Protein decomposition catalyst, protein decomposition solution, and protein decomposition method

Abstract

To provide a polyoxometalate with sulfur (S) as a central element, usable as a protein decomposition catalyst.SOLUTION: A protein decomposition catalyst contains a polyoxometalate represented by the following chemical formula (1) or the following chemical formula (2), in which sulfur (S) is included as a central element. [(SZ(Oa)W11O39)b(H2O)m]n1- (1), where Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn, or V, m: an integer of 0 to 40, n1: an integer of 1 to 16, a: an integer of 0 to 2, b: 1 or 2. [(S2Z(Oa)W17O61)b(H2O)m]n2- (2), where Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn, or V, m: an integer of 0 to 40, n2: an integer of 3 to 20, a: an integer of 0 to 2, b: 1 or 2.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a proteolytic catalyst containing polyoxometalate, a solution for proteolysis, and a proteolysis method.

Background Art

[0002] For the decomposition of conventional proteins, organometallic catalysts mimicking the structure of degrading enzymes used in vivo are used. However, such organometallic catalysts have problems such as complex synthesis, lack of mass productivity, instability of the obtained substances, and the need for special consideration for their storage.

[0003] Regarding such organometallic catalysts, for polyoxometalates composed of inorganic metals, Non-Patent Documents 1 and 2 below describe that polyoxometalates with a central atom of phosphorus (P) decompose proteins. Further, Non-Patent Document 3 below describes that as polyoxometalate, (Et2NH2) 10 [Zr(α-PW 11 O 39 )2] is used for the hydrolysis of β-casein.

[0004] Also, Patent Document 1 below proposes a protein remover for contact lenses containing an oxo acid compound and / or a polyacid compound as an active ingredient. As this polyacid compound, isopolyacid [(M m O n ) X- compounds and heteropolyacid [(L l M m O n ) X- compounds are described, and as the central atom of the heteropolyacid, vanadium (V), niobium (Nb), tantalum (Ta), molybdenum (Mo), and tungsten (W) are described. This protein remover for contact lenses is dissolved in water and used, and can remove the protein adhering to the contact lens, and can eliminate the need for washing the contact lens with water after the protein removal treatment. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 3564067 [Non-patent literature]

[0006] [Non-Patent Document 1] Angewandte international Edition 2023,e023038(1-16) [Non-patent document 2] Chemical Science,2021,12,1065-10663 [Non-patent document 3] Chemistry A European Journal 2020,26,11170-11179 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, polyoxometalates with phosphorus (P) as the central element are easier to synthesize, more suitable for mass production, and more stable than conventional organometallic catalysts. Furthermore, they have the ability to decompose proteins, and are used in protein structural analysis.

[0008] Since the physical properties of polyoxometalates, such as acidity, vary significantly depending on the central element, if any of the sulfur (S)-based polyoxometalates described above have the ability to decompose proteins, they may be useful in protein structural analysis, i.e., proteome analysis, which is often employed in drug discovery for peptide degraders, antivirals, antibacterial agents, etc., in combination with polyoxometalates with phosphorus (P) as the central element.

[0009] Accordingly, an object of the present invention is to provide a polyoxometalate having sulfur (S) as a central element that can be used as a proteolytic catalyst, and a proteolytic solution and a proteolytic method using the polyoxometalate.

Means for Solving the Problems

[0010] The proteolytic catalyst made to achieve the above object contains at least one polyoxometalate represented by the following chemical formula (1) or the following chemical formula (2) containing sulfur (S) as a central element. [(SZ(O a )W 11 O 39 ) b (H2O) m n1- ···(1) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn or V m: an integer from 0 to 40 n1: an integer from 1 to 16 a: an integer from 0 to 2 b: 1 or 2 [(S2Z(O a )W 17 O 61 ) b (H2O) m n2- ······(2) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn or V m: an integer from 0 to 40 n2: an integer from 3 to 20 a: an integer from 0 to 2 b: 1 or 2

[0011] The proteolytic catalyst can preferably decompose albumin.

[0012] It is preferable that the proteolytic catalyst is a salt of the polyoxometalate and a cation component because the handling of the proteolytic catalyst is easy.​​

[0013] The salt is preferably insoluble in water, since the proteolytic catalyst can be easily separated from a proteolytic treatment solution obtained by decomposing a protein using the proteolytic catalyst.

[0014] The present invention relates to a proteolytic liquid characterized in that it is a solution, suspension, or dispersion containing a proteolytic catalyst that contains at least one polyoxometalate represented by the above chemical formula (1) or (2) that contains sulfur (S) as a central element.

[0015] The proteolytic catalyst is preferably a salt of the polyoxometalate and a cationic component, which makes the proteolytic catalyst easier to handle.

[0016] The proteolytic catalyst is preferably a water-insoluble salt of the polyoxometalate and a cationic component, which allows the proteolytic catalyst to be easily separated from the proteolytic treatment solution.

[0017] The present invention provides a method for decomposing proteins, characterized by heat-treating a solution, suspension, or dispersion containing a protein to be decomposed and a proteolytic catalyst containing at least one polyoxometalate represented by the above chemical formula (1) or (2) containing sulfur (S) as a central element at a temperature of 20 to 60°C for 1 to 72 hours.

[0018] The use of a salt of the polyoxometalate and a cationic component as the proteolytic catalyst is preferred because it simplifies the handling of the proteolytic catalyst.

[0019] It is preferable to use a water-insoluble salt of the polyoxometalate and a cationic component as the proteolytic catalyst, and to separate the proteolytic catalyst from the aqueous solution after the protein degradation treatment is completed. [Effects of the Invention]

[0020] The polyoxometalate containing sulfur (S) as the central element, which is contained in the proteolytic catalyst according to the present invention, has the ability to degrade proteins. Also, since the physical properties of the polyoxometalate containing sulfur (S) as the central element are different from those of the polyoxometalate containing phosphorus (P) as the central element, it is presumed that the sites of the protein cleaved by the proteolytic catalyst according to the present invention are different from the cleavage sites of the protein by the polyoxometalate containing phosphorus (P) as the central element. From this, the proteolytic catalyst according to the present invention can be used in combination with the polyoxometalate containing phosphorus (P) as the central element during the structural analysis of proteins, i.e., proteome analysis, which is often employed in drug discovery, and can be used in drug discovery such as peptide degrading agents, antiviral agents, antibacterial agents, etc. Also, since it can degrade proteins, it can also be used as a degrading agent for proteins adhering to medical devices and the like.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0022] The polyoxometalate contained in the proteolytic catalyst according to the present invention contains sulfur (S) as the central element, and an example thereof can be represented by the following chemical formula (3).

[0023] [(SZ(O a )W 11 O 39 ) b (H2O) m ] n1- ···(3) Z:Zr,Hf,Ce,Co,Ni,Mn,Fe,Fe(OH),Cr,Zn or V m: an integer between 0 and 40 n1: an integer between 1 and 16 a: an integer between 0 and 2 b:1 or 2

[0024] Of the polyoxometalates represented by the above chemical formula (3), preferred are polyoxometalates represented by chemical formula (4) where a=1 and b=1, polyoxometalates represented by chemical formula (5) where a=2 and b=2, and polyoxometalates represented by chemical formula (6) where a=0 and b=1. [(SZ(O)W 11 O 39 )(H2O) m ] n11- ····(4) Z:Zr,Hf,Ce,Co,Ni,Mn,Fe,Fe(OH),Cr,Zn or V m: an integer between 0 and 40 n11: an integer between 4 and 6 [(SZ(O2)W 11 O 39 )2(H2O) m ] n12- ···(5) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Cr or Zn m: an integer between 0 and 40 n12: an integer between 10 and 16 [SZW 11 O 39 (H2O) m ] n13-········(6) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr or Zn m: an integer between 0 and 40 n13: an integer between 1 and 4

[0025] In the polyoxometalate represented by the above chemical formula (4), [SVW 11 O 40 (H2O) m ] 3- In the case of the polyoxometalate represented by the above chemical formula (5), [(SZ(O2)W 11 O 39 )2(H2O) m ] n12- (Z=Zr, Hf, Ce) is preferable. In addition, in the polyoxometalate represented by the above chemical formula (5), [SZW 11 O 39 (H2O) m ] n13- (Z=Co, Ni, Mn, Fe, Fe(OH), Cr or Zn) is preferred.

[0026] Furthermore, the proteolytic catalyst according to the present invention can also be a polyoxometalate represented by the following chemical formula (7). [(S2Z(O a )W 17 O 61 ) b (H2O) m ] n2- ······(7) Z:Zr,Hf,Ce,Co,Ni,Mn,Fe,Fe(OH),Cr,Zn or V m: an integer between 0 and 40 n2: an integer between 3 and 20 a: an integer between 0 and 2 b:1 or 2

[0027] In the polyoxometallate represented by the above chemical formula (7), a polyoxometallate represented by the chemical formula (8) in which a=2, b=2, or a polyoxometallate represented by the chemical formula (9) in which a=0, b=2 is preferred. [(S2Z(O2)W 17 O 61 )2(H2O) m n21- ······(8) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr or Zn m: An integer from 0 to 40 n21: An integer from 14 to 20 [S2ZW 17 O 61 (H2O) m n22- ······(9) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr or Zn m: An integer from 0 to 40 n22: An integer from 3 to 6

[0028] Among the polyoxometalates represented by the above chemical formula, those represented by the following chemical formula (10) can be particularly preferably used. [(SZ(O2)W 11 O 39 )2(H2O) m1 n3- ·······(10) Z: Zr, Hf, Ce m1: 0 to 20 n3: 12 or 14

[0029] The crystal structure of the polyoxometalate shown in the above chemical formula (10) is shown in FIG. 1. FIG. 1 shows the results of structural analysis of a single crystal of the polyoxometalate using X-ray structural analysis and ESI-MS, etc. As is clear from FIG. 1, metal atoms (Z, Z) arranged in each of a pair surrounding sulfur (S) as the central element with tungsten (W) are bonded via a pair of oxygen atoms (O). This Z is Zr, Hf, Ce.

[0030] The synthesis method of the polyoxometalates of the above chemical formulas (4), (5), (6), and (10) used in the present invention can be obtained in three steps as shown below. (1) The first step ​​​Dissolve an alkali tungstate such as sodium tungstate (Na2WO4·2H2O) and NH4VO3 in hot water, add acetonitrile, then dropwise add concentrated sulfuric acid (H2SO4) while stirring, and then heat at 70 - 80 °C for 12 - 24 hours. After cooling the heat-treated solution to room temperature, add (n-C4H9)4NBr, filter the resulting precipitate, and wash it. Dissolve this precipitate in acetonitrile, add (n-C4H9)4NBr again, and then heat to 70 - 80 °C. Add concentrated hydrochloric acid, stir while heating for a predetermined time, collect the resulting white precipitate by filtration, wash it with acetonitrile, and dry it. Recrystallize the obtained precipitate with acetone. (2) Second step Dissolve the precipitate obtained in the first step in acetone, add a carbonate of an alkali metal such as potassium hydrogen carbonate (KHCO3), filter the resulting precipitate, add an aqueous solution of (n-C4H9)4NB to the filtrate, filter the obtained precipitate, wash it, dry it, and then recrystallize it with acetone. (3) Third step Dissolve the recrystallized product obtained in the second step in acetone, add a stock solution of a desired metal ion selected from Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn, or V, and then preferably heat-treat at room temperature to 50 °C for 0.2 - 1 hour. Further, add (n-C4H9)4NBr to the filtrate obtained by filtering the solution cooled to room temperature of this heat-treated solution, and the resulting precipitate is the target product.

[0031] The synthesis method of the polyoxometalates of the above chemical formulas (7), (8), and (9) used in the present invention can be obtained in three steps as shown below. (1) First step Acetonitrile (CH3CN) is added to an aqueous solution of an alkali salt of tungstate, such as sodium tungstate (Na2WO4·2H2O), and concentrated sulfuric acid (H2SO4) is added dropwise while stirring. After cooling to room temperature, acetonitrile is added again, and the solution is left to stand, separating into two layers. The upper layer is then separated by heating and concentrating it to about half its original volume. A saturated solution of an alkali metal salt, such as potassium chloride (KCl), is then added, and the resulting precipitate (A4[S2W 18 O 62 ] (A: alkali metal)) is isolated and washed. (2) Second step The precipitate obtained in the first step (A4[S2W 18 O 62 ] (A: alkali metal)) in water, and then add an alkali metal carbonate such as potassium bicarbonate (KHCO3) to form a precipitate (A 32 [S2W 14 O 54 ] (A: alkali metal) is isolated and washed. The crystal structure of this precipitate has a defect in part of its framework. (3) Third step The precipitate obtained in the second step (A 32 [S2W 14 O 54 A stock solution of a desired metal ion selected from Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn, or V is added to a solution of Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn, or V, and then the mixture is heated in a pressure vessel at 100°C or higher, preferably at 120 to 130°C, for 12 to 36 hours to form a precipitate (A 32 [S2W 14 O 54 The metal in the stock solution can be coordinated to the defects in the framework of the crystal structure of (A: alkali metal). Furthermore, the heat-treated solution is cooled to room temperature, and the filtrate is filtered. Adding (n-C4H9)4NBr to the filtrate produces a precipitate, which is the target compound.

[0032] The polyoxometalate obtained in this manner is preferably handled as a salt with a cationic component. In particular, by converting it into a water-insoluble salt, the salt of the polyoxometalate and the cationic component can be easily separated from the aqueous solution obtained by the protein degradation treatment. As the salt, tetrabutylammonium [(n-C4H9)4N] + n (n=1 to 6) salts, particularly those where n=3 or 4, are preferred.

[0033] To use the polyoxometalate of formula (1) or (2) as a proteolysis catalyst, it is preferable to use a proteolysis liquid in the form of a solution, suspension, or dispersion containing a proteolysis catalyst containing at least one polyoxometalate represented by formula (3) or (7). The proteolysis treatment using this proteolysis liquid varies depending on the protein to be degraded, but can usually be carried out by heating a solution, suspension, or dispersion containing the protein to be degraded and the proteolysis liquid at a temperature of 20 to 60°C, preferably 50 to 60°C, for 1 to 72 hours, preferably 36 to 48 hours. In particular, it is preferable to use an aqueous solution, aqueous suspension, or aqueous dispersion containing a proteolytic catalyst as the proteolytic liquid, since the proteolytic catalyst is a water-insoluble salt of the polyoxometalate and a cationic component, and the proteolytic catalyst can be separated from the aqueous proteolytic treatment solution after the completion of the decomposition treatment of the protein to be decomposed.

[0034] When albumin is used as the degradation target, degradation can be performed by adding 2 mg of albumin to 1 ml of 0.1 M phosphate buffer solution (pH 7.4) and diluting the solution 50 times with distilled water to prepare an albumin solution. 2 mg of a water-insoluble salt of the polyoxometalate and a cationic component is added to 1 ml of this albumin solution, and the mixture is heated at the heating temperature described above for a predetermined time. The reactivity of albumin with the polyoxometalate in this degradation solution can be checked by electrophoresis.

[0035] The polyoxometalate (Z:Hf) shown in Figure 1 is tetrabutylammonium [(n-C4H9)4N] + The electrophoresis of albumin decomposed using salt under the above-mentioned conditions is shown in Figure 4. As is clear from Figure 4, after 3 to 4 days of heat treatment at 60°C, the band around 63 kDa, which corresponds to the molecular weight of albumin (approximately 66,000), almost disappears, but the band around 48 kDa increases, suggesting that certain sites in albumin have been selectively hydrolyzed.

[0036] The aqueous solution for protein degradation contains the water-insoluble polyoxometalate tetrabutylammonium [(n-C4H9)4N] + This insoluble component precipitates in the hydrolysis solution and can be easily separated from the supernatant, which contains the hydrolysis components of proteins. [Example]

[0037] Examples of the present invention will be described in detail below, but the scope of the present invention is not limited to these examples.

[0038] Example 1 Polyoxometalate tetrabutylammonium [(n-C4H9)4N] + The salt is (n-C4H9)8H4[(SHf(O2)W 11 O 39 )2] was synthesized in the following steps (1) to (3). (1)[(n-C4H9)4N]2[SW 12 O 40 Synthesis of Dissolve Na2WO4·2H2O (8.3 g) and NH4VO3 (1.2 g) in hot water (200 ml), and add CH3CN. While vigorously stirring the solution, add 25 ml of H2SO4 solution (10 M) drop by drop (add the next drop after the precipitate disappears). Heat the finally obtained orange solution at 70 °C for 24 hours. After cooling to room temperature, add 5 g of (n-C4H9)4NBr, and a precipitate appears. Collect it by filtration, wash it with ethanol, and dry it. This precipitate is [(n-C4H9)4N]4[SV2W 10 O 40 . Further, dissolve 12 g of this precipitate in CH3CN (300 ml), add 10 g of (n-C4H9)4NBr again, and then heat the solution to 70 °C while stirring. Add concentrated hydrochloric acid (15 ml), stir for about 1 hour while heating, collect the obtained white precipitate by filtration, wash it with acetonitrile, and dry it. Recrystallize the obtained precipitate with acetone. The obtained crystals are [(n-C4H9)4N]2[SW 12 O 40 . Incidentally, the precipitate [(n-C4H9)4N]4[SV2W 10 O 40 is presumed to be a product in which a part of [(n-C4H9)4N]4[SV2W 10 O 40 is decomposed during the heat treatment with hydrochloric acid, and V and W are replaced to be converted into [(n-C4H9)4N]2[SW 12 O 40 .

[0039] (2) Synthesis of [(n-C4H9)4N]4H2[SW 11 O 39 (1) The (n-C4H9)4N]2[SW 12 O 401.35 g of [(n-C4H9)4NB] is dissolved in 90 ml of acetone. While vigorously stirring the acetone solution, 0.9 ml of 1 M KHCO3 solution is added, and the resulting white precipitate is separated. 40 ml of an aqueous solution containing 1.5 g of (n-C4H9)4NB is added to the filtrate, yielding a pale yellow precipitate. (If the precipitate is oily, discard the aqueous solution, add diethyl ether, and wash in an ultrasonic cleaner to obtain a powdery precipitate.) This precipitate is collected by filtration, washed with water and ethanol, and dried. The precipitate is recrystallized from dry acetone. The resulting crystals are (n-C4H9)4NB]4H2[SW 11 O 39 ]. (n-C4H9)4N]2[SW 12 O 40 ] becomes weakly alkaline in the solution, the equilibrium shifts, and (n-C4H9)4N]4H2[SW 11 O 39 ] is presumed to be the main component.

[0040] (3)(n-C4H9)8H4[(SHf(O2)W 11 O 39 )2] Synthesis (2) synthesized [(n-C4H9)4N]4H2[SW 11 O 39 1.033 g of [N-CH2SO4] was dissolved in 97 mL of acetone, and 2.5 mL of a 0.2 M Hf(IV) stock solution (8.190 g of HfOCl2·8H2O dissolved in 100 mL of water) was added and stirred for 10 minutes at room temperature. The solution was heated at 70 °C for 24 hours, filtered to remove impurities, and then 1.5 g of (n-CH9)4NBr was added. 40 mL of water was added, and the resulting precipitate was collected by filtration and washed with water and ethanol. The precipitate was recrystallized by dissolving it in acetonitrile at 70 °C and leaving the solution at room temperature.

[0041] The X-ray diffraction data for the obtained crystals were analyzed using the direct method of the precision structure analysis program SHELXL-2018, and electrospray ionization mass spectrometry (ESI-MS) analysis revealed that (n-C4H9)8H4[(SHf(O2)W 11 O 39)2] was identified. The ESI-MS chart is shown in Fig. 2(a). The enlarged data of the maximum peak in Fig. 2(a) is shown as "Observed" in Fig. 2(b). This data matches the "Simulated" data of [(SHfW 11 O 39 )H2O] 2- shown in Fig. 2(b). Also, the enlarged data of the second largest peak in Fig. 2(a) is shown as "Observed" in Fig. 2(c). This data matches the "Simulated" data of [(SHfW 11 O 39 )HO] 3- shown in Fig. 2(c). Also, this crystal was insoluble in water. Its water-insolubility was visually confirmed, and the precipitate that had settled in water was confirmed by IR analysis.

[0042] Example 2 Synthesis of tetrabutylammonium [(n-C4H9)4N] + salt (n-C4H9)8H4[(SZr(O2)W 11 O 39 )2 1.033 g of [(n-C4H9)4N]4H2SW 11 O 39 synthesized in (2) of Example 1 was dissolved in 97 ml of acetone, 2.5 ml of a 0.2 M Zr(IV) stock solution (a solution prepared by dissolving 6.445 g of ZrOCl2·8H2O in 100 ml of water) was added, and the mixture was stirred at room temperature for 10 minutes. The solution was heated at 70 °C for 24 hours, impurities were removed by filtration, 1.5 g of (n-C4H9)4NB was added, 40 ml of water was added, and the resulting precipitate was collected by filtration. The precipitate was washed with water and ethanol. The precipitate was dissolved in acetonitrile at 70 °C, and the solution was left at room temperature for recrystallization. The obtained crystals, similar to those in Example 1, were analyzed by the direct method of the SHELXL-2018 precision structure analysis program for X-ray diffraction data and subjected to electrospray mass spectrometry (ESI-MS analysis). As a result, (n-C4H9)8H4[(SZr(O2)W 11 O 39)2]. The results of ESI-MS analysis are shown in Figure 3(a). The data obtained by enlarging the maximum peak in Figure 3(a) is shown in "Observed" in Figure 3(b). This data corresponds to the [(SZrW 11 O 39 )H2O] 2- The data agrees with the "Simulated" data in Fig. 3(a). The data obtained by enlarging the second largest peak in Fig. 3(a) is shown in "Observed" in Fig. 3(c). This data is consistent with the [(SZrW 11 O 39 )HO] 3- This matches the "Simulated" data. The crystals were insoluble in water, which was confirmed by visual inspection and by IR analysis of the precipitates in the water.

[0043] Example 3 Polyoxometalate tetrabutylammonium [(n-C4H9)4N] + The salt is (n-C4H9)8H4[SZW 11 O 39 ] (Z = Mn, Co, Ni, Cu, Zn, Fe) synthesis (n-C4H9)4H2SW synthesized in Example 1(2) 11 O 39 Dissolve 0.948 g of in 120 ml of acetonitrile to prepare a 0.2 M stock solution of metal ions (Mn 2+ : 5.74g Mn(NO3)2·6H2O, Co 2+ : 5.82 g Co(NO3)2·6H2O, Fe 3+ : 8.08g Fe(NO3)3·9H2O, Ni 2+ : 5.82g Ni(NO3)2·6H2O, Cu 2+ : 4.83g Cu(NO3)2·3H2O, Zn 2+: Add 3.0 ml of a solution prepared by dissolving 5.95 g of each of Zn(NO3)2·6H2O in 100 ml of water. After stirring the solution at room temperature for 10 minutes, add 70 ml of an aqueous solution in which 1.0 g of n-Bu4NBr is dissolved, and a precipitate will form. Collect the precipitate by filtration, wash it with water and ethanol, and dry it. Recrystallize the precipitate from acetone. The obtained crystals, similar to those in Example 1, are analyzed by the direct method of the X-ray diffraction data using the SHELXL-2018 of the precise structure analysis program and are subjected to electrospray mass spectrometry (ESI-MS analysis). As a result, ((n-C4H9)4[SZW 11 O 39 (Z = Mn, Co, Ni, Cu, Zn, Fe). These crystals were insoluble in water. The insolubility in water was confirmed visually and by IR analysis of the precipitate precipitated in water.

[0044] Example 4 (n-C4H9)8H4[(SHf(O)W 11 O 39 )2 obtained in Example 1 was used as a proteolytic catalyst to perform degradation treatment on albumin under the following conditions. The albumin solution was prepared by measuring 5 ml of a commercially available 0.1 M phosphate buffer solution (pH 7.4) into a 50 ml volumetric flask, adding 10 mg of albumin derived from human serum to the solution, and diluting it with distilled water. Measure 0.5 ml of the albumin solution into a sample tube, add 1 mg of (n-C4H9)8H4[(SHf(O)W 11 O 39 )2 obtained in Example 1, and heat it at a constant temperature while vibrating and stirring with a thermo block (BIOSAN LTD., TS-100C). Alternatively, a mini rotator (AS ONE Corporation, ACR-100) was placed in an incubator (AS ONE Corporation, E1-300V) to heat the sample while rotating it at a constant temperature. Incidentally, the degradation treatment solution was separated into two layers: the supernatant of the solution of the degradation components of albumin and the solution of the proteolytic catalyst in the lower layer.

[0045] Example 5 The supernatant of the albumin decomposition treatment solution under the conditions of Example 4 was subjected to electrophoresis under the following conditions. A 40-μl sample for electrophoresis was prepared by mixing 30 μl of the supernatant from the treatment solution with 10 μl of a solution containing mercaptoethanol and SDS sampling buffer. The prepared solution was heated at 98°C for 3 minutes in a thermoblock incubator. 15 μl of the heated electrophoresis sample was poured into the gel, and electrophoresis was performed for approximately 1 hour at a voltage of 248 V and a current of 0.03–0.08 A (power of 7–20 W). After electrophoresis, the gel was removed and washed three times with purified water for 5 minutes on a shaking table. After washing, the gel was transferred to a separate container and immersed in CBB staining solution for 30 minutes. After staining, the gel was removed and washed three times with purified water for 5 minutes on a shaking table. The gel was then repeatedly washed for 10 minutes until the blue color of the CBB staining solution had completely disappeared from the entire gel. Finally, after confirming that the blue color had completely disappeared from the areas without sample, the gel was photographed. The results of the electrophoresis are shown in Figure 4. As is clear from Figure 4, after 3 to 4 days of heat treatment at 60°C, the band around 63 kDa, which corresponds to the molecular weight of albumin (approximately 66,000), almost completely disappears, but the band around 48 kDa increases, suggesting that certain sites in albumin have been selectively hydrolyzed.

[0046] Example 6 (n-C4H9)8H4[(SHf(O)W 11 O 39 )2] (referred to as SHfW) and (n-C4H9)8H4[(SZr(O2)W 11 O 39 )2] (referred to as SZrW) was used as a proteolytic catalyst to decompose bovine hemoglobin under the following conditions. The bovine hemoglobin solution was prepared by placing 5 ml of commercially available 0.1 M phosphate buffer solution (pH 7.4) in a 50 ml measuring flask, adding 10 mg or 20 mg of bovine hemoglobin derived from bovine red blood cells to the solution, and diluting with distilled water. 0.5 ml of the hemoglobin solution was placed in a sample tube, and the (n-C4H9)8H4[(SHf(O)W11 O 39 )2] was added in an amount of 1 mg, and the mixture was heated at a constant temperature while being vibrated and stirred using a thermo-block (BIOSAN LTD., TS-100C). Alternatively, a mini-rotator ((stock) AS ONE Corporation, ACR-100) was placed in an incubator ((stock) AS ONE Corporation, E1-300V), and the sample was heated at a constant temperature while being rotated. In addition, the decomposition treatment solution was separated into two layers: the supernatant of the solution of the decomposition components of hemoglobin and the solution of the proteolytic catalyst in the lower layer.

[0047] Example 7 Electrophoresis was performed under the following conditions on the supernatant of the treatment solution obtained by decomposing hemoglobin under the conditions of Example 6. 30 μl of the supernatant of the treatment solution was mixed with 10 μl of a solution obtained by mixing mercaptoethanol and SDS sampling buffer to prepare 40 μl of a sample for electrophoresis. The adjusted solution was heated at 98°C for 3 minutes in a thermo-block constant temperature bath. 15 μl of the electrophoretic sample after heating was injected into the gel, and the voltage was set to 248 V and the current to 0.03 - 0.08 A (power 7 - 20 W), and electrophoresis was performed for about 1 hour. After electrophoresis, the gel was taken out and washed 3 times on a shaker with purified water for 5 minutes each. After washing, it was transferred to another container, immersed in CBB staining solution, and stained for 30 minutes. After staining, the gel was taken out and washed 3 times on a shaker with purified water for 5 minutes each. Then, washing was repeated for 10 minutes until the blue color of the CBB staining solution disappeared from the entire gel. Finally, it was confirmed that the blue color completely disappeared from the part where no sample was added, and the gel was photographed. The results of electrophoresis are shown in Fig. 5. "Lane No. 1 - 13" shown in Fig. 5(a) is the electrophoresis of the sample decomposed at a heating temperature of 40°C in Table 1 below, and "Lane No. 1 - 13" shown in Fig. 5(b) is the electrophoresis of the sample decomposed at a heating temperature of 60°C in Table 1 below.

[0048]

Table 1

[0049] As is clear from Fig. 5, when SHfW and SZrW are used as the decomposition catalyst, the band of the monomer of hemoglobin around 17 KDa decreases and the band around 11 KDa increases. Therefore, it is presumed that a predetermined site of hemoglobin has been selectively hydrolyzed. In Fig. 5, a line was drawn on the lane where the band around 11 KDa increased significantly.

Industrial Applicability

[0050] The proteolytic catalyst according to the present invention can be used for proteome analysis, peptide decomposing agents, antiviral agents, antibacterial agents, and decomposing agents for proteins attached to medical devices and the like.

Claims

1. A protein-degrading catalyst, characterized by containing at least one polyoxometalate represented by the following chemical formula (1) or the following chemical formula (2) containing sulfur (S) as a central element. [(SZ(O ａ )W 11 O ３９ ) ｂ (H ２ O) ｍ ｎ１－ ...(1)​ Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn or V m: An integer from 0 to 40 n1: An integer from 1 to 16 a: An integer from 0 to 2 b: 1 or 2 [(S ２ Z(O ａ )W １７ O ６１ ) ｂ (H ２ O) ｍ ｎ２－ ......(2) Z: Zr, Hf, Ce, Co, Ni, Mn, Fe, Fe(OH), Cr, Zn or V m: An integer from 0 to 40 n2: An integer from 3 to 20 a: An integer from 0 to 2 b: 1 or 2

2. The protein-degrading catalyst according to claim 1, characterized in that the protein is albumin.

3. The protein-degrading catalyst according to claim 1, characterized in that the protein-degrading catalyst is a salt of the polyoxometalate and a cation component.

4. The protein-degrading catalyst according to claim 3, characterized in that the salt is insoluble in water.

5. A solution, suspension or dispersion containing the protein-degrading catalyst according to claim 1, which is a liquid for protein degradation.

6. The liquid for protein degradation according to claim 5, characterized in that the protein-degrading catalyst is a salt of the polyoxometalate and a cation component.

7. The liquid for protein degradation according to claim 5, characterized in that the protein-degrading catalyst is a water-insoluble salt of the polyoxometalate and a cation component.

8. A protein degradation method, characterized by heat-treating a solution, suspension or dispersion containing a protein to be degraded and the protein-degrading catalyst according to claim 1 at a temperature of 20 to 60°C for 1 to 72 hours.

9. The protein degradation method according to claim 8, characterized in that a salt of the polyoxometalate and a cation component is used as the protein-degrading catalyst.

10. The protein degradation method according to claim 8, characterized in that a water-insoluble salt of the polyoxometalate and a cation component is used as the protein-degrading catalyst, and after the protein degradation treatment is completed, the protein-degrading catalyst is separated from the protein degradation treatment aqueous solution.

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