, imidazole 2,4,6-trimethylpyridine, triethanolamine, veronal, N-ethylmorpholine, tris, glycine, 2-amino- Salts such as 2-methyl-1,3-propanediol, diethanolamine, boric acid, glycylglycine, and carbonic acid are used. The ionic strength and PH of a salt solution is the ionic strength
2M or less with a pH of 6 to 11, more preferably,
Considering post-elution processing, etc., the ionic strength is 0.5M or less,
A pH of 9 to 10.5 is used. The composition ratio of the water-soluble organic solvent of the eluent and the buffer varies depending on the type of water-soluble organic solvent, but
The proportion of water-soluble organic solvent may be any amount within the range of 5 to 80%, but in the case of methanol it is 40 to 60%.
%, in the case of acetone 20-40% gives favorable results. Cu/Zn collected and purified by the method of the present invention
- After adjusting the pH of the SOD-containing eluate to around neutrality using inorganic and organic acids such as hydrochloric acid, sulfuric acid, and acetic acid, the solvent is desolvated under reduced pressure at room temperature by the usual method, and the solution is deionized water or a buffer solution. dialysis to remove residual salts, and purification procedures such as DEAE-carrier chromatography and gel filtration to produce highly purified Cu/Zn.
- SOD can be obtained. Next, the excellent effects of the method of the present invention will be explained using experimental examples. Experimental example Recovery rate and specific activity of SOD activity (1) Sample preparation Add 300 ml of deionized water (C) (50 ml) to 150 ml of human red blood cells for hemolysis, then add the following (A), ( Perform treatment B) or (C). Thereafter, the obtained solution was thoroughly dialyzed with 0.005M phosphate buffer (PH7.5) and used as a sample. (A) Method of the present invention The lysed blood was passed through a Diaion HP-20 column (2.3 x 25 cm, 100 ml), and almost all of the Cu.Zn-SOD contained in the lysed blood was adsorbed. The column was washed with 200ml of 0.05M sodium acetate buffer (PH5.0), followed by 100ml of water.
After washing with water, elution was performed with an eluate of 0.1 M glycine/sodium buffer (PH 10.0) containing 50% (v/v) methanol. Cu・Zn
-SOD is eluted as one peak,
Almost all the activity was found in 50 to 250 ml of eluate from the start of elution. (The activity of SOD recovered in this active fraction was 70,084 units, and the recovery rate from hemolysis was 92%.) Furthermore, the eluate containing methanol was adjusted to pH 7.0,
by evaporator in a bath heated to 30℃
Concentrate to 40ml. (B) Heat treatment method (Japanese Patent Application Laid-open No. 57-12993) 15g of sodium chloride and cupric chloride for hemolysate
15 mg was added and heat treated at 70°C for 60 minutes.
After cooling to room temperature, remove the precipitate generated by centrifugation, and add 215 mg of cupric chloride.
The pH was adjusted to 5.6, and heat treatment was performed again at 65°C for 15 minutes.
After cooling, the formed precipitate was removed by filtration,
450 ml of a slightly blue liquid was obtained. (C) Organic chlorine compound treatment method (JBC244 6051
(1969)) While stirring the lysed blood, 75 ml of ethanol and 45 ml of chloroform were added to the solution to precipitate hemoglobin. This precipitate-containing solution
After diluting with 30 ml of water and removing the precipitate by centrifugation, 70 g of dipotassium phosphate (equivalent to 30% (w/v)) was added to the resulting supernatant to separate into two phases. Ta. Collect the upper layer (ethanol-water phase) containing Cu・Zn-SOD,
After removing suspended matter by centrifugation, 0.75 times the volume of acetone was added to precipitate Cu.Zn-SOD, and then the precipitate was dissolved in 10 ml of deionized water. (2) Experimental method The SOD activity of Cu・Zn−SOD in the sample was measured using the method of Mc Cord and Fridovich (JBC 244
6049 (1969)). In other words, superoxide ion (O ^- ₂ ) generated by the enzymatic reaction of xanthine oxidase using xanthine as a substrate.
SOD activity was measured by temporal changes at a wavelength of 550 nm using a method that utilizes the fact that SOD inhibits the reducing action of oxidized cytochrome C.
In this method, the stronger the SOD activity, the weaker the absorption change. In addition, the protein in the sample was quantified according to the method of Lowry et al. (JBC 193 265 (1951)) using bovine serum albumin as a standard. That is,
A reaction in which tyrosine, tryptophan, and cysteine in proteins are reduced and colored using a phenolic reagent consisting of phosphomolybdic acid and phosphotungstic acid, and a complex is formed by Cu ²⁺ bonding with nitrogen atoms in protein chains that have lost protons in alkaline conditions. This is a method for quantifying protein content by combining the Biuretz reaction, which forms and colors the protein. For quantitative determination, a calibration curve is created using bovine serum albumin as a standard, and the protein amount is expressed as a ratio to this. (3) Experimental results The results are shown in Table 1.

[Table] As is clear from this table, B, which is a comparative method,
In method C, the recovery rate is around 70%, with a specific activity of about 400 units/mg protein, whereas in method A, which is the method of the present invention, the recovery rate is 87%, with a specific activity of 800 units/mg protein, which is more than 20% of the recovery rate. An almost double improvement in activity was seen. In this way, the method of the present invention can produce high-grade Cu・Zn−
It has the advantage of being able to obtain SOD with a high recovery rate and being easy to operate. In addition, the non-polar high porous polymer resin used is regenerated by a normal method, and the regenerated product has the same Cu/Zn-SOD adsorption ability as a new product, so it can be reused semi-permanently. Extremely economical. Next, the method of the present invention will be explained in more detail with reference to Examples. Example 1 Without removing blood, 1.15% potassium chloride solution or 0.9% sodium chloride solution of 2.5 times the weight of the placenta was added to 30 fresh human placentas, and the tissues were thoroughly disrupted using a homogenizer. . This crushed solution is centrifuged to remove solids and Cu/Zn−
A crude extract containing SOD was obtained. This is directly applied to Diaion HP-20 without any pre-treatment.
Column (12 x 70 cm, 8)
Adsorbed SOD. The column was washed with 16 ml of 0.05M sodium acetate buffer (PH5.0), and then further washed with 8 ml of water to remove red impurities adsorbed on the resin. Elution includes 50% (v/v) methanol in 0.1M glycine sodium buffer.
This was carried out using an eluent with a pH of 10.0. The fraction showing Cu・Zn−SOD activity was found in the eluate at 4 to 20 minutes after the start of elution.
The activity of SOD, which appeared during this period and was recovered in the eluate, was 91% at this stage. Further, this eluate was neutralized to pH 7.0, methanol was removed using an evaporator in a bath heated to 30°C, and further concentrated to 2. The concentrate is
The mixture was thoroughly dialyzed and equilibrated with 0.005M phosphate buffer (PH7.5). Cu・Zn− contained in dialysate
There are 2796600 units of SOD, and the recovery rate from the extract is
88%, the increase in specific activity due to this process is about 500 times
It was 608 units/mg (protein). The dialyzed concentrate is further purified by commonly used purification means to achieve a high degree of purity. That is, it was adsorbed on a DE-52 (trademark, DEAE-Cellulose, manufactured by Watmann) column (2.6 x 45 cm, 240 ml) equilibrated with 0.005M phosphate buffer (PH7.5), and 0.005M phosphate buffer (PH7.5) was used. After washing with 5), elution was performed with 0.005M phosphate buffer (PH7.5) containing 0.04M common salt. Cu・Zn−SOD
was eluted as two peaks, one large and one small. This active peak portion is collected and concentrated using an ultrafiltration membrane.
Equilibration was performed with 0.005M phosphate buffer containing 0.9% sodium chloride. This concentrated solution was further purified by gel filtration using a Sephadex G-75 (trademark, manufactured by Pharmacia) column (2.9 x 150 cm, 1) equilibrated with the same buffer. Cu obtained in this way
Zn-SOD has extremely high purity, and its specific activity is
It was 3500 units/mg (protein). In addition, the Cu・Zn−SOD recovered through all the above processes is
There were 1,620,800 units, and the recovery rate was 51%. Example 2 Following the procedure of Example 2, an experiment was conducted by changing the elution solution, non-polar high porous polymer resin, etc. after column washing. That is, 24 fresh human placentas were added with a 1.15% potassium chloride solution of 2.5 times the weight of the placenta without removing blood, and the tissues were thoroughly disrupted using a homogenizer. This crushed solution was centrifuged to remove the solid content, and 33.6 Cu/Zn−
A crude extract containing SOD was obtained. This crude extract
2.8 (equivalent to two placentas) was adsorbed onto a column (4.0 cm x 40 cm) filled with 500 ml of various non-polar high porous polymer resins, and Cu/Zn-SOD was purified according to the method of Example 2. I went there. Conditions such as column washing solution and elution solution, and obtained Cu/Zn
-SOD activity, recovery rate from crude extract, and specific activity are shown in Table 2.

[Table] Example 3 After thoroughly washing and removing blood from two fresh human placentas with a 1.15% potassium chloride solution, 2.5 times the weight of the placenta in the same solution as above was added, and the tissues were sufficiently crushed using a homogenizer to form a solid. minutes were removed by centrifugation. Diamond ion can be used as is without any treatment of the supernatant liquid.
Cu.Zn-SOD was adsorbed by passing through a HP-20 column (2.7 x 35 cm, 200 ml). Column is 0.05M sodium acetate buffer (PH5.0)
After washing with 400ml and further washing with 200ml of water, 0.1M
Glycine-sodium buffer at 50% (v/
Elution was carried out using an eluent of pH 10.0 containing methanol (v). Cu.Zn-SOD was eluted as a single peak, and almost all the SOD activity was contained within 100 ml to 500 ml from the start of elution. The recovered Cu・
The activity of Zn−SOD is 97550 units and Cu・Zn−
The yield from the SOD crude extract was 88%. The purification method of the eluate after this step is completely the same as in Example 2 except that DEAE-Toyopearl (manufactured by Toyo Soda Co., Ltd.) is used instead of DE-52 resin, and the Cu. Zn−SOD is 52100
unit, 47%, and specific activity was 3450 units/mg (protein). In this example, the placenta was not exsanguinated.
The reason why the yield and yield are lower than in Example 1 in which Cu.Zn-SOD was extracted is that Cu.Zn-SOD is lost in the red blood cell portion contained in the placenta due to blood removal. Example 4 1 kg of bovine liver was washed with a 1.15% potassium chloride solution, 5 times the liver weight of the same solution was added, and enough tissue cells were disrupted using a homogenizer.
Insoluble solids in the crushed solution were removed by centrifugation to obtain a crude extract containing Cu.Zn-SOD. This solution can be used as a diamond ionizer without further processing.
passed through a HP-20 column (6.5 x 60 cm, 2),
After washing with four parts of 0.05 sodium acetate buffer, it was further washed with one part of water. Through this adsorption and cleaning process, 13% of SOD activity was observed to leak. Elution is
50% of 0.1M glycine-sodium buffer
(v/v) methanol solution (PH10.0). The activity of SOD was concentrated in the eluate at 0.45 to 4 after the start of elution, and the activity of SOD recovered was 1,680,000 units, and the yield from the extract was 80%. Cu・
The eluate containing Zn-SOD is further purified as in Example 2 to produce pure Cu・Zn-SOD.
becomes. Example 5 1 kg of baker's yeast was suspended in twice the volume of 0.02M phosphate buffer (PH7.0), and the bacterial cells were crushed using a French press under cooling. The solid content in the crushed solution was removed by centrifugation to obtain a crude extract containing Cu.Zn-SOD. This extract was passed through a Diaion HP-20 column (2.8 x 40 cm, 250 ml) without further treatment to adsorb Cu.Zn-SOD. The column is
After washing with 500 ml of 0.05 sodium acetate buffer (PH 5.0) and further washing with 100 ml of water, elution was performed with an eluent containing 50% (v/v) methanol in 0.1 M glycine-sodium buffer at PH 10.0. did. Cu.Zn-SOD was eluted as a single peak, and almost all the SOD activity was contained within 100 ml to 500 ml from the start of elution. Recovered Cu/Zn
-The activity of SOD was 266,900 units, and the yield from the extract was 51%. Also, obtained at this stage
The specific activity of Cu.Zn-SOD was 980 units/mg (protein).

Claims (1)

[Claims] 1. After contacting a non-polar high porous polymer resin with a solution containing impure copper/zinc superoxide dismutase, the resin is washed, and then the copper/zinc superoxide dismutase adsorbed on the resin is eluted. A method for purifying copper/zinc superoxide dismutase, characterized by: