JPH01157382A - Purification of superoxide dismutase - Google Patents

Abstract

PURPOSE:To remove most of admixture protein, by treating a solution of superoxide dismutase(SOD) containing admixture protein with an acid and/or alkali and treating with a protease. CONSTITUTION:An animal corpuscle emolysis solution obtained from slaughter blood or a SOD solution containing admixture protein such as hemoglobin of most of the corpuscle emolysis solution is heattreated or treated with an acid or alkali and then with a protease. The acid or alkali treatment is carried out by treatment with an acid such as an organic or inorganic acid at pH2-6 not limited by the Food Sanitation Act and treatment with an alkali such as caustic soda at pH9-12. Pepsin, penicillopepsin, capepsin, sgthiricin, etc., may be cited as the protease.

Description

[Detailed description of the invention] [Industrial application field] Superoxide dismutase (hereinafter abbreviated as SOD)
is an enzyme that catalyzes a reaction that eliminates active oxygen O2-, which acts as a cytotoxin, and is known to have pharmacological effects on inflammation and rheumatism. Recently, 0
It is also being considered for use as a food for the purpose of eliminating SOD 2'' and replenishing SOD into the body, and also as an additive for cosmetics and the like.

Since such SOD has conventionally been extracted from animal blood cells such as slaughtered blood, which are inexpensive and available in large quantities, it is necessary to remove contaminant proteins such as hemoglobin.

The present invention relates to a method for removing contaminant proteins from an SOD solution containing contaminant proteins, and specifically relates to a drug.

This invention relates to a method for purifying SOD that can be used as food, etc.

[Prior art] When SOD is used as a medicine, especially an injection drug, etc.
Due to issues such as immunogenicity, it is necessary to extract SOD with high specific activity and extremely high purity. S like this
As a method for obtaining OD, for example, an organic solvent or heavy metal is added to a red blood cell hemolysate solution, and the mixture is heated at room temperature or 50 to 70°C.
There is a method of obtaining SOD using a known purification method such as gel filtration or ion exchange after heat treatment is performed to an extent that does not deactivate SOD in the range of 100 to 100% to remove most of the contaminant proteins.

However, since this method uses substances that are harmful to the human body, such as organic solvents or heavy metals, it requires large-scale equipment and a process to separate and confirm the organic solvents or heavy metals from SOD after purification. There are problems such as.

On the other hand, when SOD is used as a food or the like, there are less restrictions on purity than when it is used as a medicine. For example, when obtaining SOD using a red blood cell hemolysate as a material, there is generally no problem as long as the purification is carried out to the extent that red hemoglobin does not remain.As a method for obtaining such SOD, the method described above is used. It is also possible to use other methods, but in this case there are problems such as the complexity of the process and the time and cost required for implementation. Another method is to heat red blood cell hemolysate at a temperature of 50 to 70°C where the SOD is stable.
There is a method of heat treatment within the temperature range.

However, this method of performing heat treatment alone does not precipitate all of the contaminant proteins such as hemoglobin that have been denatured by the heat treatment, and the resulting SOD has a red color, making it difficult to use as a food as it is. There is. In order to remove residual hemoglobin etc. from the SOD obtained by this method and obtain SOD that can be used as food, it is necessary to further perform known purification techniques such as gel filtration and ion exchange. It does not meet the industrial demands of reducing production costs by simplifying as much as possible.

By solving the above problems, it is possible to purify SOD that can be used as food with simple operations, and by adding conventionally known techniques such as gel filtration and ion exchange, it is possible to produce SOD that can be used as a medicine. As a result of intensive research on the method for producing SOD, it was discovered that SOD is stable and that only contaminant proteins can be degraded by the action of proteolytic enzymes under conditions that denature only contaminant proteins, thereby completing the present invention.

That is, the present invention provides a method for purifying SOD, which is characterized in that an SOD solution containing contaminant proteins is treated with acidity and/or alkalinity and then treated with proteolysis, and will be described in detail below.

[Means for Solving the Problems] The present invention provides animal blood cell hemolysis solutions obtained, for example, from slaughtered blood, or most of the hemoglobin in these hemolysis solutions, by heat treatment, acid treatment, etc. that does not deactivate SOD. process,
It is applied to SOD solutions containing contaminant proteins derived from animal blood cell hemolysate solutions that have been removed by alkali-1 treatment or the like. These SOD solutions are first subjected to a treatment in which SOD is stable and only contaminant proteins are denatured.

As a means of denaturing contaminant proteins, acid treatment is used to denature only the contaminant proteins without denaturing SOD.

Although any alkaline treatment can be used without any restriction, in the present invention, acid treatment and alkaline treatment that do not use substances harmful to the human body are preferable in order to simplify the operation.

For example, p) hydrochloric acid, phosphoric acid in the range of 12-6.

#Acid treatment with organic or inorganic acids that are not restricted by the Food Sanitation Act, such as acid, carbonic acid, lactic acid, citric acid, or alkaline treatment in the pH range of 9 to 12 with salts of the aforementioned acids, chemical soda, ammonia, etc. , an acid which is not subject to restrictions under the Food Sanitation Law and is preferably employed in the present invention because it does not cause denaturation of SOD and causes denaturation of hemoglobin, etc. present in the SOD solution derived from, for example, an animal blood cell hemolysate solution.

As alkaline substances, food additive analysis method (
Please refer to Kodansha) etc.

These treatments alone are sufficient to organize contaminant proteins such as hemoglobin, but if other contaminant proteins that are difficult to denature are present, it is also possible to repeat these treatments. Furthermore, for example, after acid treatment, an enzyme as described later may be added to decompose the denatured contaminant protein, followed by alkaline treatment, and then the enzyme may be added.

The above-mentioned treatment may be carried out at about room temperature where the SOD is stable, but it may also be carried out at a temperature of 30 to 70°C in consideration of the optimum temperature of the enzyme used, which will be described later.

As a proteolytic enzyme, SOD (usually exists as a dimer and has a molecular weight of 32,000) contaminant proteins.
There is no restriction as long as it can be broken down into low molecular weight peptides that can be separated by general methods such as ultrafiltration and dialysis.
Specifically, pepsin.

benicilopepsin, cabegudin, sugetilisin.

Asparatic proteases with low substrate specificity, such as chymotrypsin, are preferred. The amount of enzyme added needs to be determined empirically depending on the conditions such as the activity of the enzyme used by the practitioner, but according to the experience of the present inventors, the amount of added enzyme is 1/10 to 1/1 of the total protein in the hemolytic solution. 1000 was appropriate.

Enzymes generally have an optimum pH at which they can effectively exert their activity.
, is known to have a suitable temperature.

For this reason, the pH and temperature of the hemolytic solution after denaturation treatment may be adjusted prior to the addition of proteolytic enzymes depending on the enzyme used, or the enzyme may be selectively employed depending on the state of the hemolytic solution. For example, when denaturation is carried out by acid treatment, an enzyme having an optimum pH in the acidic region is used, and in case of alkaline treatment, an enzyme having an optimum pH in the alkaline region is used.

Also, if necessary, adjust the temperature to 70' to match the optimum temperature of the enzyme.
Heating can also be carried out to a temperature range below 50°C.

Through the above operations, the contaminant proteins in the solution are decomposed into peptides, some of which become insolubilized, so they are removed by known techniques such as centrifugation and filtration, and low-molecular-weight peptides remaining in the solution are It can be removed by filtration or dialysis.

[Effects of the Invention] According to the present invention, most of the contaminant proteins in the SOD solution can be removed. In particular, when an SOD solution derived from an animal red blood cell hemolysate solution is produced using the present invention, no residual hemoglobin is observed, which would be an obstacle when using purified SOD as a food. In the present invention, the SOD obtained using animal red blood cell hemolysis solution as a raw material is 5% relative to the raw material.
It has a specific activity ~20 times higher, and can be used in foods and even medicines.

Since the SOD obtained by the present invention contains enzymes used to decompose contaminant proteins, it cannot be used as a medicine as it is, especially as an injection drug. However, it can be used as a material for gel filtration, ion exchange, etc. High purity SOD can be obtained extremely easily by the conventionally known purification method. In this case, since the present invention does not require the use of substances harmful to the human body, the necessary equipment, processes, etc. can be simplified compared to conventional methods. Furthermore, even when using SOD as food, the present invention does not require the use of substances harmful to the human body, so SOD that can be used as food can be obtained immediately by simple operations.

Although it is possible to obtain SOD that can be used in foods, etc. using the conventional heat treatment method for purifying SOD, the present invention uses enzyme treatment to decompose contaminant proteins into peptides, so centrifugal separation, filtration, ultraviolet filtration, etc. Since it is possible to use a simple and quick method such as filtration to separate large amounts of SOD from this peptide, it is possible to separate SOD and denatured contaminant proteins using methods such as gel filtration and ion exchange. It is possible to increase the yield and operability and reduce production costs, etc., compared to the method that requires the following steps.

EXAMPLES Below, Examples will be shown to explain the present invention in more detail, but the present invention is not limited to these Examples.

[Example] Example 1 Pig red blood cells were diluted to 5@ with water to hemolyze them, 1ON caustic soda was added dropwise to adjust the pH to 10.5, and after being left at room temperature for 1 hour, 12N hydrochloric acid was added dropwise to adjust the pH to 3.5. After 30 minutes, the hemoglobin is denatured by neutralization with ION caustic soda, the precipitate is removed, and an SOD solution (SOD activity 6
0 U / [! I+, specific activity 18U/■) 400a+
Get I. Add 12N hydrochloric acid dropwise to this solution to pH 5.0.
After adjusting the temperature to
Add 90 quarts of water and leave it overnight while stirring.

The insoluble matter generated in this operation was removed by diatomaceous filtration, and the resulting supernatant was further dialyzed against pure SOD solution. When the SOD activity and protein concentration were measured, the SOD activity was 68 U / ml, SOD specific activity was 340 U/rag, and purity increased approximately 20 times.

This solution did not exhibit the red color derived from hemoglobin.

Note that SOD activity was determined by J, M, McCord & I.

Fri dou ich (J, B t o 1.
Chem.

244.6049 (1969)),
Protein concentration was Prate i nAs5 manufactured by BioRad.
Measured with ay Kit.

[Brief explanation of the drawing]

Figure 1 shows SOD solution (1), which is a raw material obtained by treating the pig red blood cell hemolysis solution obtained in Example 1 with alkali treatment and acid treatment to remove most of the fire-retardant proteins such as hemoglobin, and the purified SOD solution (2). It is an absorption spectrum diagram. The SOD solution used as a raw material has strong absorption near 40°n Ill, which is caused by hemoglobin, which is the main contaminant protein, and is the cause of the red color.
In solution this absorption is absent. This indicates that hemoglobin in the SOD solution has been removed, and at the same time indicates that the SOD solution is colorless. During the measurement, the absorbance at 280 nm of the two SOD solutions should be adjusted so that they are almost the same (this absorption originates from tyrosine and tryptophan residues in proteins and is a measure of protein concentration). In addition, the absorbance at 280 nm is ■: 0.309. ■: 0.287.

Claims (1)

[Claims] (1) A method for purifying superoxide dismutase, which comprises acidifying and/or alkaline treating a superoxide dismutase solution containing contaminant proteins and then treating it with a protease.