JP4690027B2 - Method for measuring protease activity and reagent therefor - Google Patents

Description

  The present invention relates to a method for measuring protease activity in a test sample and a reagent therefor.

  Various methods for measuring protease activity are known. For example, a method using acid-modified hemoglobin as a substrate, a method for measuring an acid-soluble peptide generated by hydrolysis of casein, a synthetic peptide N-acetyl-L- Quantitative determination of released diiodo-L-tyrosine using phenylalanyl-3,5-diiodo-L-tyrosine (APDT) by the ninhydrin method, etc., high-performance liquid chromatography of peptides generated by the action of enzymes on synthetic peptides 4 Method of using 4 methyl coumalulu 7 amide (MCA) synthetic peptide substrate, paranitrophenyl ester synthetic peptide substrate, paranitroanilide synthetic peptide substrate, quantification of ammonia or carboxylic acid generated by hydrolysis How to combine, dyes such as Congo Red, Azo, Orcein A method using a substrate, a method using a radioactive, a method using a peptide thioester, gelatin film method and the like are known.

Japanese Patent Laid-Open No. 5-192192 JP 2002-159297 A Specialized WO97 / 008194

  However, the various measurement methods described above are complicated or require an apparatus for measurement. For this reason, for example, when trying to measure protease activity in the environment, it is necessary to bring a measuring device to the measurement site or to perform complicated processing on the site, which is not convenient.

  Accordingly, an object of the present invention is to provide a method for measuring protease activity and a reagent therefor that can easily measure protease activity without requiring a special apparatus.

  As a result of diligent research, the inventors of the present application, when a protease acts on a colored compound in which an amino acid or an oligopeptide is amide-bonded to at least one of the amino groups of a colored dye having at least one amino group, The present invention was completed by finding that the amide bond was cleaved and discolored.

That is, the present invention uses, as a substrate for a protease in a test sample, a colored compound in which an amino acid or an oligopeptide is amide-bonded to at least one amino group among the amino groups of a colored dye having at least one amino group. Provided is a method for measuring protease activity in the test sample, wherein an enzyme reaction is carried out and the protease activity is measured based on the discoloration of the colored compound. The present invention also provides a colored compound in which an amino acid or an oligopeptide is amide-bonded to at least one amino group among the amino groups of a colored dye having at least one amino group , wherein the colored dye is cresyl violet. A reagent for measuring protease activity, which is Safranin O or Methylene Violet 3RAX, is provided.

  According to the present invention, a novel method for measuring protease activity and a reagent therefor have been provided for the first time, by which it is possible to easily measure protease activity by observing discoloration with the naked eye without requiring a special device. . In the method of the present invention, protease activity can be measured by the naked eye and based on the color change. Discoloration is advantageous because it can be observed more easily than fluorescence that requires excitation light, and it is easy to discriminate even subtle changes compared to coloring (colorless coloring).

  As described above, in the method of the present invention, a colored compound in which an amino acid or an oligopeptide is amide-bonded to at least one amino group among the amino groups of a colored dye having at least one amino group is used as a substrate. It has been found that when a protease acts on the colored compound, the amide bond is cleaved and discolored. Here, “discoloration” means that the color can be seen with the naked eye both before and after the enzyme reaction, and the color changes with the naked eye. Preferred examples of coloring pigments include cresyl violet, safranin O and methylene violet 3RAX, Nile Blue A, Darrow Red, Azure A, Azure C, Brilliant Cresyl Blue, Rhodamine 123, and Thionine having an amino group in the conjugate system. Examples thereof include, but particularly preferably, cresyl violet, safranin O, and methylene violet 3RAX having the following chemical structures, but are not limited thereto.

  When the colored pigment has a plurality of amino groups like cresyl violet, at least one of them may be amidated. The amino acid to be subjected to amidation may be one amino acid molecule or an oligopeptide (preferably about 2 to 10 amino acids). The type of amino acid or oligopeptide is appropriately selected according to the type of protease. For example, in order to measure the protease activity of Dermatophagoides farinae extract or Dermatophagoides pteronyssinus extract, which is a mite antigen, a leucine, methionine or lysine molecule having an amide bond is preferred, and in particular, one or two of cresyl violet Those having a single amino group and an amide bond with leucine are preferred.

  The amide bond between a coloring dye and an amino acid is, for example, a reaction between a coloring dye molecule and an amino acid whose amino group is protected with Boc in methylene chloride or DMF at room temperature for 1 day using carbonyldiimidazole as a condensing agent. And can be achieved by deprotection with trifluoroacetic acid, and detailed methods are described in the examples below. Oligopeptides can also be amide-bonded by adding a dye appropriately protected with a protecting group to the same method or existing peptide synthesis methods such as the F-moc method.

The substrate can be used in the form of a solution, or can be used by supporting the substrate on a support. In the case of a solution, water and an aqueous buffer can be preferably used as the solvent. As the support, a porous support capable of increasing the amount of the substrate to be supported can be preferably used. Examples of the porous support include gels such as agarose gel, polyacrylamide gel, and silica gel, and filter paper made of glass fibers. And a porous film made of a synthetic resin. In the case of using a substrate solution, the substrate concentration in the solution is appropriately set according to the type of substrate used, the range of predicted protease activity, etc., but is usually about 10 μM to 50 mM, preferably about 100 μM to 10 mM. Degree. Also, when the substrate is supported on the support, it is appropriately set according to the type of the substrate to be used, the predicted protease activity range, etc., but when applied to the support surface, it is usually 1 per 1 cm ^2. In the case of impregnating the support in the range of nmol to 100 nmol, it is usually about 0.1 μmol-100 μmol.

  Measurement can be performed simply by bringing the substrate into contact with a test sample. If it does so, protease activity can be measured based on discoloration of a coloring compound. “Measurement” includes detection, quantification and semi-quantification. Semi-quantification is possible by observing the degree of color change, and if necessary, it can also be quantified by measuring absorbance. The reaction temperature is not particularly limited and is not limited as long as the protease to be measured is a temperature at which the protease activity can be exerted. Usually, it is convenient and preferable to measure at room temperature. Also, the measurement time is not particularly limited and can be appropriately set, but is usually about 1 minute to 24 hours.

  The sample used in the method of the present invention is not limited in any way, and examples thereof include samples containing proteases of environmental allergens (such as mites and pollen), clinical specimens such as foods and various body fluids, and the like. However, it is not limited to these.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

  A substrate that causes a color change by enzymatic cleavage was synthesized. Leucine or methionine was bonded to the amino group of cresyl violet, safranin O, and methylene violet 3RAX by an amide bond by the following method. That is, a dye molecule (final concentration 0.1 M) and an amino acid whose amino group is protected with Boc (final concentration 0.1 M), dimethylaminopyridine (final concentration 0.2 M) as a catalyst, and carbonyldiimidazole (final concentration 0). .1M) as a condensing agent and reacted in methylene chloride or DMF at room temperature for 1 day to form amide bonds. After the reaction, deprotection was performed using trifluoroacetic acid (50%) / methylene chloride (50%).

  Cresyl violet has two amino groups. From the above synthesis, both amino groups contribute to amide bonds with amino acids (yellow, maximum absorption wavelength: 440 nm) or only one of them. The one contributing to the amide bond (orange, 490 nm) was isolated. Each absorption wavelength was measured using a plate reader (SPECTRA Max).

  Furthermore, aminopeptidase M was added to the amide bond compound of leucine and cresyl violet, and an experiment was conducted to cleave the amide bond. That is, 50 microliters of an aminopeptidase M, which is an enzyme that cleaves an amino acid bonded to the N-terminal by an amide bond, appropriately diluted with PBS, 5 microliters of a substrate dissolved in ethanol at about 10 mM, and 30 The sample was allowed to stand and its absorption spectrum was measured using a 96-well plate. As a result, the substrate in which only one amino group contributed to the amide bond changed its absorption spectrum from orange (maximum absorption wavelength: 490 nm) to purple (maximum absorption wavelength: 590 nm) by enzymatic digestion. The compound in which the group contributed to the amide bond was shown to exhibit purple (maximum absorption wavelength: 590 nm) from yellow (maximum absorption wavelength: 450 nm) to green and blue. As a result of mass spectrometry, it was confirmed that the substance showing blue color was obtained by binding one leucine to cresyl violet. Moreover, the purple absorption spectrum finally exhibited by enzyme digestion completely coincided with the absorption spectrum of cresyl violet itself, and it was confirmed from the results of thin phase chromatography that it was cresyl violet itself. When one leucine is bound to cresyl violet, the two colors of blue or orange are shown because the molecular structure of cresyl violet is not symmetrical and the absorption spectrum differs depending on which amino group is involved. is there. The respective absorption spectra are shown in FIG. It was confirmed that the same color tone change occurred when methionine or lysine was used in addition to leucine as an amino acid.

  When methylene violet 3RAX has one amino group and contributes to an amide bond with an amino acid, it exhibits a light purple color (maximum absorption wavelengths: 550 nm and 590 nm). When digested with aminopeptidase M, A bright pink color (maximum absorption wavelength: 550 nm) was exhibited. The extinction spectrum after digestion completely matches the extinction spectrum of methylene violet 3RAX. FIG. 2 shows how the amide conjugate of methylene violet 3RAX and leucine undergoes a spectrum change by aminopeptidase M. In FIG. 2 and FIG. 3 described later, “Apase” means aminopeptidase M.

  The amide conjugate of safranin-O and leucine had two amino groups, and the conjugate of leucine and these showed a red color. Digestion with aminopeptidase M gave an orange color. The extinction spectrum after digestion is completely in agreement with that of safranin-O. FIG. 3 shows how the amide conjugate of safranin O and leucine undergoes a spectrum change by aminopeptidase M.

  In fact, it was confirmed that the amide bond (CV-Leu) of cresyl violet and leucine changes color according to the amount of mite antigen in house dust. That is, for the ag samples shown in Table 1, the amount of mite antigen was quantified based on protease activity using a commercially available ELISA kit for indoor allergen measurement according to the attached protocol (Table 1). When the above-mentioned CV-Leu was added to these a-g samples and left overnight, a purple color was observed in the a and b samples, indicating that the amount of mite antigen was large. This result correlates with the ELISA result. Therefore, it became clear that the protease activity of mite antigen can be measured by changing the color of CV-Leu as a substrate.

It is an absorption spectrum of a starting material or a reaction mixture before and after an enzyme reaction when amide peptidase M is allowed to act on an amide conjugate of cresyl violet and leucine. It is an absorption spectrum of a starting material or a reaction mixture before and after an enzymatic reaction when aminopeptidase M is allowed to act on an amide conjugate of methylene violet and leucine. It is an absorption spectrum of a starting material or a reaction mixture before and after an enzymatic reaction when aminopeptidase M is allowed to act on an amide conjugate of safranin-O and leucine.

Claims (4)

  An enzymatic reaction is carried out using a colored compound in which an amino acid or oligopeptide is amide-bonded to at least one amino group of the colored dyes having at least one amino group as a substrate for a protease in a test sample. The method for measuring protease activity in the test sample, wherein the protease activity is measured based on the discoloration of the colored compound.   The method according to claim 1, wherein the substrate is a colored compound in which an amino acid is amide-bonded to at least one amino group among the amino groups of a colored dye having at least one amino group.   The method according to claim 1 or 2, wherein the coloring pigment is cresyl violet, safranin O or methylene violet 3RAX. At least one amino group of the amino group of the colored dye having at least one amino group, Ri coloring compound amino acid or oligopeptide is amide bond formation, the substrate is colored with at least one amino group A reagent for measuring protease activity, which is a colored compound in which an amino acid is amide-bonded to at least one amino group among the amino groups of the dye, wherein the colored dye is cresyl violet, safranin O, or methylene violet 3RAX .

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