WO1998048043A1 - Procede de dosage enzymatique d'une proteine de saccharification - Google Patents
Procede de dosage enzymatique d'une proteine de saccharificationInfo
- Publication number
- WO1998048043A1 WO1998048043A1 PCT/JP1998/001904 JP9801904W WO9848043A1 WO 1998048043 A1 WO1998048043 A1 WO 1998048043A1 JP 9801904 W JP9801904 W JP 9801904W WO 9848043 A1 WO9848043 A1 WO 9848043A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protease
- faod
- protein
- glycated
- melleus
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
- C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
- Y10S435/929—Fusarium
Definitions
- the present invention relates to a method for measuring a glycated protein using fructosyl amino acid oxidase (hereinafter, referred to as “FAOD”).
- FOD fructosyl amino acid oxidase
- the present invention relates to a method for measuring saccharified protein which can be used for the present invention, and a protease which can be suitably used for the method.
- a glycated protein is a substance produced by non-enzymatically and irreversibly binding of an amino group of an amino acid constituting a protein and an aldehyde group of a reducing sugar such as aldose. Since such a non-enzymatic and irreversible binding reaction is also referred to as “Amadori rearrangement”, the glycated protein described above is sometimes referred to as “Amadori compound”.
- the production rate of such a glycated protein generally depends on the concentration of the protein and the reducing sugar, which are the “raw materials” to which the glycated protein is to be provided, the contact time between the two, or the temperature during the saccharification reaction. Naturally, the higher the amount of the protein and the reducing sugar, the longer the contact time between the two, and the higher the temperature (to the extent that protein denaturation does not occur), the higher the rate of production of glycated protein as a reaction product. In addition, the amount of generation increases.
- the concentration of the glycated protein varies depending on the half-life of the protein as a raw material of the saccharification reaction. Therefore, various information on the living body can be obtained by measuring the concentration of the glycated protein.
- a fructosylamine derivative in which blood hemoglobin is saccharified is glycohemoglobin
- an albumin in which glycated albumin is glycoalbumin and a derivative in which blood protein is saccharified has low reducing ability.
- the blood concentration of these glycated protein derivatives reflects the average blood glucose level in the past period in the living body
- the measured blood concentration of the glycated protein derivative is used for diagnosis of diabetes symptoms. And an important indicator of the management of symptoms. Therefore, establishing a means to measure blood levels of glycated proteins is extremely useful from a clinical perspective.
- glycated proteins in a sample are prepared by, for example, reacting a glycated protein with an oxidoreductase and measuring the amount of oxygen consumed or the amount of product (eg, hydrogen peroxide) produced based on the action of the oxidoreductase. It is known that the measurement can be performed by the measurement (for example, JP-B 5-33997, JP-B 6-65300, JP-A 2-195900, JP-A 3-155780, JP-A 4-48 74, JP-A-5-192193, JP-A-6-46846, JP-A-7-289253, JP-A-8-154672, and JP-A-8-336386.
- the measurement can be performed by the measurement (for example, JP-B 5-33997, JP-B 6-65300, JP-A 2-195900, JP-A 3-155780, JP-A 4-48 74, JP-A-5-192193, JP-A-6-468
- 150 A JP-A-6-46846 (see EP 0 576 838 A), JP-A-7-289253, JP-A-8-154672, and JP-A-8-336386).
- an enzymatic reaction using a glycated protein as a substrate is represented by the following formula.
- R 1 represents an aldose residue of a reducing sugar
- R 2 represents an amino acid
- FADs fructtosyl amino acid oxidases
- glycated protein as a substrate.
- the present applicant has already obtained FAODs derived from bacteria belonging to the genera Fusarium, Gibberella, Penicilli dish, etc., and these are useful for the measurement of glycated proteins. (See Japanese Patent Application Laid-Open No. 7-289253, Japanese Patent Application Laid-Open No. 8-154672, and Japanese Patent Application No. 8-336386).
- F AOD produced by Fusarium oxysporum S-1F 4 (hereinafter referred to as “FAOD-S”)
- FOD-G Gibberella fujikuroi FAOD
- E.coli has activity against fructosyl lysine and / or fructosyl polylysine, and is therefore useful for the measurement of human serum or human glycated albumin.
- the above-mentioned FAOD method can be adapted to a general-purpose testing device, and it is less expensive than conventional methods such as the HPLC method and the antibody method. It is expected that measurement will be performed in a short time. Furthermore, since it is possible to accurately determine the glycated protein in a biological component by utilizing the specificity of the FAOD enzyme, the measurement of glycated protein by the FAOD enzyme method can It is expected to be used as a mass screening test in diagnosis and as a treatment marker for diabetic patients.
- the glycated protein substrate efficiently binds to the substrate binding site of FAOD, which is an enzyme (catalyst). Therefore, in order to increase the rate of the enzymatic reaction, it is important to design a substrate for increasing the efficiency of the binding.
- FAOD has a higher activity on glycated peptides (smaller molecular weight) than glycated proteins, This is because they tend to have a higher activity on glycated amino acids than glycated peptides (even smaller in molecular weight).
- F AOD F AOD
- F AOD F AOD
- fragmenting decreasing the molecular weight of a glycated protein present in a biological component using a protease.
- glycated amino acids are most preferable in terms of affinity with the substrate of FAOD, and it is theoretically possible to use a protease that digests and fragments all glycated proteins into amino acids.
- the fragmentation treatment to the amino acid requires an extremely long time.
- a protease that selectively cleaves a glycated amino acid site present in a glycated protein.
- proteases are known to be useful in combination with certain FAODs, and these proteases can be broadly divided into end-type and exo-type proteases. I have.
- the former end-type protease is an enzyme that decomposes from the inside of the protein.
- XXI lysyl endopeptidase, proleather, bromelain: F and the like.
- the latter exo-type protease is an enzyme that sequentially decomposes from the end of the peptide chain, and examples thereof include aminopeptidase and carboxypeptidase.
- JP-A-5-192193 discloses a protein useful for measuring glycated proteins.
- proteases proteinase, pronase E, ananain, thermolysin, subtilisin, and bovine lentil proteases are described.
- the protease described in Japanese Patent Application Laid-Open No. 5-192193 has been subjected to a fragmentation treatment of saccharified protein present in a sample, and then 55 ° to inactivate the activity of the protease. Incubate for 30 minutes at C.
- the reason for inactivating the protease in this way is to prevent or prevent the fragmentation of FAOD itself, which is the "catalyst" in the process of the next glycated protein-FAOD reaction, by the protease. That's why.
- FAOD itself, which is a “catalyst”
- the amount of oxygen consumed or the amount of hydrogen peroxide produced based on the action of FAOD on glycated proteins will decrease, resulting in saccharification. Decreases protein detection sensitivity.
- the protease inactivation treatment is usually essential. Operation.
- Japanese Patent Publication No. 5-33997 discloses no specific protease
- Japanese Patent Application Laid-Open No. 5-192193 discloses a proteases used together with ketoamine oxidase derived from a bacterium belonging to the genus Debryomyces. Only I (Proteinase K, Protease II) are disclosed.
- the glycated protein present in the sample was actually treated using the protease described in JP-A-5-192193, After inactivating the protease for 30 minutes, the FAOD was reacted.As a result, the amount of hydrogen peroxide or the amount of oxygen consumed was small, and as a result, the detection sensitivity was not sufficient. It is known.
- protease can be inactivated by the addition of an inhibitor, but some combinations of proteases and inhibitors inhibit the inactivation of the target protease. Some combinations do not achieve complete activation (remaining some protease activity).
- the inactivation of protease described above is based on the binding of inhibitor to the active center of the protease enzyme to which the substrate is to be bound.
- the inhibitor In order for the inhibitor to bind to the active center of the enzyme, after the protease reaction is completed, the inhibitor must be added and reacted for a certain period of time at a constant temperature. Therefore, the time required for the entire measurement of glycated protein becomes longer by the inactivation reaction time.
- An object of the present invention is to provide a method for enzymatically measuring glycated protein, which can solve the above-mentioned problems of the prior art, and an enzyme that can be suitably used for the method.
- Another object of the present invention is to provide a method for measuring a glycated protein, which can measure a glycated protein more accurately and with high sensitivity, and an enzyme which can be suitably used in the method. Disclosure of the invention
- the present inventors have conducted intensive studies and as a result, it has been found that a specific protease can accurately and precisely provide FAOD suitable for measurement of glycated protein (for example, glycated albumin) in a biological component in various respects. It has been found that highly sensitive glycated protein measurement is possible, and is extremely effective in achieving the above-mentioned object.
- glycated protein for example, glycated albumin
- the protease of the present invention is based on the above findings, and more specifically, is a protease used in combination with FAOD (fructosyl amino acid oxidase) in measuring a glycated protein in a sample. It is characterized by According to the present invention, there is further provided a method for measuring a glycated protein in which FAOD acts on a sample containing the glycated protein, wherein the glycated protein is subjected to protease treatment under acidic conditions. Measurement methods are provided o
- a method for measuring a glycated protein in which a protease and a FAD are allowed to act on a sample containing the glycated protein, wherein the protease comprises a protease derived from the genus Aspergillus as the protease.
- the present invention provides a method for measuring a glycated protein, characterized by using zeolites.
- a method for measuring a glycated protein in which a protease and FAOD are allowed to act on a sample containing the glycated protein, wherein the protease XIV is used as the protease.
- a method is provided. BRIEF DESCRIPTION OF THE FIGURES
- FIG. 1 is a graph showing the results of determining the relationship between glycated albumin concentration and absorbance (glycated albumin measurement-albumin reagent) in Examples described later.
- FIG. 2 is a graph showing the results of determining the pH dependence of the relative activity of protease (optimal pH of A. melleus BP-6277 protease) in Examples.
- FIG. 3 is a graph showing the results of determining the temperature dependence of the protease relative activity (temperature dependence of A. melleus elle-62777 ° --IT) in the examples.
- Fig. 4 shows the results of measuring the saccharification rate of HSA using the combination of commercially available protease "Sumitim MP" and FAO DS in the examples (saccharification by Sumitim MP / FAOD-S). (Measurement of rate).
- FIG. 5 shows the results of measuring the saccharification rate of HSA using the combination of the protease and FAOD-S obtained in the examples (measurement of the saccharification rate by A. melleus BP-6277 / FAOD-S). It is a graph.
- FIG. 6 is a graph in which the activity value at each pH is shown as a relative value with the activity value at pH 5 (optimal pH) of the acidic protease obtained in the example as 100.
- FIG. 7 is a graph in which the activity value at 50 ° C. (optimal temperature) of the acidic protease obtained in the example at 100 ° C. is set as 100, and the activity value at each temperature is shown as a relative value.
- FIG. 8 is a graph showing the measurement results of the absorbance based on the FAOD reaction with respect to HSA (human serum albumin) having different saccharification rates in Examples.
- any sample containing a glycated protein (typically, a sample derived from a living body) can be used.
- a sample typically, a sample derived from a living body
- a biological sample such as blood (whole blood, plasma or serum), and urine.
- the treatment of the above-mentioned sample with protease described below may be generally performed according to the instructions of the supplier of each enzyme.
- the temperature is 50 ° C
- protease XIV trade name, manufactured by Sigma
- the temperature is 37 ° C. C, preferably incubate for about 30 minutes in Tris-HC1 buffer (pH 8.0).
- the detection sensitivity it is preferable to select a protease that digests and fragments a glycated protein in a sample according to the FAOD to be used.
- the protease one kind of protease may be used, or a plurality of kinds of proteases may be used in combination or in combination.
- the detection sensitivity can be improved by performing the fragmentation treatment of the glycated amino acid more specifically by using a plurality of types of proteases.
- protease XIV trade name, manufactured by Sigma
- protease derived from the genus Aspergillus from the viewpoint that accurate glycated protein measurement can be easily performed.
- Aspergillus Aspergillus it is particularly preferable to use a protease derived from Aspergillus melleus (hereinafter referred to as "A.melleus").
- a preferred example of such a protease derived from A.melleus is a specific A.melleus strain (depositary institution name ⁇ address: ⁇ 305—8566 1-3-1 Tsukuba-Higashi, Ibaraki, Japan National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, Deposit date: March 3, 1998, deposit number: FERM BP-6277) Protease produced from The protease was confirmed to have the same enzymatic properties as "1MP" (the same level of matching with FAOD or the same level of activity; see Example 4 described later).
- proteases derived from A. melleus there are a protease having an optimal pH on the basic side and an protease on the acidic side, and any of them can be used in the present invention.
- a protease derived from A. melleus that can be suitably used in the present invention
- Sumiteam MP (trade name, manufactured by Shin Nippon Chemical Industry Co., Ltd.) used in the field of food processing can be exemplified.
- This Sumiteam MP is used as a food enzyme, and is inexpensive and can be supplied in large quantities. Therefore, it can be advantageously used in clinical tests such as screening.
- the glycated protein has a site to be glycated depending on the type of the protein, and more specifically, on the type and position of the amino acids constituting the protein.
- the protein is albumin
- glycine is most likely to be glycated at the 525th lysine from the N-terminus, and in most glycated albumin, the 525th lysine site from the N-terminus is glycated. Therefore, the detection sensitivity can be improved if a protease capable of suitably fragmenting the glycated lysine can be used.
- Proteases derived from the genus Aspergillus such as "Proteases XIV" or A.
- melleus such as "Sumitim MP" can also be suitably used in this regard.
- a protease which can be easily inactivated by a simple treatment after the protease treatment.
- simple treatment include, for example, pH change, heating, addition of an inhibitor, and the like.
- glycated amino acids at the glycation site of a glycated protein can be efficiently cleaved, and the FAOD increases the absolute amount of consumed oxygen or reaction products (eg, hydrogen peroxide) to improve detection sensitivity.
- Proteases having an optimum pH on the acidic side may be used as long as it is a protease that enables the above.
- the method of changing the pH eliminates the need for heat denaturation or the addition of an inhibitor to inactivate the protease.
- protease having an optimal pH on the acidic side for example, a protease having an optimal pH on the acidic side, which is separated and produced from the protease produced by A. me 1 leus, can be used. is there. (Inactivation of protease)
- the FAOD reaction can be started by inactivating the protease and adding FAOD.
- protease inactivation it is preferable to suppress or prevent the protease fragmentation treatment of FAOD.
- the site to be glycated is determined by the relationship with the biological component (for example, the type and position of the amino acid that constitutes the protein), so the optimal FAOD is selected according to the type of the glycated protein to be measured. Is preferred.
- Examples of the FAOD that can be used in the method of the present invention include bacteria belonging to the genus Fusarium, Gibberella, Penicillium, Aspergillus, etc., fructosyl lysin and / or fructosyl lysine.
- An enzyme derived by culturing in the presence of Tosyl N-Hi Z-Lysine can be mentioned.
- Such a FAOD can be obtained, for example, by a method disclosed in Japanese Patent Application Laid-Open No. 7-289253, Japanese Patent Application Laid-Open No. 8-154672, Japanese Patent Application Laid-Open No. 8-336386, or the like.
- the above-mentioned Fusarium oxysporum S-1 is considered to have activity against fructosyl lysine and / or fructosyl poly lysine (which is a site which is easily saccharified from human serum or human glycated albumin).
- FAOD—S derived from F 4 (Fusarium oxysporum S-1F 4) and FAOD—G derived from Gibberella 'Fujikuroi AKU 3802 (Gibberella fujikuroi) (JP-A-7-289253) can be used particularly preferably. It is.
- the titer of FAOD used in the present invention can be suitably measured by the following method.
- the glycated protein in the sample is treated using the protease described above to a state in which FAD can easily react, and Alternatively, it is preferable that after treatment with protease, FAOD is allowed to act and the amount of oxygen consumed or the amount of reaction products in the FAOD-substrate reaction is measured.
- the reaction of glycated protein (or its protease-treated product) with F AOD produces hydrogen peroxide and glucosone. Any of these hydrogen peroxide and glucosone can be used as F AOD reaction products to be measured in a later step.
- the method for measuring these F AOD reaction products is not particularly limited, and can be appropriately selected from known methods and used.
- the amount of hydrogen peroxide can be determined by a method known in the art, for example, a colorimetric or chromogenic method (using a chromogen that produces a dye upon decomposition with a catalyst having peroxidase or peroxidase-like activity). Measurement method, etc.), electrochemical measurement method (method using hydrogen peroxide electrode, etc.), measurement of the amount of aldehyde generated (by hydrogen peroxide) in the presence of hydrogen peroxide and alcohol. The amount of generated hydrogen peroxide can be quantified by any method.
- glycated protein may be quantified using the above-described method and based on a calibration curve prepared in advance using a sample containing a known amount of glycated protein.
- the amount of F AOD activity is always constant. If necessary, it is preferable to dilute a sample composed of a biological component or the like with a buffer solution before performing the measurement.
- the color development system for the colorimetric method of hydrogen peroxide includes couplers such as 4-aminoaminopyrine (4AA), 3-methyl-12-benzothiazolinone hydrazone (MBTH) and phenol in the presence of peroxidase.
- couplers such as 4-aminoaminopyrine (4AA), 3-methyl-12-benzothiazolinone hydrazone (MBTH) and phenol in the presence of peroxidase.
- a system that generates a dye by oxidative condensation with a chromogen such as the above can be used.
- chromogens As chromogens, phenol derivatives, aniline derivatives, toluidine derivatives, etc. Can be used. Specific examples of the chromogen include, for example, N-ethyl-N- (2-hydroxy-13-sulfopropyl) -m-toluidine, N, N-dimethylaniline, ⁇ , ⁇ -getylaniline, 2,4-dichloropheno 1-, 2-ethyl-1- (2-hydroxy-13-sulfopropyl) -1,3-dimethoxyaniline, ⁇ -ethyl-1- (3-sulfopropyl) -1,3-dimethylaniline (MAPS) , N-ethyl-1-N- (2-hydroxy-13-sulfopropyl) -13,5-dimethylaniline (MAOS) and the like.
- N-ethyl-N- (2-hydroxy-13-sulfopropyl) -m-toluidine N,
- a known leuco-type coloring reagent which is oxidized in the presence of peroxidase to give a color can also be used as the chromogen.
- a coloring reagent include o-dianicidin, o_tolidine, 3,3-diaminobenzidine, 3,3,5,5-tetramethylbenzidine, and N- (carboxymethylaminocarbonyl) one. 4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -13,7-bis (dimethylamino) phenothiazine (DA67), and the like.
- a fluorescence method and a chemical luminescence method can be used.
- a compound that emits fluorescence by oxidation for example, homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivative and the like can be used.
- peroxidase, potassium ferricyanide, hemin or the like can be used as a catalyst, and luminol, lucigenin, isorluminol, pyrogallol, or the like can be used as a substrate.
- a known aldose reagent such as diphenylamine can be used to measure glucosone, which is a FAD reaction product other than hydrogen peroxide.
- the electrode When measuring hydrogen peroxide, which is a FAOD product, using an electrode, the electrode is not particularly limited as long as it is a material that can transfer electrons to and from hydrogen peroxide. Silver or the like can be suitably used.
- the electrode measurement can be carried out by a method known to those skilled in the art, such as diametry, potentiometry, and chromometry.
- Glycated proteins can also be measured by interposing an electron carrier in the reaction between the FAOD or the substrate and the electrode, and measuring the resulting oxidation and reduction currents or the amount of electricity.
- an electron carrier any substance having an electron transfer function can be used, and examples thereof include substances known to those skilled in the art, such as Hua's cene derivatives and quinone derivatives.
- a glycated protein can also be measured by interposing an electron carrier between hydrogen peroxide generated by the FAOD reaction and an electrode, and measuring the resulting oxidation or reduction current or its electric quantity.
- glycated protein was fragmented with each protease, and then FAOD-G was reacted as FAOD, and the amount of hydrogen peroxide produced was measured.
- Substrate human serum albumin (trade name: A1b, manufactured by SI GMA)
- F AOD F AOD -G (F AOD -G isolated and purified from Gibberella fujikuroi by the method described in JP-A-7-289253)
- Chromogen 4-aminoaminopyrine
- a “FAOD reaction solution” having the following composition was prepared.
- proteases that obtained high absorbance with both human serum albumin (HSA) and human serum substrates.
- Proteinase K 0.0496 0.0501 0.0217 0.0216
- a sample for measurement was prepared by diluting with a buffer so as to obtain a concentration of 1 and FAOD-G or FAOD-S was digested and fragmented with Sumizyme MP in the same manner as in the screening of Example 2.
- the result of measuring the amount of hydrogen peroxide generated when the compound is acted on as an absorbance is shown in the graph of FIG.
- the vertical axis in FIG. 1 shows the absorbance at 555 nm, and the horizontal axis shows the albumin concentration. From the results shown in FIG. 1, when Sumizyme MP was used as a protease, the color development based on the action of FAOD on the above sample showed a good proportional relationship to the saccharified albumin concentration. It was confirmed that the proteinase was useful for measuring glycated human albumin.
- the five strains obtained were ampoules in a lyophilized state, they were reconstituted with designated reconstituted water 200/1 having the following composition, and then inoculated with GPY M slant having the following composition. After culturing at 30 ° C for 2 days, growth was observed in all strains. This slant was stored at 4 ° C, and when culturing, the slant was subcultured to each medium.
- each strain was inoculated into 10 ml of a GPYM medium having the following composition, cultivated with shaking at 30 ° C. for 2 days, and then subcultured in 50 Oml of the same culture solution, followed by culturing for 2 days. The shaking speed was set at 11 rpm for both.
- the inner solution of the dialysis was centrifuged in a microtube (4 ° C, 12000 rpm, 20 minutes), the supernatant was recovered, and the volume was measured. The results obtained are shown below.
- the volume of the culture supernatant concentrate after dialysis was 1.6 to 3.8 ml, which was several tens times higher than the volume of 100 ml before the addition of ammonium sulfate. Becomes
- the protease activity of each of the culture supernatant concentrates (5 types) obtained above was measured.
- the culture supernatant concentrate 201 described above 20% of 5% by weight HSA (pH 8) as a substrate, and 0.1 M Tris-HC1 buffer ( (pH 8.0)
- HSA 5% by weight
- Tris-HC1 buffer pH 8.0
- TCA 0.6M trichloroacetic acid
- A.melleus IF 04420 0.5144mg / ml 113.5U / ml
- the optimal pH was measured by changing the pH of the buffer constituting the reaction solution for the activity measurement used in the above “Protease activity measurement”.
- the optimum pH was confirmed to be approximately 8 to 9 similar to that of Sumizyme MP.
- the pH dependence of the protease activity from A. melleus FERM BP-6277 is shown in the graph of FIG. In this graph, the enzyme activity was shown as a relative activity value based on the activity at the optimum pH (the above activity value was converted to 100).
- the pH of the buffer solution was fixed at 8.5, and the measurement was performed in the range of 15 to 70 ° C in increments of 5 ° C.
- the activity was measured in the same manner as in the measurement of protease activity after a digestion reaction for 30 minutes.
- Injection volume 50 1
- HSA saccharification rate
- S1GMA Alubumin Human Fraction V
- KDK GAA-2000 HPLC method Was dissolved to a concentration of 5% (pH 8).
- 11.7% and 18.85% of HSA was prepared by mixing 11.7% of HSA with the other two.
- the reaction was performed by adding 100 1001 of 0.1M Tris-HC1 buffer and 100 ⁇ 1 of Sumizyme MP (1 Omg / ml) to 200 ⁇ 1 of 5% by weight of HSA, and performing a closed-door reaction at 37 ° C for 4 hours. After that, the activity was confirmed using the following 4AA-TOOS color developing system using FAOD-S (0.5602 U / m 1).
- A.melleus was a strain A.melleus KDK 3001 (FERM BP-6277) stored by Kyoto Daiichi Kagaku Co., Ltd., and four A.melleus strains obtained from the Fermentation Research Institute (IFO).
- IFO Fermentation Research Institute
- the GPYM medium used here had the composition shown below.
- ammonium sulfate in the above ammonium sulfate precipitation was performed under ice cooling, and after the completion of the addition of ammonium sulfate, stirring was performed at 4 ° C for 1 hour. After stirring, centrifuge at 4 ° C, 10,000 rpm for 40 minutes, collect the precipitate, dissolve in the minimum amount of distilled water, and distill at 4 ° C overnight. Dialysis (semi-permeable membrane: dialysis membrane, manufactured by Sanko Junyaku Co., Ltd.) was performed with water. After completion of the dialysis, the inner solution of the dialysis was centrifuged in a microtube (for centrifugation) (4 ° C, 12000 rpm, 20 minutes), and the supernatant was collected.
- a microtube for centrifugation
- the protease activity of each culture supernatant concentrate was measured.
- the above-mentioned ⁇ Sumitim MP '' is a weakly alkaline protease having an optimum pH around 8, but based on the experiments of the present inventors, in addition to the weakly alkaline protease, there is an optimum pH on the acidic side. Since the presence of the protease was predicted, the measurement of the activity of acid protease was confirmed.
- HSA 5% by weight
- the culture supernatant concentrated solution obtained above was 20 ⁇ ⁇ 1, 5% by weight of HSA201 and 0.1M Tris-HC1 buffer solution.
- PH 5 60 zl were mixed and reacted at 37 ° C for 30 minutes.
- 100 ml of 0.6 M trichloroacetic acid (TCA) was added, and the mixture was allowed to stand under ice-cooling for 15 minutes or more, followed by centrifugation (TCA precipitation) at 4 ° C and 12000 rpm for 0 minutes.
- the amount of free amino acids in the supernatant was determined using a commercially available Bio-Rad DC Protein assay kit (Bio-Rad) using the supernatant 25-1 obtained after removing the protein by this TCA precipitation. (The kit was used according to the supplier's instructions).
- Bio-Rad DC Protein assay kit Bio-Rad
- the kit was used according to the supplier's instructions.
- HSA solution adjusted to pH 1.6 and 8 using 0.1M Tris-HC1 buffer as a substrate solution, and mix these two solutions. Then, substrate solutions (total of 10 types) were prepared within the pH range of 1.6 to 6.8. Further, hydrochloric acid was added to HSA at pH 1.6 to prepare a substrate solution at pH 1.
- the substrate solution 201, the protease-containing solution 20 ⁇ 1, and the distilled water 601 adjusted to the above-mentioned pHs were added, and reacted at 37 ° C. for 30 minutes.
- the pH of the reaction solution was measured again, and the pH during the protease reaction (immediately after mixing the three solutions) was measured again. 6. It was within the range of 5.
- the protein was removed by TCA precipitation, and the amount of free amino acids in the supernatant was measured using a Bio-Rad DC Protein assay kit in the same manner as in the measurement of the protease enzyme activity described above.
- the acidic protease derived from A. melleus KDK 3001 has an optimal region between pH 4.7 and 5.6, and shows a high activity especially at pH 5. It was confirmed that it was.
- the above-mentioned optimal temperature of the acidic protease derived from A. melleus KDK 3001 was measured by fixing the pH of the reaction solution to 5 and measuring the temperature in the range of 15 to 70 ° C at intervals of 5 ° C. After a 30-minute incubation, the protease activity was measured in the same manner as described above, and it worked practically at 35-65 ° C as shown in Fig. 7 and became highly active at 40-50 ° C. However, it was found that an optimum temperature exists in this range. The highest activity was at 50 ° C.
- GP C GP C
- the column uses Superdex 200 pg l 6/60 (Pharmacia), and every 30 seconds Fractions were collected.
- the GPC conditions were determined by measuring the protease activity of the collected fraction, and estimating the molecular weight of the protease from the elution time of the fraction.
- the measurement conditions of GPC used here were as follows. ⁇ GPC measurement conditions>
- the amount of sample added 50 ⁇ 1 Protease activity was determined by taking into account the dilution in the course of GPC, adding 2 parts by weight of 5% HSA and 80-1 of each fraction, and incubating overnight at 37 ° C. , And the protease activity was measured in the same manner.
- the protease activity of the fraction obtained by the GPC was measured by the same measurement method as described above, it was inferred that the molecular weight of the protease derived from this bacterium was 18382 to 22130.
- Example 5 taking advantage of the fact that the protease obtained in Example 5 has an optimal pH on the acidic side, and the optimal pH of FAOD is 8, The proteinase activity was inactivated by adjusting the pH of D to an optimal pH (pH 8) (without heating or adding an inhibitor).
- HSA human serum albumin with different saccharification rates
- the saccharification rates of these HSA were 11.7%, 22.5%, and 26.0%, respectively.
- GAA-2000 HP LC method manufactured by Kyoto Daiichi Kagaku Co., Ltd.
- HS A was used after adjusting the concentration to 5% by weight and pH5.
- HS A with different saccharification rates were mixed to prepare samples with saccharification rates of 17.1% and 18.85%.
- saccharified HS A was measured using the protease-containing solution obtained in Example 5.
- the reaction was carried out by adding 200% of the protease-containing solution of Example 5 to 200% of 5% by weight of HSA, and the reaction time was 5 hours. After this protease reaction, color development was confirmed using FAOD (FAOD-S).
- FAOD FAOD-S
- As the FAOD FAOD, FAOD-S (0.5602 U / ml) prepared according to JP-A-7-289253 was used.
- the titer of FAOD used in this example was measured by the above-described rate method.
- the composition of the reaction solution of the FAOD color developing system used for the above titration was as follows.
- protease which is used in combination with FAOD (fructosyl amino acid oxidase) in measuring a glycated protein in a sample.
- a method for measuring a glycated protein in which FAOD acts on a sample containing the glycated protein, wherein the glycated protein is subjected to protease treatment under acidic conditions is provided.
- a method for measuring glycated protein in which a protease and a FAOD are allowed to act on a sample containing a glycated protein, wherein the protease comprises Aspergillus-derived proteases.
- the present invention provides a method for measuring saccharified protein, wherein the method comprises using zeolites.
- a method for measuring a glycated protein in which a protease and a FAOD are allowed to act on a sample containing a glycated protein, wherein a protease XIV is used as the protease. How to measure glycated protein A law is provided.
- a glycated protein in a biological component can be measured with high sensitivity and accuracy by using an appropriate protease which exhibits a useful enzymatic action in combination with FAOD suitable for measuring glycated albumin. can do. Therefore, according to the present invention, there are provided a method for measuring a glycated protein which can contribute to the management and prevention of the symptoms of diabetes, and a protease suitable for use in the method.
- the pH can be adjusted to easily and quickly inactivate the protease, so that it can be applied to a wider range of clinical tests and the like.
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98917678A EP0921198B1 (en) | 1997-04-24 | 1998-04-24 | Method for enzymatically assaying saccharification protein |
US09/202,641 US6127138A (en) | 1997-04-24 | 1998-04-24 | Method of enzymatically measuring glycated protein |
DE69835268T DE69835268T2 (de) | 1997-04-24 | 1998-04-24 | Verfahren zum enzymatischen nachweis eines verzuckerungsproteins |
JP54548198A JP4061348B2 (ja) | 1997-04-24 | 1998-04-24 | 糖化タンパク質の酵素的測定方法 |
Applications Claiming Priority (6)
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JP10710697 | 1997-04-24 | ||
JP9/107106 | 1997-04-24 | ||
JP9083798 | 1998-03-18 | ||
JP9083698 | 1998-03-18 | ||
JP10/90837 | 1998-03-18 | ||
JP10/90836 | 1998-03-18 |
Publications (1)
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WO1998048043A1 true WO1998048043A1 (fr) | 1998-10-29 |
Family
ID=27306556
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1998/001904 WO1998048043A1 (fr) | 1997-04-24 | 1998-04-24 | Procede de dosage enzymatique d'une proteine de saccharification |
Country Status (5)
Country | Link |
---|---|
US (1) | US6127138A (ja) |
EP (1) | EP0921198B1 (ja) |
JP (1) | JP4061348B2 (ja) |
DE (1) | DE69835268T2 (ja) |
WO (1) | WO1998048043A1 (ja) |
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US7235378B2 (en) * | 2000-07-14 | 2007-06-26 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
WO2007072941A1 (ja) * | 2005-12-22 | 2007-06-28 | Kyowa Medex Co., Ltd. | 糖化タンパク質の測定方法 |
EP2107376A2 (en) | 2001-01-31 | 2009-10-07 | Asahi Kasei Pharma Corporation | Composition for assaying glycated proteins |
EP2639586A1 (en) | 2012-03-15 | 2013-09-18 | ARKRAY, Inc. | Measurement method using enzymes |
US11262345B2 (en) | 2015-09-02 | 2022-03-01 | Ultizyme International Ltd. | Method for measuring glycated protein using interdigitated electrode |
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USRE45626E1 (en) | 2001-10-11 | 2015-07-28 | Arkray, Inc. | Method of pre-treating sample for measuring saccharified amine and method of measuring saccharified amine |
USRE46073E1 (en) * | 2001-10-11 | 2016-07-19 | Arkray, Inc. | Method of pre-treating sample for measuring saccharified amine and method of measuring saccharified amine |
WO2003033601A1 (fr) * | 2001-10-11 | 2003-04-24 | Arkray, Inc. | Procede destine a stabiliser l'oxydation d'un chromogene |
EP1477569B1 (en) * | 2002-01-31 | 2007-07-18 | Arkray, Inc. | Method of quantifying glycosylated protein using redox reaction and quantification kit |
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US8715942B2 (en) * | 2007-11-20 | 2014-05-06 | Siemens Healthcare Diagnostics Inc. | Methods for the detection of glycated hemoglobin |
US8673646B2 (en) * | 2008-05-13 | 2014-03-18 | General Atomics | Electrochemical biosensor for direct determination of percentage of glycated hemoglobin |
DE102009026651A1 (de) | 2009-06-02 | 2011-02-17 | Großmann, Kay, Dr. rer. nat. (Chemie) | Verfahren und Messvorrichtung zur qualitativen und quantitativen Analyse von Körperflüssigkeiten |
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JP7307959B2 (ja) * | 2018-05-18 | 2023-07-13 | 株式会社Provigate | 糖化タンパク質センサ、測定方法、プログラム及びセンサの製造方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61280297A (ja) * | 1985-06-04 | 1986-12-10 | Noda Sangyo Kagaku Kenkyusho | アマドリ化合物の定量法及びその定量用試薬 |
JPH05192193A (ja) * | 1991-07-29 | 1993-08-03 | Genzyme Ltd | 非酵素的グリコシル化タンパク質の検定法 |
JPH07289253A (ja) * | 1994-03-03 | 1995-11-07 | Kyoto Daiichi Kagaku:Kk | フルクトシルアミノ酸オキシダーゼ及びその製造方法 |
JPH08154672A (ja) * | 1994-10-05 | 1996-06-18 | Kdk Corp | フルクトシルアミノ酸オキシダーゼ及びその製造方法 |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3492204A (en) * | 1965-03-31 | 1970-01-27 | Meiji Seika Co | Process for the production of acid protease by cultivation of microorganisms |
JPH02195899A (ja) * | 1989-01-25 | 1990-08-02 | Sanyo Chem Ind Ltd | 糖化蛋白の測定法および測定試薬 |
JPH02195900A (ja) * | 1989-01-25 | 1990-08-02 | Sanyo Chem Ind Ltd | 糖化蛋白の測定法および測定試薬 |
JP2601356B2 (ja) * | 1989-11-14 | 1997-04-16 | 財団法人野田産業科学研究所 | フルクトシルアミン・オキシダーゼ、その製造法、該酵素を用いたアマドリ化合物の定量法及びその試薬 |
JPH044874A (ja) * | 1990-04-20 | 1992-01-09 | Nakano Vinegar Co Ltd | フラクトシルアミノ酸分解酵素、その製造法及びその利用 |
JPH0533997A (ja) * | 1991-07-26 | 1993-02-09 | Nippondenso Co Ltd | フイルムドアユニツト |
JP3157622B2 (ja) * | 1992-06-05 | 2001-04-16 | 株式会社ミツカングループ本社 | フルクトシルアミンデグリカーゼ、その製造法及び該酵素を用いたアマドリ化合物の定量方法 |
JP3118522B2 (ja) * | 1992-07-30 | 2000-12-18 | 品川白煉瓦株式会社 | レススラグ操業転炉用熱間補修吹付材 |
JP3409337B2 (ja) * | 1992-08-24 | 2003-05-26 | 和光純薬工業株式会社 | 新規な糖誘導体及びこれを基質として用いる生理活性物質の活性測定法 |
US5712138A (en) * | 1994-10-05 | 1998-01-27 | Kyoto Daiichi Kagaku Co., Ltd. | Fructosyl amino acid oxidase |
JP4004081B2 (ja) * | 1995-04-11 | 2007-11-07 | アークレイ株式会社 | フルクトシルアミノ酸オキシダーゼおよびその製造方法 |
-
1998
- 1998-04-24 JP JP54548198A patent/JP4061348B2/ja not_active Expired - Fee Related
- 1998-04-24 US US09/202,641 patent/US6127138A/en not_active Expired - Fee Related
- 1998-04-24 DE DE69835268T patent/DE69835268T2/de not_active Expired - Fee Related
- 1998-04-24 EP EP98917678A patent/EP0921198B1/en not_active Expired - Lifetime
- 1998-04-24 WO PCT/JP1998/001904 patent/WO1998048043A1/ja active IP Right Grant
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61280297A (ja) * | 1985-06-04 | 1986-12-10 | Noda Sangyo Kagaku Kenkyusho | アマドリ化合物の定量法及びその定量用試薬 |
JPH05192193A (ja) * | 1991-07-29 | 1993-08-03 | Genzyme Ltd | 非酵素的グリコシル化タンパク質の検定法 |
JPH07289253A (ja) * | 1994-03-03 | 1995-11-07 | Kyoto Daiichi Kagaku:Kk | フルクトシルアミノ酸オキシダーゼ及びその製造方法 |
JPH08154672A (ja) * | 1994-10-05 | 1996-06-18 | Kdk Corp | フルクトシルアミノ酸オキシダーゼ及びその製造方法 |
Non-Patent Citations (2)
Title |
---|
LUISETTI M., ET AL.: "SOME PROPERTIES OF THE ALKALINE PROTEINASE FROM ASPERGILLUS MELLEUS", INTERNATIONAL JOURNAL OF TISSUE REACTIONS., BIOSCIENCE EDIPRINT, GENEVA., CH, vol. 13., no. 04., 1 January 1991 (1991-01-01), CH, pages 187 - 192., XP002912905, ISSN: 0250-0868 * |
See also references of EP0921198A4 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
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US6797503B1 (en) * | 1999-02-22 | 2004-09-28 | Arkray, Inc. | Enzyme |
USRE46105E1 (en) | 2000-07-14 | 2016-08-16 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
US7235378B2 (en) * | 2000-07-14 | 2007-06-26 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
USRE46130E1 (en) | 2000-07-14 | 2016-08-30 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
USRE46118E1 (en) | 2000-07-14 | 2016-08-23 | Arkray, Inc. | Method of selectively determining glycated hemoglobin |
EP2107376A2 (en) | 2001-01-31 | 2009-10-07 | Asahi Kasei Pharma Corporation | Composition for assaying glycated proteins |
EP2248909A1 (en) | 2001-01-31 | 2010-11-10 | Asahi Kasei Pharma Corporation | Compositions for assaying glycoprotein |
EP2236618A2 (en) | 2001-01-31 | 2010-10-06 | Asahi Kasei Pharma Corporation | Compositions for assaying glycoprotein |
EP2107123A2 (en) | 2001-01-31 | 2009-10-07 | Asahi Kasei Pharma Corporation | Composition for assaying glycated proteins |
WO2007072941A1 (ja) * | 2005-12-22 | 2007-06-28 | Kyowa Medex Co., Ltd. | 糖化タンパク質の測定方法 |
EP2639586A1 (en) | 2012-03-15 | 2013-09-18 | ARKRAY, Inc. | Measurement method using enzymes |
US8802366B2 (en) | 2012-03-15 | 2014-08-12 | Arkray, Inc. | Measurement method using enzyme |
US11262345B2 (en) | 2015-09-02 | 2022-03-01 | Ultizyme International Ltd. | Method for measuring glycated protein using interdigitated electrode |
Also Published As
Publication number | Publication date |
---|---|
US6127138A (en) | 2000-10-03 |
DE69835268D1 (de) | 2006-08-31 |
EP0921198A4 (en) | 2003-09-24 |
EP0921198A1 (en) | 1999-06-09 |
EP0921198B1 (en) | 2006-07-19 |
DE69835268T2 (de) | 2006-11-23 |
JP4061348B2 (ja) | 2008-03-19 |
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