JP3232542B2 - Human Cu, Zn type superoxide dismutase derivative and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a human Cu, Zn superoxide dismutase derivative useful for superoxide-related diseases and a method for producing the same (hereinafter referred to as "superoxide dismutase").
Is referred to as “SOD”, and “human Cu, Zn-type superoxide dismutase” is referred to as “human SOD”. ). More specifically, the present invention retains an enzymatic activity of decomposing a superoxide harmful to a living body, and has a charge and molecular weight by introducing an alkylthio group into a mercapto group of cysteine at position 111 of human SOD constituent amino acid by a disulfide exchange reaction. Human SO that is homogeneous and stable in SOD activity
The present invention relates to a D derivative and a method for producing the same.

[0002]

2. Description of the Related Art SOD is widely present in living organisms such as animals, plants and microorganisms and has a function of decomposing superoxide harmful to living organisms. Therefore, SOD is used as a preventive agent for various diseases caused by superoxide. , Therapeutics (eg, inflammation, osteoarthritis, rheumatoid arthritis,
Disorders due to radiation, ultraviolet rays, oxygen retinopathy of premature infants, cataracts, side effects of anticancer drugs such as adriamycin, disorders associated with resumption of blood flow to ischemic areas, etc.).

[0003] When SOD is used as a pharmaceutical product in humans, it is preferable to use human SOD due to the problem of antigenicity. Examples of human SOD include natural human SOD produced from human tissues and recombinant human SOD having an amino acid sequence substantially the same as human SOD produced using a microorganism by a genetic engineering technique. When human SOD is isolated from human tissues or microbial cells produced by genetic engineering techniques, conventional methods cannot obtain human SOD in a uniform form in terms of charge and molecular weight. This is a major problem in developing human SOD as a drug that requires homogeneity in quality.

[0004] W. Hartz and H.M. F. Deut
sch [J. Biol. Chem. , 224 , 4565
(1965)] focused on the reactivity of cysteine, the 111st amino acid of human SOD. And human S
The cause of the non-uniform charge and molecular weight of OD was thought to be that this cysteine had undergone various conversions due to its high reactivity, but the inference has not been verified. Also, J.I. R. Jabusch, D.M. L. F
arb, D.A. A. Kerschensteiner and H.S. F. Deutsch [Biochemistry, 19 , 231]
0 (1980)] reports that the activity stability of human SOD is improved by alkylating the mercapto group of cysteine at position 111 of human SOD.

However, as in the present invention, one of human SODs
By introducing an alkylthio group into the mercapto group of cysteine, which is the eleventh amino acid, it is possible to obtain a human SOD derivative which not only improves the stability of human SOD activity but also is uniform in charge and molecular weight. What was possible was not previously known.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a human SOD derivative which is uniform in charge and molecular weight and has a stable activity, and which is useful as a pharmaceutical and a method for producing the same.

[0007]

The present inventors have conducted studies to solve the above-mentioned problems, and as a result, introduced an alkylthio group into the mercapto group of cysteine at position 111 of the human SOD constituent amino acid by a disulfide exchange reaction. As a result, they have found that a human SOD derivative that is uniform in charge and molecular weight and has stable SOD activity can be produced, and the present invention has been completed. That is, the present invention is as follows. The first invention provides the following formula (I):

[0008]

Embedded image

Wherein (human SOD ') is human SOD
X represents the amino group, an alkyl group having 1 to 10 carbon atoms or an acyl group having 2 to 10 carbon atoms, which is obtained by removing a hydrogen atom from the mercapto group of the cysteine residue at position 111; R ¹ and R ² represent a hydrogen atom or a carbon atom having 1
Represents 10 to 10 alkyl groups. ] Human SOD
It relates to derivatives.

The second invention relates to human SOD and the following formula (II):

[0011]

Embedded image

Wherein X, R ¹ and R ² have the same meanings as defined above, and reacting with an alkyl disulfide represented by the formula (I). The present invention relates to a method for producing a derivative. Hereinafter, the present invention will be described in detail.

The human SOD derivative as the above-mentioned target, and alkyl disulfide [the compound (I
X), R ¹ , and R ² represented in (I)] are as follows.

X is an amino group, having 1 to 1 carbon atoms.
Examples thereof include an amino group and a hydroxyl group substituted with zero alkyl groups or acyl groups having 2 to 10 carbon atoms. The amino group substituted with the alkyl group of X is preferably an amino group substituted with a linear or branched alkyl group having preferably 1 to 6, more preferably 1 to 4 carbon atoms. The amino group substituted with the acyl group of X is preferably an amino group substituted with a linear or branched acyl group having preferably 1 to 6, more preferably 1 to 4 carbon atoms.

Examples of R ¹ and R ² include a hydrogen atom and an alkyl group having 1 to 10 carbon atoms. The alkyl group for R ¹ and R ² is preferably a linear or branched one having 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms.

[0016] The SOD derivative can be produced by reacting human SOD with alkyl disulfide in a buffer at a temperature not higher than room temperature. Examples of human SOD include natural human SOD and recombinant human SOD. And (human SOD ')
It represents the result of removing the hydrogen atom from the mercapto group of the cysteine residue at position 111 of human SOD. Natural human SOD
Can be obtained from human tissues such as erythrocytes and placenta by a conventional method, or can be obtained as a commercial product. Recombinant human SOD is a natural human SOD.
It has substantially the same amino acid sequence as that of the present invention, and can be obtained, for example, by the method described in JP-A-61-111690, or it can be obtained as a commercial product.

When the human SOD lacks all or a part of its constituent copper ions and zinc ions (apohuman SOD), a process for incorporating the required amount of the missing metal. is necessary. The timing of the treatment is not particularly limited as long as the desired human SOD derivative can be obtained, but it is preferable that the treatment is performed after the human SOD is reacted with the alkyl disulfide.
The treatment of Apohuman SOD with metal ions is carried out in a buffer solution.
The reaction can be performed by reacting Apohuman SOD with metal ions at a temperature not higher than room temperature. Apohit SOD
Examples thereof include a reaction with an alkyl disulfide; centrifugation, ammonium sulfate precipitation, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, metal chelating affinity chromatography, hydrophobic interaction chromatography, etc. Each treatment; those obtained by a treatment in which the above treatment methods are appropriately combined, and the like.

The metal ions include copper ions and zinc ions. In the reaction with Apohuman SOD, metal ions are supplied to the reaction system in the form of a salt. Examples of copper ions include divalent copper salts (eg, cupric chloride, cupric nitrate, cupric sulfate, cupric acetate, etc.). Examples of zinc ions include zinc salts (for example, zinc chloride, zinc nitrate, zinc sulfate, zinc acetate, and the like).

The concentration of copper salt and zinc salt is 0.5 to 20.
mM, preferably 1 to 10 mM. Examples of the buffer component in the metal ion treatment include sodium acetate, sodium hydrogen carbonate, sodium phosphate, and the like. And the concentration of the buffer is 5-5
00 mM, preferably 10-100 mM; pH
Is 4.5 to 9, preferably 4.5 to 8.0.
The concentration of Apohuman SOD is 1 to 100 mg / ml, preferably 5 to 50 mg / ml. The reaction temperature in the metal ion treatment is preferably a temperature of room temperature or lower, and more preferably a temperature of 0 to 10 ° C. The reaction time in the metal ion treatment is 10 minutes to 1 day.

The most preferred compound (II) [alkyl disulfide] used for producing the SOD derivative includes, for example, bis (2-aminoethyl) disulfide,
Bis (2-acetylaminoethyl) disulfide, bis (2-hydroxyethyl) disulfide, bis (2-hydroxypropyl) disulfide and the like can be mentioned.

The components of the buffer used at this time include:
For example, tris (hydroxymethyl) aminomethane hydrochloride, sodium carbonate, sodium hydrogen carbonate, sodium phosphate, triethanolamine hydrochloride and the like can be mentioned. And the concentration of the buffer is 5-100 m
M, preferably 10 to 50 mM; the pH is preferably pH 6 to 10 since a reaction slows down when the pH is lower than 6 and a change in mercapto group occurs to generate various by-products when the pH is higher than 9. 9, more preferably pH 7 to
8 is good.

The reaction temperature is preferably room temperature or lower, more preferably 0 to 10 ° C. The concentration of human SOD is
Although not particularly limited, preferably 1 to 100 mg / ml
Is good. The concentration of the alkyl disulfide is 5 to 300 times, preferably 10 to 200 times the molar concentration of human SOD.

The thus prepared human SOD derivative is obtained by centrifuging the reaction mixture, ammonium sulfate precipitation, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, metal chelating affinity chromatography, hydrophobicity. It can be obtained by purifying by appropriately combining purification methods such as interaction chromatography. The SOD derivative thus obtained is uniform in charge and molecular weight and has stable SOD activity.

[0024]

EXAMPLES The present invention will be described below with reference examples, examples and analysis examples.
Therefore, it is shown. Note that these examples are not intended to limit the scope of the present invention.
It is not limited. Reference Example (1) Culture of recombinant E. coli and induction synthesis of human SOD Sterile medium [Soy protein hydrolyzate (45 g), glucose
(60 g), tetracycline (0.188 g), Na
_TwoHPO_Four(90g), KH_TwoPO_Four(45 g), Na
Cl (7.5 g), NH_FourCl (15 g), MgSO_Four
(3.6 g), CaCl_Two(0.167 g), CuCl
_Two・ 2H_TwoO (80mg) and ZnSO _Four・ 7H_TwoO
(132mg)] (12,000ml)
Bacteria 545πHR (pHT351)
No. 5), and perform stirring and aeration.
And cultured overnight at 37 ° C.

This culture solution (5,000 ml) was added to a sterilized medium
[Soy protein hydrolyzate (2830 g), glucose (2
025 g), tetracycline (1.7 g), Na_TwoH
PO _Four(810g), KH_TwoPO_Four(405 g), Na
Cl (67.5 g), NH_FourCl (135 g), MgS
O_Four(32.4 g), CaCl_Two(1.5 g), CuC
l_Two・ 2H_TwoO (725 mg), ZnSO_Two・ 7H_TwoO
(1.188 g), FeSO_Four・ 7H_TwoO (5.4
g), MnSO_Four・ 5H_TwoO (1.35 g), AlCl
_Three・ 6H_TwoO (1.35 g) and H_ThreeBO_Three(68m
g)) (110,000 ml) and dissolved oxygen
Stirring to maintain the volume above 20% of saturated oxygen concentration
Klet 200, 40 while performing aeration
ZnSO at 0,600_Four・ 7H_Two2.38 g each of O,
An aqueous solution (500 ml) containing 3.57 g and 4.76 g was added.
Then, the cells were cultured at 37 ° C. at pH 7.1 to 7.4. Cress
After culturing to 1200, glucose (2025 g)
Aqueous solution (3,000ml)
Aqueous solution (500 ml) containing
In addition, induction synthesis is performed at 37 ° C. for 4 hours with aeration and stirring.
I let you. After induced synthesis, the culture mixture was centrifuged (10,80
OG for 60 minutes) to obtain 4.5 Kg of wet cells.

(2) Disruption of human SOD-producing cells and preparation of a crude extract thereof The wet cells (50 g) obtained in the same manner as in the above (1) were mixed with 1
0 mM triethanolamine buffer (pH 7.0, 25
0 ml) and crushed with a French press. The homogenate was centrifuged (28,400 G, 15 minutes) to obtain a supernatant (225 ml).

(3) Purification of Crude Human Apo SOD The wet cells (200 g) obtained in the same manner as in the above (1) were treated with a 10 mM triethanolamine buffer (pH 7.0, 5).
00 ml) and crushed with a French press, and the crushed liquid was centrifuged (10,800 G, 60 minutes) to obtain a supernatant. To this solution was added 10 mM triethanolamine buffer (pH 7.0) to make the total volume 2,000 ml.
To this was added ammonium sulfate (722 g) and the mixture was stirred at about 4 ° C. overnight. The resulting precipitate was centrifuged (13,
(400 G, 15 minutes), ammonium sulfate (516 g) was added to the obtained supernatant, and the mixture was stirred at about 4 ° C. for 3 hours, and the resulting precipitate was centrifuged (13,400 G,
15 minutes). The resulting precipitate was dissolved in a 10 mM triethanolamine buffer (pH 7.0), and the solution was dissolved in a 10 mM triethanolamine buffer (pH 7.0, pH 7.0).
(2,500 ml).

This dialysis solution is passed through a Q-Sepharose FF (Pharmacia, 200 ml) column equilibrated with 10 mM triethanolamine buffer (pH 7.0), and the column is passed through a 10 mM triethanolamine buffer (pH 7.0). 0, 1,000 ml), then 30 mM
NaCl-10 mM triethanolamine buffer (p
H7.0) and the human SOD fraction was collected. This S
Ammonium sulfate (320) was added to the OD fraction (460 ml).
g) was added and the mixture was stirred at about 4 ° C. for 6 hours, after which the resulting precipitate was collected by centrifugation (10,800 G, 30 minutes). The obtained precipitate was dissolved in water, and the solution was dissolved in water (2,500 m
Dialyzed 3 times against l). The dialysate was filtered through a 0.2 μm filter and freeze-dried to obtain 1.76 g of a colorless solid (recombinant human apo SOD).

(4) Preparation of Human SOD Treated with CuCl _{2 To} the recombinant human apo SOD (1 g) obtained in the above (3), 2M acetate buffer (pH 5.0, 1 ml) and water (14 ml) were added and stirred. Then, a 0.1 M CuCl ₂ aqueous solution (1.2
5 ml) and stirred at about 4 ° C. for one day. This liquid
0.5 M NaCl-0.1 M acetate buffer (pH 5.0)
G25 equilibrated with (Pharmacia, 100 ml)
The mixture was passed through a column and developed with a 0.5 M NaCl-0.1 M acetate buffer (pH 5.0) to collect a human SOD fraction.

This was concentrated using an ultrafiltration membrane, and 10 m
Pass through a G25 (Pharmacia, 100 ml) column equilibrated with M triethanolamine buffer (pH 7.0) and 10 mM triethanolamine buffer (pH 7.0).
0,500 ml), 25 mM NaCl-10 mM
The human SOD fraction was collected by developing with a triethanolamine buffer (pH 7.0). This was concentrated using an ultrafiltration membrane, a 0.5 M NaCl aqueous solution was added, and the mixture was concentrated again, and G25 (manufactured by Pharmacia, 100 ml) equilibrated with water.
The SOD fraction was collected by passing through a column and developing with water. This was concentrated using an ultrafiltration membrane to obtain 49 human recombinant SOD.
0 mg (calculated from the absorbance measured with an ultraviolet spectrophotometer) was obtained.

(5) Reaction of human SOD with 2,2'-dithiobis (benzothiazole) Apohuman SOD (0. 0) obtained in the same manner as in the above (3).
5 g) in 10 mM triethanolamine buffer (pH
7.0, 100 ml) and adjusted the pH to 7.0. 2,2'-dithiobis (benzothiazole)
(333 mg) was added, the pH was adjusted to 7.0, and the mixture was stirred at about 4 ° C. After 77 hours, NaCl (2.92
g), 2 M acetate buffer (pH 5.0, 5 ml) and 0.1 M
A 1M CuCl ₂ aqueous solution (1.5 ml) was added, and the mixture was stirred at about 4 ° C. for 17 hours. After stirring, the pH was adjusted to 7.0, ammonium sulfate (77 g) was added, the mixture was stirred at about 4 ° C. for 3 hours, and the resulting precipitate was centrifuged (28,400 G, 15 g).
Min).

This precipitate was dissolved in 10 mM triethanolamine buffer (pH 7.0) and dialyzed three times against 10 mM triethanolamine buffer (pH 7.0, 1,000 ml). The dialysate is passed through a Q-Sepharose FF (Pharmacia, 10 ml) column equilibrated with a 10 mM triethanolamine buffer (pH 7.0), and then passed through a 10 mM triethanolamine buffer (pH 7.0).
0, 50 ml), followed by 5 mM, 10 mM,
10 mM containing 20 mM and 50 mM NaCl, respectively
The human SOD fraction was collected by developing with a triethanolamine buffer (pH 7.0). The SOD fraction was saturated with ammonium sulfate, stirred at about 4 ° C. for 6 hours, and the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes) and dialyzed three times against water (1,000 ml). . The obtained dialysate was filtered through a 0.2 μm filter and freeze-dried to obtain 250 mg of a human SOD derivative.

(6) Reaction of human SOD with 5,5'-dithiobis (nitrobenzoic acid) Apohuman SOD (7) obtained in the same manner as in the above (3)
8.5 mg) was dissolved in 10 mM triethanolamine buffer (pH 7.0, 5 ml), and the pH was adjusted to 7.0. 5,5'-dithiobis (nitrobenzoic acid)
(9.9 mg) was added, the pH was readjusted to 7.0, and the mixture was stirred at about 4 ° C. for 14 hours. NaCl (0.16
g) and 2M acetate buffer (pH 5.0, 0.26 ml)
Was added to adjust the pH to 5.0, an aqueous 0.1 M CuCl ₂ solution (0.1 ml) was added, and the mixture was stirred at about 4 ° C. for 12 hours.

After adjusting the pH to 7.0, a 10 mM triethanolamine buffer (pH 7.0, 1,000 m
1) was dialyzed three times and passed through a Q-Sepharose FF (Pharmacia, 10 ml) column equilibrated with a 10 mM triethanolamine buffer (pH 7.0).
10 mM triethanolamine buffer (pH 7.0, 5
0ml), and then sequentially washed with 5mM, 10mM, 20m
M and 50 mM NaCl, respectively, and developed with 10 mM triethanolamine buffer solution (pH 7.0).
The OD fraction was collected. The human SOD fraction was saturated with ammonium sulfate and stirred at about 4 ° C. for 6 hours, and the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes). This was dissolved in water, dialyzed three times against water (1,000 ml), filtered through a 0.2 μm filter, and lyophilized to obtain 60 mg of a human SOD derivative.

(7) Reaction of human SOD with 6,6'-dithiobis (nicotinic acid) Apohuman SOD (0. 0) obtained in the same manner as in the above (3).
5 g) in 10 mM triethanolamine buffer (pH
7.0, 100 ml) and adjusted the pH to 7.0. To this, 6,6′-dithiobis (nicotinic acid) (30
8 mg), readjust the pH to 7.0, and add
Stir for 7 hours. To this, NaCl (2.92 g) and 2 M acetate buffer (pH 5.0, 5 ml) were added to adjust the pH to 5.0, and a 0.1 M CuCl ₂ aqueous solution (0.1 m
l) was added and the mixture was stirred at about 4 ° C for 17 hours. pH 7.0
, And ammonium sulfate (77 g) was added.
After stirring for 3 hours, the resulting precipitate was centrifuged (28.4
00G, 15 minutes). This precipitate was dissolved in 10 mM triethanolamine buffer (pH 7.0),
mM triethanolamine buffer (pH 7.0, 1,0
(00 ml).

Q-Sepharose FF obtained by equilibrating the dialysate with 10 mM triethanolamine buffer (pH 7.0)
(Pharmacia, 10 ml)
50 ml of M triethanolamine buffer (pH 7.0)
After washing with 5mM, 10mM, 20mM, 50mM
The human SOD fraction was collected by developing with a 10 mM triethanolamine buffer (pH 7.0) containing mM NaCl. After the SOD fraction was saturated with ammonium sulfate and stirred at about 4 ° C. for 6 hours, the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes). The obtained precipitate was dissolved in water, dialyzed against water (1,000 ml) three times, filtered through a 0.2 μm filter, and lyophilized to obtain 365 mg of a human SOD derivative.

(8) Reaction of human SOD with dithiobis (glycolic acid) Apohuman SOD (0. 0) obtained in the same manner as in (3) above.
5 g) in 10 mM triethanolamine buffer (pH
7.0, 100 ml) and adjusted the pH to 7.0. Dithiobis (glycolic acid) (182mg)
Was added to adjust the pH to 7.0, followed by stirring at 4 ° C. for 77 hours. After stirring, NaCl (2.92 g), 2 M acetate buffer (pH 5.0, 5 ml) and 0.1 M CuCl ₂ aqueous solution (1.5 ml) were added, and the mixture was stirred at 4 ° C for 17 hours.
The pH was adjusted to 7.0, saturated with ammonium sulfate, stirred at about 4 ° C. for 6 hours, and the resulting precipitate was centrifuged (2.
(8,400 G, 15 minutes).

This was dissolved in a 10 mM triethanolamine buffer (pH 7.0), dialyzed against a 10 mM triethanolamine buffer (pH 7.0), and desalted.
This dialysate was added to a 10 mM triethanolamine buffer (p
H7.0) and passed through a column of Q-Sepharose FF (Pharmacia, using 1 ml of resin for 10 mg of sample). The column was washed with 10 mM triethanolamine buffer (pH 7.0), and then washed with 5 mM,
10m containing 10mM, 20mM, 50mM NaCl
The human SOD fraction was collected by developing with an M triethanolamine buffer (pH 7.0). The SOD fraction was saturated with ammonium sulfate, and the resulting precipitate was centrifuged (28,4).
00G for 15 minutes). This precipitate was used as an aqueous solution, and
It was dialyzed against a 5M aqueous solution of NaCl, then dialyzed against water, and freeze-dried to obtain 350 mg of a human SOD derivative.

(9) Reaction of human SOD with dithiobis (propionic acid) Apohuman SOD (0. 0) obtained in the same manner as in (3) above.
5 g) in 10 mM triethanolamine buffer (pH
7.0, 100 ml) and adjusted the pH to 7.0. Dithiobis (propionic acid) (210mg)
Was added to adjust the pH to 7.0, followed by stirring at 4 ° C. for 77 hours. After stirring, NaCl (2.92 g), 2 M acetate buffer (pH 5.0, 5 ml), and 0.1 M CuCl ₂
An aqueous solution (1.5 ml) was added, and the mixture was stirred at 4 ° C for 17 hours. After adjusting the pH to 7.0, saturated with ammonium sulfate, and stirred at about 4 ° C. for 6 hours, the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes). This is 10
It was dissolved in a 10 mM triethanolamine buffer (pH 7.0) and dissolved in a 10 mM triethanolamine buffer (pH 7.0).
0) was desalted by dialysis.

The dialysate was equilibrated with 10 mM triethanolamine buffer (pH 7.0) and Q-Sepharose FF (manufactured by Pharmacia Co., Ltd .;
ml used). The column was washed with a 10 mM triethanolamine buffer (pH 7.0), and then sequentially washed with 5 mM, 10 mM, 20 mM, and 50 mM NaCl.
10 mM triethanolamine buffer solution (pH 7.
The human SOD fraction was collected by developing in step 0). After the SOD fraction was saturated with ammonium sulfate and stirred at about 4 ° C. for 6 hours, the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes). This precipitate was used as an aqueous solution, and 0.5 M NaC
After dialysis against 1 aqueous solution and then against water, lyophilization gave 125 mg of human SOD derivative.

(10) Fractionation of CuCl ₂ -treated human SOD by Q-Sepharose FF 10 mM triethanolamine buffer solution (pH 7.0, 20 ml) was added to the supernatant (54 ml) obtained in the above (2). Make up to 74 ml. To this was added NaCl (2.34 g) and 2M acetate buffer (pH 5.0, 4 ml),
Was adjusted to 5.1, and a 0.1 M CuCl ₂ aqueous solution (1.6
ml), and the mixture was stirred at about 4 ° C for 1 hour. Bis (2-hydroxyethyl) disulfide (0.123
g), adjust the pH to 7.5, stir at about 4 ° C. for 20 hours, adjust the pH to 7.0, add water and add 100 ml
And ammonium sulfate (18.05 g) was added,
Stirred at C overnight. The resulting precipitate is centrifuged (28, 40).
0G, 15 minutes) and the resulting supernatant (110 ml)
Ammonium sulfate (26.28 g) was added to the mixture, and the mixture was stirred at about 4 ° C. for 3 hours.
OG, 15 minutes).

This precipitate was dissolved in 10 mM triethanolamine buffer (pH 7.0) and dialyzed three times against 10 mM triethanolamine buffer (pH 7.0, 2,000 ml). The dialysate was passed through a Q-Sepharose FF (Pharmacia, 20 ml) column equilibrated with 10 mM triethanolamine buffer (pH 7.0).
The column was treated with a 10 mM triethanolamine buffer (p
H7.0, 100 ml), and then washed with 20 mM NaCl.
-10 mM triethanolamine buffer (pH 7.0)
And human SOD fractions were collected. Ammonium sulfate (84.0 g) was added to the obtained SOD fraction (120 ml), the mixture was stirred at about 4 ° C. overnight, and the resulting precipitate was centrifuged (2
(8,400 G, 15 minutes). This precipitate was dissolved in water, dialyzed three times against 2,000 ml of water, and lyophilized to obtain 143 mg of a human SOD derivative.

Example (1) Reaction of human SOD with cystamine Apohuman SOD (106) obtained in the same manner as (3) in Reference Example
mg) in 1 M triethanolamine buffer (pH 7.
(0.1 ml), water (5 ml) and cystamine dihydrochloride (11.3 mg) were added to adjust the pH to 7.0, and water was added to make 10 ml, followed by stirring at 4 ° C. for 24 hours.
After stirring, 1 M acetate buffer (pH 5.0, 2 ml), 0.1 mL
1M CuCl ₂ aqueous solution (0.2 ml) and NaCl
(0.58 g) and then water to make a total volume of 20 m.
After adjusting the pH to 5.0, the mixture was stirred at 4 ° C. for 24 hours. The pH was adjusted to 7.0 and dialyzed against 10 mM triethanolamine buffer (pH 7.0) to desalinate.

(2) Reaction of human SOD with bis (2-acetylaminoethyl) disulfide Apohuman SOD (106) obtained in the same manner as in (3) of Reference Example
mg) in 1 M triethanolamine buffer (pH 7.
0, 0.1 ml), water (5 ml) and bis (acetylaminoethyl) disulfide (11.8 mg).
After adjusting H to 7.0, water (10 ml) was added, and
Stir for 4 hours. After stirring, 1 M acetate buffer (pH 5.
0, ₂ ml), 0.1 M CuCl ₂ aqueous solution (0.2 m
l) and NaCl (0.58 g) were added, followed by water to bring the total volume to 20 ml and after adjusting the pH to 5.0,
Stirred at 4 ° C. for 24 hours. The pH was adjusted to 7.0,
The solution was dialyzed against 10 mM triethanolamine buffer (pH 7.0) and desalted.

(3) Reaction of human SOD with bis (2-hydroxypropyl) disulfide Apohuman SOD (0.1%) obtained in the same manner as (3) of Reference Example
5 g) in 10 mM triethanolamine buffer (pH
7.0, 30 ml), adjust the pH to 7.0,
Bis (2-hydroxypropyl) disulfide (54.
7 mg) and the pH was readjusted to 7.0, followed by stirring at 4 ° C. for 24 hours. NaCl (0.92 g), 2
M acetate buffer (pH 5.0, 1.5 ml) and 0.1
Add an aqueous solution of MCuCl ₂ (0.47 ml) and add
Stir for 4 hours. After stirring, the pH was adjusted to 7.0, saturated with ammonium sulfate, stirred at about 4 ° C. for 6 hours, and the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes). This precipitate was dissolved in a 10 mM triethanolamine buffer (pH 7.0) and dialyzed against a 10 mM triethanolamine buffer (pH 7.0) to desalinate.

(4) Reaction of human SOD with bis (2-hydroxyethyl) disulfide Apohuman SOD (0.1%) obtained in the same manner as in (3) of Reference Example
5 g) in 10 mM triethanolamine buffer (pH
7.0, 30 ml), adjust the pH to 7.0,
Bis (2-hydroxyethyl) disulfide (23 m
g) was added and the pH was readjusted to 7.0, followed by 24 ° C at 4 ° C.
Stirred for hours. This was mixed with NaCl (0.92 g), 2 M acetate buffer (pH 5.0, 1.5 ml) and 0.1 M Cu
An aqueous solution of Cl ₂ (0.47 ml) was added, and the mixture was stirred at 4 ° C. for 13 hours. After stirring, the pH was adjusted to 7.0, saturated with ammonium sulfate, stirred at about 4 ° C. for 6 hours, and the resulting precipitate was collected by centrifugation (28,400 G, 15 minutes).
This precipitate is washed with a 10 mM triethanolamine buffer (pH
7.0) and dialyzed against 10 mM triethanolamine buffer (pH 7.0) to desalinate.

(5) Human S obtained by the above (1) to (4)
Purification of OD fraction The SOD fraction obtained in the above (1) to (4) was Q-Sepharose FF (Pharmacia, 10 m sample) equilibrated with 10 mM triethanolamine buffer (pH 7.0).
(1 ml of resin was used per g). The column was treated with a 10 mM triethanolamine buffer (pH 7.
After washing with 0), 5 mM, 10 mM, 20 mM,
The SOD fraction was collected by developing with a 10 mM triethanolamine buffer (pH 7.0) containing 50 mM NaCl. The SOD fraction was saturated with ammonium sulfate, stirred at about 4 ° C. for 6 hours, and the resulting precipitate was centrifuged (2
(8,400 G, 15 minutes). This precipitate was used as an aqueous solution, dialyzed against a 0.5 M NaCl aqueous solution, then dialyzed against water, and freeze-dried to give a human SOD derivative [(1) 74 mg, (2) 74 mg, (3) 105 m2].
g, (4) was 125 mg].

(6) Reaction of crude human SOD with bis (2-hydroxyethyl) disulfide A 10 mM triethanolamine buffer solution (pH 7.0, 20 ml) was added to the supernatant (54 ml) obtained in (2) of Reference Example. Add 74 ml, add NaCl (2.34 g), bis (2-
After adding (hydroxyethyl) disulfide (0.123 g) to adjust the pH to 7.5, the mixture was stirred at about 4 ° C for 20 hours. To this was added a 2M acetate buffer (pH 5.0, 4 ml) to adjust the pH to 5.1, a 0.1 M CuCl ₂ aqueous solution (1.6 ml) was added, and the mixture was stirred at about 4 ° C. for 1 hour. p
H was adjusted to 7.0, water was added to make the total volume 100 ml, ammonium sulfate (18.05 g) was added,
And stirred overnight. The precipitate formed is centrifuged (28, 40).
0G, 15 minutes) and the resulting supernatant (110 ml)
Ammonium sulfate (26.28 g) was added to the mixture, and the mixture was stirred at about 4 ° C. for 3 hours.
00G, 15 minutes).

This precipitate was dissolved in 10 mM triethanolamine buffer (pH 7.0), and the solution was dissolved in 10 mM triethanolamine buffer (pH 7.0, 2,000 ml).
Was dialyzed three times. The dialysate was passed through a Q-Sepharose FF (Pharmacia, 20 ml) column equilibrated with 10 mM triethanolamine buffer (pH 7.0). The column was washed with 10 mM triethanolamine buffer (pH 7.0, 100 ml), and then washed with 20 mM N
aCl-10 mM triethanolamine buffer (pH
7.0), and the SOD fraction was collected. Ammonium sulfate (84.0 g) was added to the SOD fraction (120 ml), the mixture was stirred at about 4 ° C. overnight, and the resulting precipitate was centrifuged (2
(8,400 G, 15 minutes). The precipitate was dissolved in water, dialyzed three times against 2,000 ml of water, and lyophilized to obtain 160 mg of a human SOD derivative.

[Analysis Examples] (1) Analysis by Anion Exchange Chromatography Anion exchange of the human SOD derivative obtained in (5) to (9) of Reference Example and (1) to (5) of Example The analysis by the chromatographic method was performed by the following A or B method. (Method A) 20 mM Tris-HCl buffer (pH 8.5) 1
For 100 μl of a solution containing about 1 mg of sample per ml,
Analysis was carried out by liquid chromatography under the following conditions.

Detector: UV absorption spectrophotometer (measuring wavelength 280 nm) Column: DEAE 5PW (manufactured by Tosoh) Column temperature: room temperature Flow rate: 0.8 ml / min Mobile phase: (I) solution [20 mM Tris-HCl Buffer (pH 8.5)] (II) solution [solution (I) containing potassium fluoride at a concentration of 0.5 M] According to the gradient program shown in the following Table 1, (I)
Solution and solution (II) are sent.

[0052]

[Table 1]

(Method B) The conditions for transferring the solution (I) and the solution (II) were the same as those in the method A except that the gradient program shown in Table 2 was used.

[0054]

[Table 2]

The results are shown in FIGS. 1 to 5 (Aa).
To (A-i). (2) Peptide mapping analysis 6 M guanidine hydrochloride solution (0.5 M trishydroxyaminomethane-hydrochloric acid buffer, 10 mM EDTA, pH 6.
8) (0.4 ml) was placed in a test tube and replaced with nitrogen.
Samples ((5) to (9) in Reference Example, (1) to (5) in Example)
(4 mg) was added and the mixture was added under a nitrogen atmosphere.
-Vinyl pyridine (2 µl) was added, left at 25 ° C for 1 hour, dialyzed against water, and lyophilized. This lyophilized product (1 mg) was mixed with a 50 mM trishydroxyaminomethane-hydrochloric acid buffer (pH 6.8) (0.19 ml) and a lysyl endopeptidase solution (1 mg / ml) (10 μm).
1), heated at 40 ° C. for 9 hours, then added 50 mM Trishydroxyaminomethane-hydrochloric acid buffer (pH 6.8) (0.89 ml), and heated at 40 ° C. for another 15 hours. . The mixture (80 μl) was analyzed by liquid chromatography under the following conditions.

Detector: UV absorption spectrophotometer (measuring wavelength: 220 nm) Column: TSK-gel (ODS-80TM, manufactured by TOSOH) Column temperature: about 35 ° C. Flow rate: 1.0 ml / min Mobile phase: ( III) Solution [0.1 W / V% trifluoroacetic acid solution] (IV) Solution [0.1 W / V% trifluoroacetic acid mixed solution of acetonitrile and water (4: 1)] According to the gradient program shown in Table 3 below , (III)
The solution and the solution (IV) are sent.

[0057]

[Table 3]

The results are shown in FIGS. 6 to 11 (Ba).
To (Bj). (3) Metal analysis A sample (as described in (5) to (9) of Reference Example and (1) to (5) of Example) (about 20 mg) was ashed with sulfuric acid and nitric acid, and 0.05N The solution dissolved in nitric acid to make 50 ml was used as a copper measurement sample solution. 0.05N nitric acid was added to 25 ml of this solution to make 50 ml, and this was used as a zinc measurement sample solution. Using these sample solutions, the amounts of copper and zinc were analyzed by atomic absorption spectrometry. Table 4 shows the results.

[0059]

[Table 4]

(4) Activity measurement Using samples (described in (5) to (9) of Reference Examples and (1) to (5) of Examples), McCord and Fridvich were used.
[J. Biol. Chem. , 244,6049
(1969)], the human SOD activity was measured. Table 5 shows the results.

[0061]

[Table 5]

(5) Determination of SH group A solution (250 μl) containing water (1 ml) containing a 2-hydroxyethylthio group-modified human SOD derivative (about 5 mg) synthesized in the same manner as in (4) of Example. The dithiothreitol solution (2 mg / ml, 25 mM EDTA, 7.
4 mM sodium phosphate buffer, pH 8.1) (250
μl) and heated at 50 ° C. for 1 hour. After cooling, 10
% Trichloroacetic acid aqueous solution (1 mM EDTA) (500 μ
l) was added and the resulting precipitate was removed by centrifugation. Supernatant (3
0 μl), 1 mM EDTA aqueous solution (120 μl),
2.5 M borate buffer (5 mM EDTA, pH 9.5)
(600 μl) and 7-fluorobenzo-2-oxa-
A solution of ammonium 1,3-diazole-4-sulfonate (2 mg / ml, 2.5 M borate buffer) was added and 60
After heating at 1 ° C. for 1 hour and allowing to cool, the obtained solution was used as a sample solution.

Instead of the human SOD derivative, a 2-mercaptoethanol aqueous solution (1 mg / 10 ml) (250 μm)
The same operation was performed using l) to obtain a standard solution. Human S
The amount of 2-mercaptoethanol in the OD derivative was determined from the ratio of the peak area of the sample solution to the peak area of the standard solution by analyzing the sample solution or the standard solution (20 μl) by liquid chromatography under the following conditions. . As a result, the amount of 2-mercaptoethanol with respect to the human SOD derivative was 0.47% (the theoretical value when two molecules of 2-mercaptoethanol were bound to one molecule of the human SOD derivative was 0.48%). %.).

Detector: Fluorescence detector (excitation wavelength 38
5 nm, detection wavelength 515 nm) Column: TSK-gel (ODS-120T, 2
Column temperature: room temperature Flow rate: 1.0 ml / min Mobile phase (V) solution [0.1 M sodium phosphate buffer (pH 6.
(0) / methanol mixture (95: 5)] (VI) solution [0.1 M sodium phosphate buffer (pH 6.
0) / methanol mixture (60:40)] (V) according to the gradient program shown in Table 6 below.
Solution and (VI) solution are sent.

[0065]

[Table 6]

(6) Stability test Stability of human SOD (obtained in reference example (4)) and human SOD derivative [oxyethylthiolated human SOD obtained in examples (4) and (5)] The properties were examined by storage in an aqueous solution at 30 ° C. for 3 months. Changes in quality due to storage were analyzed by anion exchange chromatography as shown below (C
Method).

(Method C) 100 μl of a solution containing about 1 mg of a sample per 1 ml of 10 mM triethanolamine hydrochloride-NaOH buffer (pH 7.0) was analyzed by liquid chromatography under the following conditions. Detector: UV absorption spectrophotometer (measurement wavelength: 280 nm) Column: MonoQ HR5 / 5 (manufactured by Pharmacia) Column temperature: room temperature Flow rate: 1.0 ml / min Mobile phase: (VII) solution [10 mM triethanolamine Hydrochloric acid-NaO
H buffer solution (pH 7.0)] (VIII) solution [Sodium chloride is 0.5M (VII) solution] According to the gradient program shown in the following Table 7, (VII)
The solution and the solution (VIII) are sent.

[0068]

[Table 7]

As a result, it was found that although a remarkable change was observed in the quality of human SOD, the quality of the human SOD derivative hardly changed. The results are shown in (Ca) to (Cd) of FIGS.

[0070]

The human SOD derivative of the present invention is uniform in charge and molecular weight and has stable SOD activity.
It can be expected as an excellent drug for various diseases caused by superoxide.

[Brief description of the drawings]

FIG. 1 (Aa) shows human S described in (5) of Reference Example.
It is the result of the anion exchange chromatography analysis (method A) of the human SOD derivative obtained by the reaction of OD and 2,2'-dithiobis (benzothiazole). (A-b) is
It is the result of the anion exchange chromatography analysis (method B) of the human SOD derivative obtained by the reaction of human SOD and 6,6′-dithiobis (nicotinic acid) described in (7) of Reference Example.

FIG. 2 (Ac) shows human S described in (6) of Reference Example.
It is the result of the anion exchange chromatography analysis (method B) of the human SOD derivative obtained by the reaction of OD and 5,5'-dithiobis (nitrobenzoic acid). (Ad) shows the results of anion exchange chromatographic analysis (method B) of the SOD derivative obtained by the reaction of human SOD and dithiobis (glycolic acid) described in (8) of Reference Example.

FIG. 3 (Ae) shows human S described in (9) of Reference Example.
Anion exchange chromatographic analysis of the SOD derivative obtained by the reaction of OD with dithiobis (propionic acid) (B
). (Af) shows (1) and
FIG. 9 shows the results of anion exchange chromatographic analysis (method A) of the SOD derivative obtained by the reaction between human SOD and cystamine described in (5).

FIG. 4 (Ag) shows the anion exchange chromatography of the SOD derivative obtained by the reaction of human SOD with bis (2-acetylaminoethyl) disulfide described in Examples (2) and (5). It is a result of a graph method analysis (Method A). (A-
h) is anion exchange chromatographic analysis (method A) of the SOD derivative obtained by the reaction of human SOD with bis (2-hydroxypropyl) disulfide described in Examples (3) and (5). The result.

FIG. 5 (Ai) is an anion exchange chromatograph of a SOD derivative obtained by the reaction of human SOD with bis (2-hydroxyethyl) disulfide described in Examples (4) and (5). It is a result of a method analysis (Method A).

FIG. 6 (Ba) shows human S described in (4) of Reference Example.
It is mapping analysis of OD.

FIG. 7 (Bb) shows human S described in (5) of Reference Example.
It is a result of mapping analysis of the human SOD derivative obtained by the reaction of OD and 2,2'-dithiobis (benzothiazole). (Bc) is the result of mapping analysis of a human SOD derivative obtained by the reaction of human SOD with 6,6′-dithiobis (nicotinic acid) described in (7) of Reference Example.

FIG. 8 (Bd) shows human S described in (6) of Reference Example.
It is a result of mapping analysis of the human SOD derivative obtained by the reaction of OD and 5,5'-dithiobis (nitrobenzoic acid). (Be) is the human S described in (8) of Reference Example.
It is a result of mapping analysis of the human SOD derivative obtained by the reaction of OD and dithiobis (glycolic acid).

FIG. 9 (Bf) shows human S described in (9) of Reference Example.
It is a result of the mapping analysis of the human SOD derivative obtained by reaction of OD and dithiobis (propionic acid). (B
-G) is the human SOD obtained in Examples (1) and (5).
9 shows the results of mapping analysis of a human SOD derivative obtained by the reaction between cystamine and cystamine.

FIG. 10 (Bh) shows a mapping analysis of a human SOD derivative obtained by reacting human SOD obtained in Examples (2) and (5) with bis (2-acetylaminoethyl) disulfide. Is the result of (Bi) is (3) of the embodiment.
And human SOD obtained by the reaction of human SOD obtained in (5) with bis (2-hydroxypropyl) disulfide
It is the result of mapping analysis of a derivative.

FIG. 11 (Bj) shows the results of mapping analysis of the human SOD derivative obtained by the reaction of human SOD obtained in Examples (4) and (5) with bis (2-hydroxyethyl) disulfide. It is.

FIG. 12 shows (Ca) the human SOD obtained in Reference Example (4).
Is a result of analysis by anion exchange column chromatography (method C).

FIG. 13 (Cb) shows the human SOD obtained in Reference Example (4).
Is the result of analyzing by anion exchange column chromatography (method C) after storing at 30 ° C. for 3 months. (Cc) is the result of analyzing the oxyethylthiolated human SOD of Example (4) by anion exchange column chromatography (method C).

FIG. 14 shows (Cd) that the oxyethylthiolated human SOD of (4) in Example was stored at 30 ° C. for 3 months,
It is the result analyzed by the anion exchange column chromatography (method C).

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/02 C07K 1/06-1/08 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. The following formula (I): [In the formula, (human SOD ′) represents a product obtained by removing a hydrogen atom from the mercapto group of the cysteine residue at position 111 of human Cu, Zn-type superoxide dismutase;
Represents an amino group, an amino group substituted with an alkyl group having 1 to 10 carbon atoms or an acyl group having 2 to 10 carbon atoms, or a hydroxyl group; R ¹ and R ² represent a hydrogen atom, or Represents an alkyl group having 1 to 10 carbon atoms. A human Cu, Zn-type superoxide dismutase derivative represented by the formula: 2. A human Cu, Zn superoxide dismutase having the following formula (II): (Wherein X, R ¹ and R ² have the same meanings as defined in claim 1), and are reacted with an alkyl disulfide represented by the formula (1). A method for producing a Cu, Zn superoxide dismutase derivative.