JP4586181B2 - Protein stabilization method, protein stabilizer and protein-containing solution - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a protein stabilization method, a protein stabilizer, and a protein-containing solution, and in particular, a protein stabilization method and a protein stabilizer mainly focusing on means for allowing a peptide having a specific amino acid sequence or a salt thereof to coexist. And a protein-containing solution.

The inventors of the present application have been continuing research on the active ingredients of shijimi. Although the freshwater clam extract contains useful components showing a physiological activity, including various vitamins such as vitamin B _12, what is of particular interest among them is ornithine. Shijimi has long been known to be effective for so-called hangovers or to reduce the symptoms, but ornithine is thought to be involved in its action. The inventors of the present application have already proposed a method for increasing the in vivo content by promoting and promoting the action of ornithine, which is an active ingredient, in vivo (Patent Documents 1 and 2). In addition, in the process of studying the elucidation of the mechanism for increasing the ornithine content, a tripeptide, a novel useful substance having a peptide bond in the order of β-alanine, ornithine and ornithine from the N-terminus, was discovered and A method for producing a tripeptide has also been proposed (Patent Document 3).

  Furthermore, it has been found from the studies by the inventors of the present application that the tripeptide content in the living body of the rainbow is increased or decreased according to the change in the environmental temperature of the rainbow. This suggests that the tripeptide plays an important role in the survival of shijimi, and it is estimated from the molecular structure containing ornithine that the stability of the protein responsible for the enzyme reaction that is particularly important in life. Expected to be involved in the transformation. This is because ornithine contained in the tripeptide is a compound having an amino group similar to arginine and polyamines known as protein stabilizers, as will be described later.

  Proteins are substances that directly take charge of the structure and function of cells, but there are many types, and they exist specifically in the cell nucleus and cytoplasm and perform their functions. Various enzymes, peptide hormones, immune antibodies, etc. are proteins and are important players in life phenomena. In addition, since it is possible to measure or detect the target substance with high accuracy by using a specific reaction between a protein and a substance, clinical diagnosis, food analysis, Environmental analysis is conducted.

  As described above, proteins are still widely used, but on the other hand, proteins are generally weak against heat, often aggregate when stored at room temperature, and a decrease in activity is observed. Therefore, in order to use proteins industrially, it is necessary to maintain the expected function (activity) for a certain period of time, and protein stabilization is an important research subject. As small molecule stabilizers, guanidine hydrochloride, urea, arginine and the like have been used for a long time, but recent knowledge suggests a method in which an amino acid ester and a polyamine coexist (Patent Document 4).

Japanese Patent No. 3573676 “Method for treating swordfish shells and method for producing the swordfish extract” Japanese Patent Application Laid-Open No. 2003-274907 Japanese Patent Application Laid-Open No. 2005-200377 “New Tripeptide and Method for Producing the Same” JP 2004-108850 “Method for Stabilizing Proteins”

  However, there are still no sufficient protein stabilizers including those disclosed in the above patent documents, and there is a demand for the development of more effective stabilizers. Under such circumstances, the present invention suppresses aggregation of a protein bound to an insoluble carrier such as a protein or a polymer, stabilizes the protein, and improves the stability of a solution containing the protein. Accordingly, an object of the present invention is to provide a measurement reagent and a standard solution with high measurement accuracy and excellent long-term stability. That is, the problem to be solved by the present invention is to provide a method, a protein stabilizer, and a protein-containing solution excellent in protein stabilizing effect, excluding the problems of the prior art.

  As a result of studying the above problems, the inventor of the present application uses a tripeptide in which β-alanine, ornithine and ornithine in the order of β-alanine, ornithine, which are useful components in swordfish extract, are used as a protein stabilizer. First, the effectiveness exceeding the conventional stabilizers was found, and based on this, the present invention was achieved. That is, the invention claimed in the present application, or at least the disclosed invention, as means for solving the above-described problems is as follows.

(1) A reagent composition comprising a peptide having a peptide bond in the order of β-alanine and ornithine from the N-terminal, or ornithine and ornithine in the order of the N-terminal, or a salt thereof, and at least one of a protein and a protein-binding carrier. A method for stabilizing a protein, characterized by coexisting with a product.
(2) A protein stabilizer comprising a peptide having a peptide bond or a salt thereof in the order of β-alanine and ornithine from the N terminus, ornithine and ornithine from the N terminus.
(3) A protein stabilizer comprising a dipeptide having a peptide bond in the order of β-alanine and ornithine, or a dipeptide having a peptide bond in the order of ornithine and ornithine or a salt thereof.
(4) Reagent composition containing a peptide having a sequence in which β-alanine and ornithine are sequenced from the N-terminus, or a peptide-bonded sequence in the order of ornithine and ornithine from the N-terminus, or a salt thereof, and at least one of protein and protein-binding carrier A protein-containing solution characterized by coexisting with a product.
(5) A biological component measuring reagent comprising the protein-containing solution according to (4).
(6) A standard solution used for biological component measurement, comprising the protein-containing solution according to (4).

  Since the protein stabilization method, the protein stabilizer and the protein-containing solution of the present invention are configured as described above, according to this, the aggregation of the protein bound to an insoluble carrier such as a protein or a polymer is effective. And an excellent protein stabilization effect can be obtained. And since the stability of the solution containing a protein can be improved, it is possible to provide a measurement reagent and a standard solution having high measurement accuracy and excellent long-term stability.

  For example, a tripeptide or the like, which is an example of a protein stabilizer according to the present invention and is peptide-bonded in the order of β-alanine, ornithine, ornithine from the N-terminus, is described in Patent Document 4 as described in detail in Examples. It has a protein aggregation inhibitory effect and a stabilizing effect that greatly exceed the amino acid esters and polyamines shown. In addition, since the tripeptide is a natural component contained in the swordfish extract, which is a food, it is highly safe and can be used in a wide range of applications including foods and pharmaceuticals as well as analytical reagents.

  Further, other examples of the protein stabilizer according to the present invention, such as dipeptides that are peptide-bonded in the order of β-alanine and ornithine from the N-terminus, have the same or higher protein aggregation inhibitory effect as the above tripeptide, Has a stabilizing effect.

Hereinafter, the present invention will be described in more detail.
First, the peptide or salt thereof used in the protein stabilization method of the present invention is a peptide having a peptide bond sequence in the order of β-alanine and ornithine from the N terminus, ornithine and ornithine in order of N terminus, or a salt thereof. These are allowed to coexist in a reagent composition containing at least one of a protein and a protein-binding carrier, thereby obtaining a protein stabilizing action.

  That is, a peptide containing any of these sequences or a salt thereof can be used as a protein stabilizer. For example, a peptide bond in the order of β-alanine and ornithine, or a peptide bond in the order of ornithine and ornithine. Any of the above-described dipeptides or salts thereof, or tripeptides or salts thereof that are peptide-bonded in the order of β-alanine, ornithine, ornithine can be preferably used.

  However, the protein stabilizer according to the present invention is not limited to these dipeptides, tripeptides or salts thereof, and is not limited to the number of constituent amino acids, but widely includes peptides or salts thereof containing such sequences.

  Examples of the salt of the peptide include hydrochloride, acetate, trifluoroacetate and the like, but are not particularly limited thereto.

  A protein-containing solution obtained by coexisting the above-mentioned peptide that can act as a protein stabilizer or a salt thereof in a reagent composition containing at least one of a protein and a protein-binding carrier is also within the scope of the present invention. Here, the pattern of the substance contained in the reagent composition includes (I) protein, (II) protein binding carrier, (III) protein and protein binding carrier.

  Further, a reagent for measuring a biological component using the protein-containing solution or a standard solution for measuring a biological component is also within the scope of the present invention.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
Example 1. Protein aggregation inhibition effect (tripeptide)
Chicken lysozyme (Sigma) was dissolved in 50 mM phosphate buffer (pH 7.1) to prepare a 1 mg / ml protein solution. To this solution, arginine ethyl ester which showed the highest aggregation inhibitory effect in Patent Document 4 above, and a tripeptide (hereinafter referred to as β-Ala-) having peptide bonds in the order of β-alanine, ornithine and ornithine from the N-terminal according to the present invention. Orn-Orn) was added so that each concentration became 20 mM or 60 mM, and heat treatment was performed at 98 ° C. for 10, 20 and 30 minutes. The protein solution to which the sample was not added was similarly heat-treated and classified as no addition. Centrifugation at 15,000 rpm for 20 minutes after heat treatment (HIMAC CR15T: Hitachi, Ltd.) Using a spectrophotometer (U-3310: Hitachi, Ltd.) for the solution obtained by diluting the resulting supernatant 10 times with distilled water, the wavelength Absorbance at 280 nm was measured to determine the amount of protein remaining in the solution.

  Table 1 shows the change in aggregation over time due to the heat treatment of the protein in Example 1. The change in aggregation over time is shown as the amount (%) of aggregated protein obtained by subtracting the amount of remaining protein (%) from the amount of protein obtained from the absorbance before the start of heating as 100%. As a result, 90% or more of the coagulation modification was observed in the non-added section when heated for 20 minutes or more, but the arginine ethyl ester 20 mM-added section was 79.3% when heated for 30 minutes, and the 60 mM-added section remained at 18.8%. The inhibitory effect was confirmed. On the other hand, in the 20 mM addition section of β-Ala-Orn-Orn, 49.1% was observed after heating for 30 minutes, and almost no coagulation denaturation was observed in the 60 mM addition section. From this, it was shown that β-Ala-Orn-Orn has a significantly higher aggregation inhibitory effect than arginine ethyl ester.

Example 2 Residual activity of protein (tripeptide)
Chicken lysozyme (Sigma) was dissolved in 50 mM phosphate buffer (pH 7.1) to prepare a 1 mg / ml protein solution. To this solution, arginine ethyl ester and β-Ala-Orn-Orn according to the present invention were added so that each concentration was 20 mM or 60 mM, and heat treatment was performed at 98 ° C. for 10 minutes, 20 minutes, and 30 minutes. . The protein solution to which the sample was not added was similarly heat-treated and classified as no addition. Next, 10 ml of protein solution after heat treatment was added to 2 ml of 0.5 mg / ml Micrococcus lysodeikticus (Sigma) solution (100 mM phosphate buffer, pH 6.5), respectively, and a spectrophotometer (U-3310: Hitachi, Ltd.) The lysozyme activity was measured at an absorbance of 600 nm.

  Table 2 shows changes in residual enzyme activity due to the heat treatment of the protein in Example 2. The change in the residual enzyme activity is shown with the enzyme activity of the protein solution before heating as 100%. In the non-addition section, the enzyme activity was almost lost after 10 minutes of heating, and the enzyme activity was almost lost.However, in the 20-ppm addition section of arginine ethyl ester, the 30-minute heating was 13.5% and the 60-mM addition section was 61.8%. Indicated. On the other hand, in the 20-mM addition section of β-Ala-Orn-Orn, heating for 30 minutes was 32.5%, and the enzyme activity more than twice that of the 20-mg arginine ethyl ester addition section remained. Furthermore, in the 60 mM addition section, almost no enzyme inactivation was observed even when heated for 10 minutes, and nearly 80% residual activity was observed even when heated for 30 minutes. That is, β-Ala-Orn-Orn showed a significantly higher enzyme activity after heat treatment of the protein solution than arginine ethyl ester.

  From these results, it was found that the addition of β-Ala-Orn-Orn to the protein solution not only suppresses protein aggregation due to heat denaturation, but also retains the function (activity) of the protein. Further, it has been clarified that the effect of β-Ala-Orn-Orn is further superior to various stabilizers disclosed in Patent Document 4.

Example 3 FIG. Anti-aggregation effect of protein and residual activity of protein (dipeptide)
Furthermore, for comparison, spermine, one of the polyamines, and dipeptides (β-Ala-Orn, Orn-Orn) that form part of the tripeptide (β-Ala-Orn, Orn-Orn) are similarly aggregated and denatured. Rate and residual enzyme activity were examined.

Table 3 shows the change in aggregation over time due to the heat treatment of the protein in Example 3. The change in aggregation over time is shown as the amount (%) of aggregated protein obtained by subtracting the amount of remaining protein (%) from the amount of protein obtained from the absorbance before the start of heating as 100%. Also,
Table 4 shows changes in residual enzyme activity due to the heat treatment of the protein in Example 3. The change in the residual enzyme activity is shown with the enzyme activity of the protein solution before heating as 100%.

  As a result, β-Ala-Orn and Orn-Orn, which are dipeptides that form part of the tripeptide (β-Ala-Orn-Orn), also have the effect of suppressing the aggregation of proteins equivalent to or higher than the tripeptide. It was shown that there is an effect of retaining the function (activity) of the protein.

  It should be noted that the tripeptide and dipeptide constituent amino acids β-Ala (β-alanine)) and Orn (Ornithine) shown in the above examples have strong effects as observed in the present invention in each example. No effect was observed.

According to the protein stabilization method, protein stabilizer and protein-containing solution of the present invention, protein aggregation can be effectively suppressed, and an excellent protein stabilization effect can be obtained. And since the stability of the solution containing protein can be improved, it is possible to provide a measurement reagent and a standard solution with high measurement accuracy and excellent long-term stability. In addition, it can be used in a wide range of applications including foods and pharmaceuticals as well as analytical reagents, and has high utility value.

Claims (6)

Coexist with a peptide or a salt thereof having a peptide-bonded sequence in the order of β-alanine and ornithine from the N-terminal or in the order of ornithine and ornithine from the N-terminal in a protein composition or at least one of protein-binding carriers. A method for stabilizing a protein, characterized by comprising: A protein stabilizer comprising a peptide having a peptide bond or a salt thereof in the order of β-alanine and ornithine from the N-terminus, or ornithine and ornithine from the N-terminus. A protein stabilizer comprising a dipeptide which is peptide-bonded in the order of β-alanine and ornithine, or a dipeptide which is peptide-bonded in the order of ornithine and ornithine or a salt thereof. Coexist with a peptide or a salt thereof having a peptide-bonded sequence in the order of β-alanine and ornithine from the N-terminal or in the order of ornithine and ornithine from the N-terminal in a protein composition or at least one of protein-binding carriers. A protein-containing solution, wherein A reagent for measuring biological components, comprising the protein-containing solution according to claim 4. A standard solution used for biological component measurement, comprising the protein-containing solution according to claim 4.