JP2001054398A - Selective fragmentation of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To selectively fragmentize a protein for the clinical diagnosis, etc., by adding a protease and a specific protein selective protease inhibitor to a specimen liquid and fragmentizing the proteins other than the specific protein. SOLUTION: A protease and a protease inhibitor (e.g. protein G and/or protein A) selective to a specific protein such as globulin component are added to a specimen liquid containing a globulin component, a protein other than the globulin component, etc., to fragmentize the proteins mainly other than the specific protein such as globulin component. The protein can be selectively fragmentized by the process usable for the determination of saccharizing protein important for the diagnosis and control of diabetes, etc., and usable for clinical examination, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for fragmenting a protein into proteins other than a specific protein, and a composition for fragmenting a protein. The present invention also relates to a method for measuring a glycated protein which is not affected by a specific protein, and a composition for measuring a glycated protein. Further, the present invention provides
The present invention relates to a protease inhibitor that selectively inhibits the action of a protease on a specific protein. The protease inhibitor of the present invention is used in a method for selective fragmentation of the protein of the present invention and a method for measuring a glycated protein. A method for selective fragmentation of a protein according to the present invention,
The methods for measuring glycated proteins and the compositions used for these methods are used in clinical tests.

[0002]

2. Description of the Related Art Measurement of glycated protein is very important in diagnosing and managing diabetes, and glycated hemoglobin, which reflects the average blood glucose level in the past about 1 to 2 months, and the average blood glucose level in the past about 2 weeks, Reflected glycated albumin and fructosamine, which is a collective term for glycated proteins showing a reducing ability in serum, are routinely measured. In glycated hemoglobin, the α-amino group of the N-terminal valine of the β-chain of hemoglobin is glycated, and in glycated albumin, glycated globulin components, and fructosamine, the ε-amino group of lysine residue is glycated.

[0003] It is also known that in diabetic patients, the amount of globulin protein changes and affects the value of fructosamine [Rodrigues S et al. Clin. Chem. 35: 134-138 (1
989)], There is a need for a glycated protein measurement system that is not affected by globulin components in diagnosing and managing diabetes. Similarly, there is a need for a method of fragmenting a protein that is not affected by a specific protein that changes in various diseases, and a method of quantifying a glycated protein using the same.

[0004] As a method for quantifying glycated protein, the following method is used.
The methods (a) to (e) and the enzyme method are known. (a) Method using chromatography [J. Clin. Chim. Cli
n. Biochem. 19: 81-87 (1981)] (b) Electrophoresis (Clin.chem. 26: 1958-1602 (1980)) (c) Immunoassay (JCCLA 18: 620 (1993)) (d) A method for measuring fructosamine using the reducing property of glycated protein in alkaline conditions [Clin. Chim. Acta 12
7: 87-95 (1982)] (e) Method using thiobarbituric acid (Clin. Chim. Act
a 112: 197-204 (1981))

However, the methods (a) and (b) have many problems such as operability, accuracy, and the necessity of an expensive dedicated device, and the method (c) is not always accurate. The methods (d) and (e) were affected by coexisting substances in the sample, and had a problem in specificity.

[0006] An enzymatic method is a highly accurate, simple and inexpensive quantification method. As enzymes used for the measurement of glycated proteins, the following enzymes (f) to (k) that act on at least glycated amino acids are known. (f) Fructosyl amino acid oxidase: from the genus Corynebacterium (Japanese Patent Publication No. 6-65300), from the genus Aspergillus (Japanese Unexamined Patent Publication No.
No. 155780), from the genus Gibberella
No. 7-289253). (g) Fructosylamine deglycase (JP-A-6-46846)
No.). (h) Fructosyl amino acid degrading enzyme (JP-A-4-4874). (i) Ketoamine oxidase (JP-A-5-192193). (j) Alkyl lysinase: (JP-A-2-195900). (k) NAD and / or NADP using CH-OH group as hydrogen donor
Oxidoreductase using as a hydrogen donor: (JP-A-2-95899).

[0007] Corynebacterium (Corynebacterium ) of the above (f)
tertium) -derived fructosyl amino acid oxidase (Japanese Patent Publication No. 5-33997) is an enzyme specific to α-amino glycated amino acids which does not act on amino acids whose ε-amino group is glycated, but has a thermal stability of 45. ℃ 90 for 10 minutes heat treatment
% Or more, it is difficult to use practically.
On the other hand, the other enzymes (f) to (k) described above have an ε-amino group and an α-amino group that act well on both glycated amino acids and have excellent thermal stability and reactivity. β
It can be used for measurement of glycated hemoglobin in which the α-amino group of the N-terminal valine in the chain is glycated, glycated albumin in which the ε-amino group of the lysine residue is glycated, and fructosamine.

The present inventors have also created a substantially pure transformed microorganism which produces an enzyme that acts on at least glycated amino acids from the above microorganisms, which has high thermostability, ε-amino groups and A method for efficiently producing fructosylamine oxidase, which has high reactivity with both saccharified amino acids having a saccharified α-amino group, has been developed (JP-A-10-201473). However, all of these enzymes have a strong action on glycated amino acids, but hardly have an action on a target glycated protein. Therefore, in order to measure a glycated protein, it is necessary to fragment a glycated protein using a chemical method or an enzymatic method to produce a glycated amino acid or a glycated peptide, and subsequently to act on the enzyme acting on at least the glycated amino acid. . As a method for fragmenting the glycated protein, an enzymatic method is preferable.

The following examples (1) to (o) show examples in which a protease is allowed to act on a glycated protein, and then the measurement is performed using an enzyme that acts on at least a glycated amino acid. (l) Pronase treatment-Fructosamine was measured using fructosylamine deglycase (Japanese Patent Laid-Open No. 6468/1994). (m) Protease treatment and detection with fructosyl amino acid oxidase (JP-A-5-192193). (n) Detection with lysine residue releasing reagent-ε-alkyl lysinase (JP-A-2-195900). (o) NAD using lysine residue free reagent -CH-OH group as hydrogen donor
And / or detected with an oxidoreductase using NADP as a hydrogen donor (Japanese Patent Application Laid-Open No. 2-195899).

The use of the above-mentioned methods (l) to (o) enables the quantification of glycated proteins in serum, but does not fragment only specific proteins. That is, a protease that does not act on globulin components but acts on albumin in plasma or hemoglobin in whole blood has not been known so far.

As an enzymatic method for measuring glycated albumin,
A method of decomposing proteins with Aspergillus-derived protease or protease type XIV and quantifying with fructosamine oxidase (WO98 / 48043) is known, but in the examples, albumin samples are used as samples, and the effect of globulin components is affected. No indication is given. Furthermore, according to the experiments of the present inventors, these proteases also acted well on globulin components, and it was not possible to measure only glycated albumin without being affected by globulin components when using serum and plasma samples. .

A method for measuring glycated albumin or glycated hemoglobin using an exo-type protease or an endo-type protease as a method for measuring an Amadori compound (WO9)
7/13872), but similarly, in the examples, albumin and hemoglobin standards were used as samples, and it was not shown that there was no effect of globulin components. Further, according to the experiments of the present inventors, proteases that act on glycated albumin and proteases that act on glycated hemoglobin that can be actually used have an action on globulin components in both exo-type and endo-type, and are not affected by globulin components. It was difficult to measure glycated albumin or glycated hemoglobin.

[0013]

SUMMARY OF THE INVENTION In measuring glycated proteins in a test solution, particularly serum and plasma, accurate measurement without being affected by various diseases requires the determination of specific proteins that vary with the disease. The effects need to be avoided.

An object of the present invention is to solve such a problem, and is intended to fragment a target protein without being affected by a specific protein present in a test solution. It is an object of the present invention to provide a method for quantifying the fragmented protein, if necessary, and a composition for fragmenting or quantifying the protein used in the method. Furthermore, specifically, the present invention fragments an objective glycoprotein without being affected by globulin components present in test liquids such as serum and plasma, and further, if necessary, fragments this glycoprotein. An object of the present invention is to provide a method for quantifying a glycoprotein and a composition for fragmenting or quantifying a protein used in the method. Another object of the present invention is to provide a protease inhibitor selective for globulin components used in these fragmentation methods, quantification methods, and compositions used in these methods.

[0015]

As described above, in order to avoid the influence of a specific protein that changes due to a disease, specificity is given to protease treatment to selectively fragment proteins other than the specific protein mainly. Alternatively, quantification may be performed using an enzyme that does not act on fragments generated from a specific protein and acts on glycated amino acids. However, it has been difficult to selectively fragment specific proteins, especially proteins other than globulin components, using various types of proteases. Furthermore, enzymes that are stable enough to withstand practical use and act on glycated amino acids are characterized by the fact that ε-amino groups and α-amino groups work well on both glycated amino acids, and fragments produced from specific proteins, especially globulin components, It was also difficult to distinguish from the protein-derived fragment.

As a result of intensive studies, the present inventors have found that the presence of certain metal ions, protein G or protein A in the protease reaction solution can selectively inhibit the action of the protease on specific proteins, especially globulin components. Further, even when an enzyme acting on a glycated amino acid is directly acted on the reaction solution, the enzymatic action is not inhibited, and the glycated protein can be measured with good reproducibility, accuracy, and convenience. The present inventors have found that a protease inhibitor selective for a specific protein other than the above can be screened, and have completed the present invention.

That is, the present invention has been made to achieve such an object, and relates to a method for selective fragmentation of a protein and a composition for protein fragmentation as described below. 1. A selective protein fragmentation method comprising adding a protease and a specific protein-selective protease inhibitor to a test solution, and fragmenting proteins other than the specific protein. 2. A composition for protein fragmentation comprising a protease and a protease inhibitor selective for a specific protein.

Examples of the test solution in the present invention include a test solution containing a globulin component as a specific protein and further containing a protein other than the globulin component, such as collected blood, serum, and plasma. . As the protease inhibitor specific to a specific protein, a protease inhibitor selective to a globulin component, for example, a metal ion, in particular, a transition metal, or an ion of a metal belonging to Group III or IV of the Periodic Table is used alone or in combination. Preferably, ions of zinc, nickel, iron, copper, cobalt, aluminum, potassium, tin, lead and the like are used alone or in combination. Further, protein G and / or protein A are used as protease inhibitors that are selective for globulin components.

Further, the present invention relates to a method for quantifying glycated protein and a composition for quantifying glycated protein as described below. 1. Quantification of glycated protein by adding a protease and a specific protein-selective protease inhibitor to a test solution, fragmenting mainly only the specific protein, and quantifying glycated amino acids and / or glycated peptides generated from the protein. Law. 2. A composition for glycated protein quantification, comprising a protease, a protease inhibitor selective for a specific protein, and an enzyme acting on glycated amino acids. The present invention also relates to a protease inhibitor selective for globulin components as described above (a protease inhibitor that specifically inhibits the action of proteases on globulin components).

In the present invention, the same test liquid as described above is used as the test liquid, and the same protein-selective protease inhibitor is used as the above-mentioned inhibitor. Also,
It is preferable that the protein is glycated albumin or glycated hemoglobin, and that an enzyme that acts on glycated amino acid, for example, fructosylamine oxidase is used to generate a glycated amino acid and / or a glycated peptide from the protein.

These are used as a method for selective fragmentation of proteins in clinical biochemical tests, compositions for fragmentation, and quantitative methods and compositions useful for measuring glycated proteins.

Hereinafter, the structure and preferred embodiments of the present invention will be described in more detail. The test solution to be measured according to the present invention may be any test solution containing a specific protein and a protein other than the specific protein, and preferably, at least a globulin component and a protein other than the globulin component. A test solution containing
More preferably, blood components such as serum, plasma, blood cells, whole blood and the like can be mentioned. The separated red blood cells can also be used as a more preferable test liquid because globulin components may be mixed depending on separation conditions and affect the measured value.

As the protease which can be used in the method for selective fragmentation of the protein of the present invention, any protease can be used as long as it effectively acts on the protein contained in the test solution. Derived protease. Specific examples are shown below.
It is merely an example and is not intended to be limiting.

[0024] Examples of animal-derived proteases, e <br/> Rasutaze (Elastase), trypsin (trypsin is), Kimoto <br/> trypsin (Chymotripsin), pepsin (Pepsin), bovine pancreatic protease, cathepsin (Cathepsin) , Calpain (Cal
pain), protease type-I, protease type-X
X (manufactured by Sigma), aminopeptidase M ( Aminopep
tidaseM) , carboxypeptidase A (Carboxypeptidas
e A) (all manufactured by Boehringer-Mannheim), pancreatin (Pancreatin: and the like is manufactured by Wako Pure Chemical Industries, Ltd.), and the like.

Examples of plant-derived proteases include kallikrein, ficin, and papain.
(Papain), Chymopapain, Bromelain
(Bromelain) (manufactured by Sigma), Papain W-40, and Bromelain F (manufactured by Amano Pharmaceutical).

Examples of microorganism-derived proteases include:
The following (1) to (14) are mentioned. (1) Bacillus (Bacillus) from genus protease; subtilisin (Subtilisin), protease - type -VIII, -IX
, -X, -XV, -XXIV, -XXVII, -XXXI (all manufactured by Sigma), thermolysin, nagase (Nagar
se) (Wako Pure Chemical Industries, Ltd.), orientase-90N, -10N
L, -22BF, -Y, -5BL, Nucleysin (from Hankyu Bio-Industry), Proleather, Protease-
N, -NL, -S "Amano" (above, Amano Pharmaceutical), GODO-BN
P, -BAP (Purchased by Joint Sake Seishin), Protin-A, -P,
Deskin, Depireisu, Biosoak, Samoases (all manufactured by Daiwa Kasei), Toyo Team NEP (Toyobo),
Neutrase, Esperase, Savinase, Durazyme, Biofeed Pro, Alcalase, NUE, Pyrase, Clear Lens Pro, Evalase, Novozyme-F
M, Novolan (above, manufactured by Novo Nordisk Bio Industries), Entilon-NBS, -SA (above, manufactured by Rakuto Kasei Kogyo), Alkaline protease GL440, Opticlean -M375 Plus, -L1000, -ALP440 (above, Kyowa Hakko Co., Ltd.), Bioprase APL-30, SP-4FG, XL-416F, AL-15F
G (all manufactured by Nagase Seikagaku Corporation), Aloase AP-10,
Protease Y (produced by Yakult Yakuhin Kogyo), Corolase-N, -7089, Veron W (produced by Higuchi Trading Company), Kirazyme P-1 (produced by Roche) and the like.

(2) Protease derived from genus Aspergillus ; protease types -XIII, -XIX, -XXI
II (all made by Sigma), Sumiteam -MP, -AP, -LP
, -FP, LPL, Enzyme P-3 (above, manufactured by Nippon Chemical Co., Ltd.), Orientase-20A, -ONS, -ON5, Tetrase S (above, manufactured by Hankyu Bio-Industry), Newase A, protease -A, -P, -M "Amano" (above, Amano Pharmaceutical Co., Ltd.), IP enzyme, morsin F, AO protease (above, Kikkoman), Protin-F, -FN, -F
A (Daiwa Kasei Co., Ltd.), Denapsin 2P, Dena Team
-SA-7, -AP, Denzyme AP (from Nagase Seikagaku Corporation), protease YP-SS, punchase -NP-2, -P
(Above, manufactured by Yakult), Sakanase (manufactured by Kaken Pharma), flavorzyme (manufactured by Novo Nordisk Bioindustry), Veron PS (manufactured by Higuchi Trading Company) and the like.

(3) Protease derived from the genus Rhizopus ; protease type XVIII (manufactured by Sigma), peptidase R, neurase F (manufactured by Amano Pharmaceutical), XP
-415 (manufactured by Nagase Seikagaku Corporation) and the like. (4) a protease derived from the genus Penicillium ;
PD enzyme (manufactured by Kikkoman) and the like. (5) Protease derived from Streptomyces sp .; protease-type XIV; also known as Pronase, -XXI
(The above, manufactured by Sigma), actinase -AS, -AF (the above,
Kaken Pharma Inc.), Tasinase (Kyowa Hakko), al
kalofilicproteinase (Toyobo) and the like.

(6) Staphylococcus
Genus-derived protease; protease type XVII (manufactured by Sigma) and the like. (7) Clostridium (Clostridium) derived from genus protease; clostripain (Clostripain), non-specific neutral protease (Nonspesificprotein
ase) (all manufactured by Sigma). (8) Lysobacter- derived protease; endoproteinase Lys-c (manufactured by Sigma) and the like.

(9) A protease derived from the genus Grifola ; metalloendopeptidase (Metalloendopeputida)
se; manufactured by Sigma). (10) Yeast- derived protease; proteinase A (Proteinase A; manufactured by Sigma), carboxypeptidase Y (carboxypeptidase Y; manufactured by Boehringer Mannheim) and the like. (11) Protein derived from the genus Tritirachium ; proteinase K (Sigma)
etc.

(12) A protease derived from the genus Thermus ; Aminopeptidase T (manufactured by Boehringer Mannheim) and the like. (13) Pseudomonas (Pseudomonus) derived from genus protease; endoproteinase Asp-N (EndoproteinaseAsp-
N; manufactured by Wako Pure Chemical Industries, Ltd.). (14) Achromobacter genus protease; LysylEndopeputida
se), achromopeptidase (all manufactured by Wako Pure Chemical Industries, Ltd.) and the like.

The specific protein-selective protease inhibitor which can be used in the protein fragmentation method of the present invention is prepared by allowing a protease to act on a test solution in the presence of a specific protein-selective protease inhibitor, and mainly Any inhibitor may be used as long as it can fragment proteins other than proteins. For example, a globulin component-selective inhibitor capable of causing a protease to act on a test solution containing a globulin component and a protein other than a globulin component in the presence of a globulin component-selective protease inhibitor, thereby mainly fragmenting proteins other than the globulin component. Is preferred. Examples thereof include metal ions, protein A, and protein G. The effects of these substances can be confirmed by the screening method for a specific protein-selective protease inhibitor described later, and other substances selected by the screening method for a specific protein-selective protease inhibitor described later. If so, any inhibitor can be used.

As the metal ion, for example, a transition metal,
Periodic table group III, group IV metal ions are preferred, transition metal ions are more preferably zinc, nickel, iron, copper, cobalt ions, group III metal ions are more preferably aluminum and gallium ions, group IV metals As the ions, tin and lead ions are more preferable. Further, in consideration of the toxicity of the metal, the formation of a precipitate due to the interaction with serum, and the like, aluminum or nickel ions are most preferable. Incidentally, these metal ions may be used alone or in combination.

The concentration of the metal ion that can be used may be any concentration as long as it is a metal ion concentration that can inhibit the protease action on a specific protein. The concentration of metal ions that can inhibit protease action
About 10 μM to 1.0 M is preferable, and about 50 μM to 100 mM is more preferable. To add metal ions, for example,
An aqueous solution of a salt having the metal ion releasing ability may be used.

[0035] Regarding the liquid composition of the reaction for selectively fragmenting proteins other than the specific protein, the pH and the concentration of the protease are determined so that the reaction proceeds efficiently in consideration of the optimum pH of the protease to be used. Then, a specific protein-selective protease inhibitor may be appropriately prepared and added so as to have an effective concentration.

For example, the protease-type-XXVI
I (manufactured by Sigma) has a strong proteolytic activity when the pH is around 7 to 10, and the reaction pH can be selected from 7 to 10. Further, the protease addition concentration may be a concentration capable of sufficiently decomposing the protein in the test solution during the reaction time actually used.

When an aluminum ion, which is a protease inhibitor selective for globulin components, is used as a protease inhibitor specific for a specific protein, glycated amino acids and / or glycated amino acids in a fragmentation solution of globulin components using fructosylamine oxidase are used. Alternatively, when a glycated peptide is detected, a protease inhibitory action selective for globulin components is confirmed at 0.05 mM or more, and further, at 0.3 mM or more, glycated amino acids and / or glycated peptides cannot be substantially detected from globulin components, and 10 mM or more. Is preferably 0.05 mM to 100 mM, and more preferably 0.3 mM to 10 mM, since cloudiness of the sample is observed in the above.

The glycated protein to be measured in the glycated protein quantification method of the present invention includes, for example, glycated albumin or glycated hemoglobin, but the glycated protein to be measured is not limited to these. Any glycated protein derived from a protein other than the specific protein, for example, any glycated protein derived from a protein other than the globulin component may be measured.

The protease that can be used in the glycated protein quantification method of the present invention effectively acts on the glycated protein contained in the test solution and effectively produces glycated amino acids and / or glycated peptides derived from the protein. Any one may be used as long as it can be used, for example, animals, plants,
Microbial proteases and the like can be mentioned. When the glycated protein to be measured is glycated albumin, the genus Bacillus and Streptomyces ( Streptomyces ) are used.
eptomyces) microorganism-derived protease is more preferable because of its large effect on human albumin, and when the glycated protein to be measured is glycated hemoglobin, Bacillus (Bacillus) , Aspergillus (Aspergillus )
s) A protease derived from the genus Streptomyces or the genus Tritirachium is more preferred because of its large effect on human hemoglobin.

The protease activity in the method for quantifying a glycated protein in the present invention is more practically determined by quantifying the resulting glycated amino acids and glycated peptides than by using a general protease activity measuring method. It is. The activity of the protease on glycated albumin in human albumin and on glycated hemoglobin in human hemoglobin was measured by the following method.

<< Method of Measuring Protease Activity on Glycated Protein >> Substrate The following commercially available standard product was used as a substrate, and the substrate concentration was set in consideration of the standard human serum concentration. The content of glycated albumin or glycated hemoglobin contained in the standard product is measured in advance by a known method. HSA substrate solution; Albumin Human; Essentially Globul
in Free; 25 mg / ml, saccharified albumin ratio = 31.9%, fructosamine value = 265 μmol / L [manufactured by Sigma; albumin concentration in the substrate solution was determined using an albumin measurement kit (albumin
II-HA Test Wako; Wako Pure Chemical Co., Ltd.), saccharified albumin ratio was measured with a saccharified albumin meter (GAA-2000; Kyoto Daiichi Kagaku), and fructosamine value was measured using a fructosamine measurement kit (Auto Hb substrate solution; Hemoglobin Human; 55 mg / ml, glycated hemoglobin ratio; HbA1c = 4.5% [Sigma; HbA1c values were measured using a glycated hemoglobin meter (Hiauto A-1C). HA-8150
; Kyoto Daiichi Kagaku). ]

<Method of preparing protease reaction sample> HSA
Substrate solution 200μl, protease solution 40μl, and 1M
Tris buffer solution (pH 8.0, 10 μl) is mixed well, reacted at 37 ° C. for 30 minutes, filtered with Ultrafree MC (fraction molecular weight: 10,000; manufactured by Millipore), and the filtrate is used as a protease reaction sample.
The same operation is performed using distilled water instead of the HSA substrate solution to obtain a blank sample. On the other hand, in the case of the Hb substrate solution, 150 μl of the substrate solution and 60 μl of the protease solution are used.
And 1M Tris buffer pH 8.0, 5μl, and mix well at 37 ℃
The reaction is carried out for -60 minutes, and the same operation as the HSA substrate solution is performed.

<Method for measuring glycated amino acid and glycated peptide in protease reaction sample><Composition of reaction solution> 50 mM Tris buffer, pH 8.0 0.02% 4-aminoantipyrine (manufactured by Wako Pure Chemical Industries;
Abbreviated as 4-AA. ) 0.02% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (manufactured by Dojin Chemical Laboratory; hereinafter, referred to as TOOS
Abbreviated. 2U / ml fructosamine oxidase (manufactured by Asahi Kasei Corporation; hereinafter, abbreviated as FOD) 5U / ml peroxidase (manufactured by Sigma, abbreviated as POD)

<Reaction Procedure> 300 μl of the above reaction solution is dispensed into a cell, incubated at 37 ° C. for 3 minutes, and photometry is performed at 555 nm (A ₀ ). Then add 30 μl of protease reaction sample.
Incubate at 37 ° C for 5 minutes and measure 555 nm (A
₁ ). The A ₀ blank subjected to the same operation using a blank sample instead of the protease reaction samples, measuring the A ₁ blank. The effect of the protease on the glycated protein is represented by the following equation when represented by a change in absorbance. _{ΔA = (A 1 -A 0)} - (A 1 blank -A ₀ Blank)

The activity was 1 μM per minute at 37 ° C. from the substrate solution.
The protease activity to produce a glycated amino acid is 1 U,
When the following HSA substrate solution is used, from the formula 1, Hb
When a substrate solution is used, the activity is calculated from Equation 2, and the obtained activities are respectively expressed as U (glycated albumin; hereinafter abbreviated as GA) and U (glycated hemoglobin; hereinafter abbreviated as GH).

[0046]

(Equation 1) ΔA: Absorbance change (Abs) 40: Protease solution volume (μl) 250: Total protease reaction volume (μl) 30: Protease reaction time (min) 30: Protease reaction solution volume (μl) 330: Saccharified amino acid measurement reaction solution Total volume (μl) 39.2: 4-AA, number of millimolecules of TOOS in millimolecules 2: Number of hydrogen peroxide necessary to produce 1 molecule of dye

[0047]

(Equation 2) ΔA: Absorbance change (Abs) 60: Protease solution volume (μl) 215: Total protease reaction volume (μl) 60: Protease reaction time (min) 30: Protease reaction solution volume (μl) 330: Saccharified amino acid measurement reaction solution Total volume (μl) 39.2: Number of millimolecules of 4-AA, TOOS, 2: 1 Number of hydrogen peroxide required to generate 1 molecule of dye

The enzyme acting on the glycated amino acid which can be used in the method for quantifying glycated protein of the present invention can be effectively used for the glycated amino acid and glycated peptide produced from the glycated protein contained in the test solution by the action of the protease. Any enzyme may be used as long as it acts and acts on a glycated amino acid capable of substantially measuring a glycated protein, but an enzyme having high stability is preferred.

Examples of preferred enzymes include Gibberella (Gib
berella) or Fructosamine oxidase derived from the genus Aspergillus (eg IFO-6365, -4242, -5710 etc.), fructosylamine deglycase derived from the genus Candida, genus Penicillium (eg IFO-4651, -6581, 7905, -5748, -7994, -4
897, -5337 etc.)
Fusarium ( for example, IFO-4468, -4471, -6
384, -7706, -9964, -9971, -31180, -9972 etc.), oxidases acting on glycated amino acids such as ketoamine oxidase from genus Acremonium (Acremonium) or genus Debryomyces (Debaryomyces). A preferred example is a recombinant fructosamine oxidase (manufactured by Asahi Kasei Kogyo Co., Ltd.) which has a sufficient activity even in the presence of a protease.

The activity of oxidase acting on glycated amino acids was measured by the following method. << Method for measuring the activity of oxidase acting on glycated amino acids >><Composition of reaction solution> 50 mM Tris-HCl buffer pH7.5 0.03% 4-AA (Wako Pure Chemical Industries) 0.02% Phenol (Wako Pure Chemical Industries, Ltd.) 4.5 U / ml POD (manufactured by Sigma) 1.0 mM α-carbobenzoxy-ε-D-fructosyl-L-lysine (synthesized and purified based on the method of Hashiba et al. Hashiba H, J. Agric. FoodChem. 24:70, 1976 or less, abbreviated as ZFL.)

Put 1 ml of the above reaction solution into a small test tube,
After pre-warming for 5 minutes, appropriately dilute enzyme solution 0.02ml
And stirred to start the reaction. Exactly 10 minutes reaction
After 2 minutes, 2 ml of 0.5% SDS was added to stop the reaction.
 Measure absorbance at 500 nm (A _s). Also as blank
Perform the same operation using 0.02 ml of distilled water instead of the enzyme solution.
And measure the absorbance (A_b). Absorbance of this enzyme action
(A_s) And blind absorbance (A_b) Absorbance difference (A_s− A_b)
Determine the enzyme activity. Separately, standard for hydrogen peroxide
The relationship between the absorbance and the generated hydrogen peroxide was investigated using a solution.
Keep it. Generates 1 μM hydrogen peroxide per minute at 37 ° C
The enzyme amount is defined as 1 U, and the calculation formula is as follows. Enzyme activity (U / ml) = (A_s− A_b) X 1.16 x enzyme dilution

The liquid composition in the glycated protein quantification method of the present invention is mainly determined by using the liquid composition of the reaction for selectively fragmenting proteins other than the specific protein or proteins other than the globulin component. Or selectively fragment proteins other than globulin,
The glycated amino acid and / or glycated peptide generated from the protein may be quantified using a known method.

As a method for quantifying the glycated amino acid and / or glycated peptide produced from the protein, the protein may be deproteinized and then quantified by coloring a dye or the like using the reducing ability of the glycated amino acid or glycated peptide. May be determined by means of chromatography, antigen-antibody reaction, enzymatic reaction or the like without deproteinization, and most preferably by using an enzyme acting on glycated amino acids.

When an enzyme that acts on glycated amino acids is used, some types of protease inhibitors that are selective for globulin components may inhibit the action of the enzyme. Is preferred. For example, the protease-type-XXVII (manufactured by Sigma) has a strong proteolytic activity when the pH is around 7 to 10, and the reaction pH can be selected from 7 to 10. Further, the protease addition concentration may be a concentration capable of sufficiently decomposing the glycated protein in the test solution during the reaction time actually used, and is 0.1 mU to 100 U.
(GA) / ml or U (GH) / ml, preferably 1.0 mU to 50 U
(GA) / ml or U (GH) / ml is more preferred. Further, when using, for example, aluminum ion as a globulin component-selective protease inhibitor, it is preferably at least 0.05 mM, more preferably at least 0.3 mM which substantially eliminates protease action on the globulin component, and 0.5 g or less of an enzyme acting on glycated amino acids. Inhibition starts at mM or higher, and turbidity at the time of addition of the test solution becomes significant from 1 mM or higher.
It is preferably from 05 mM to 1 mM, more preferably from 0.1 mM to 0.5 mM.

With respect to the liquid composition of the reaction for measuring the fragmented glycated protein using the enzyme acting on the glycated amino acid, the reaction proceeds efficiently in consideration of the optimum pH of the enzyme acting on the glycated amino acid to be used. Thus, the pH may be selected, and then the amount of the enzyme acting on the glycated amino acid may be determined. For example, in the case of recombinant fructosamine oxidase (manufactured by Asahi Kasei Corporation), the region showing 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the reaction pH can be selected from 6.5 to 10. The enzyme addition concentration may be a concentration that can sufficiently decompose the glycated protein in the test solution during the reaction time actually used, and is preferably 0.01 U to 1000 U / ml, and 0.1 U to 500 U / m.
l is more preferred, and 0.5 U and 100 U / ml are most preferred.

From the above, the protein fragmentation composition of the present invention may be prepared as containing a protease and a protease inhibitor selective for a specific protein or a protease inhibitor selective for a globulin component. A composition for quantifying a glycated protein that is not affected by a specific protein or globulin component is prepared as containing a protease, a specific protein-selective inhibitor or a globulin component-selective protease inhibitor, and an enzyme that acts on glycated amino acids. Good,
These compositions can be provided, for example, as a liquid product, a frozen product of the liquid product, or a lyophilized product.

Further, in the protein fragmentation composition and the enzyme reaction composition for quantifying glycated protein according to the present invention, for example, surfactants, salts, buffers, pH adjusters, preservatives and the like may be appropriately selected and added. good.

In appropriate additives, surfactants include polyoxyethylene alkyl ethers [eg, polyoxyethylene (10) octyl phenyl ether (Triton X-100), polyoxyethylene (23) lauryl ether (above, Nacalai Tesque, Inc.)], polyoxyethylene sorbitan fatty acid esters [; polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40),
Polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan trioleate (Tween 85) (Kanto Chemical Co., Ltd.)), polyoxyethylene glycerin fatty acid esters , Polyoxyethylene sorbite fatty acid esters, polyoxyethylene alkylphenylformaldehyde condensate, polyoxyethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine・ Fatty acid amides, polyoxyethylene alkyl ether phosphoric acid / phosphates, polyoxyethylene alkyl ether sulfates, polyglycerin fatty acid ester , Glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, N acyl amino acid salts, alkyl ether carboxylate salts, alkyl phosphate, N acyl taurate, sulfonate, alkyl sulfate, betaine acetate type Amphoteric surfactant, imidazoline-type amphoteric surfactant, lecithin derivative (above, manufactured by Nikko Chemicals Co., Ltd.), ADEKATOL 720N, ADEKATOL B-795, ADEKATOL SO
-120, Adecanol B-795 (from Asahi Denka Kogyo), polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctyl phenyl ether, polypropylene glycol, polyvinyl alcohol, Triton X-305, Triton X-114, Triton X 0.01 to 10%, preferably 0.05 to 5%, such as -405, Triton WR-1339 (all manufactured by Nacalai Tesque), and various metal salts such as lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, 1 mM to 5 M, preferably 10 mM to 1 M, such as zinc chloride and calcium chloride, various buffers,
For example, 10 mM to 2 M, preferably 20 mM, such as Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good's buffer, etc.
~ 1M, various preservatives, e.g. 0.01-10 of sodium azide
%, Preferably 0.05 to 1%.

In the method for quantifying a glycated protein according to the present invention, the enzymatic action acting on the glycated amino acid is detected, for example, by using the amount of change in the coenzyme when dehydrogenase is used, for example, by using NAD as the coenzyme. The amount of change produced was directly quantified using a known technique, such as measuring reduced NAD, which is a reduced coenzyme, at a wavelength near its maximum absorption wavelength of 340 nm using a colorimeter, or was directly quantified. Various reduced diaphorases or electron carriers such as phenazine methosulfate (hereinafter abbreviated as PMS), methoxy PMS, dimethylaminobenzophenoxazinium chloride (Meldra Blue) and nitrotetrazolium, WST-1, WST- Indirectly quantified using reducing color reagents such as various tetrazolium salts represented by 8 (above). And it may also directly by a known method other than this may be measured indirectly.

For example, when oxidase is used, it is preferable to measure the amount of consumed oxygen or the amount of reaction products. When fructosamine oxidase is used as a reaction product, for example, hydrogen peroxide and glucosone are produced by the reaction, and both hydrogen peroxide and glucosone can be measured directly and indirectly by a known method.

The amount of the above hydrogen peroxide may be determined by, for example, producing a dye or the like using peroxidase or the like and determining the color, emission, or fluorescence, or may be determined by an electrochemical method. An aldehyde may be generated from an alcohol using the method described above, and the amount of the generated aldehyde may be determined. The color development system of hydrogen peroxide generates a dye by oxidative condensation between a coupler such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (MBTH) and a chromogen such as phenol in the presence of peroxidase. And a leuco-type reagent that directly oxidizes and develops color in the presence of peroxidase.

As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used, and specific examples thereof include N, N-dimethylaniline,
N, N-diethylaniline, 2,4-dichlorophenol, N-
Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl
-3,5 dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAO
S), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m
-Toluidine (TOOS), N-ethyl-N-sulfopropyl-m-
Anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3, 5-dimethoxyaniline (DAPS), N-sulfopropyl-3, 5-dimethoxyaniline (HDAPS ), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2 -Hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS) And the like).

Specific examples of leuco-type reagents include o-
Dianisidine, o-tolidine, 3,3 diaminobenzidine, 3,3,5,5-tetramethylbenzidine; and N- (carboxymethylaminocarbonyl)-
4, 4-bis (dimethylamino) biphenylamine (DA6
4), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67);

Further, in the fluorescence method, compounds emitting fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivatives, etc., are used. In the chemiluminescence method, luminol, lucigenin is used as a catalyst. , Isoluminol, pyrogallol and the like can be used. As a method of producing aldehyde from alcohol using catalase or the like and quantifying the generated aldehyde, a method using the Hunch reaction,
Examples thereof include a method of developing a color by a condensation reaction with MBTH, a method of using aldehyde dehydrogenase, and the like.
Glucosone can be quantified using a known aldose reagent such as diphenylamine.

When measuring hydrogen peroxide using an electrode, the electrode is not particularly limited as long as it is a material capable of exchanging electrons with hydrogen peroxide. For example, platinum, gold, silver, etc. Known methods such as amperometry, potentiometry, and coulometry can be used as the electrode measurement method.Furthermore, the oxidation between the oxidase or substrate and the electrode can be carried out by interposing an electron carrier in the reaction between the electrode and the electrode. Alternatively, the reduction current or the amount of electricity may be measured. Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. Further, an oxidation or reduction current or an amount of electricity obtained by interposing an electron carrier between hydrogen peroxide generated by the oxidase reaction and the electrode may be measured.

In order to quantify glycated protein in a test solution by the thus prepared composition for quantifying glycated protein of the present invention, the composition for quantifying glycated protein is added to the composition for quantifying glycated protein.
When a rate assay is carried out by adding 0.001 to 0.5 ml and reacting at a temperature of 37 ° C., a few minutes to tens of minutes between two points after a certain time after the start of the reaction, for example, 3 minutes and 4 minutes The amount of altered coenzyme, dissolved oxygen, hydrogen peroxide or other products in one minute after one minute or every five minutes after eight minutes may be measured directly or indirectly by the above method. In the case of the point assay, the amounts of the changed coenzyme, dissolved oxygen, hydrogen peroxide or other products after a certain time from the start of the reaction may be measured in the same manner. In this case, the amount of glycated protein in the test solution can be determined by comparing the change in absorbance or the like when measured using a glycated protein of known concentration.

<< Screening Method for Specific Protein-Selective Protease Inhibitor >> To screen for a specific protein-selective protease inhibitor, a specific protein of interest, for example, a human globulin component, other than a specific protein of interest, Proteins, for example, human albumin and human hemoglobin, each dissolved alone, were reacted with the protease in the presence and absence of a candidate substance, and subsequently or simultaneously produced using an enzyme that acts on glycated amino acids. The glycated amino acid or glycated peptide is quantified, and the quantitative value of the specific protein, for example, the quantitative value of the human globulin component is significantly reduced as compared with the change in the quantitative value of the protein other than the specific protein of interest, for example, human albumin or human hemoglobin. What you do It may be selected.

The specific protein of interest in the present screening method, for example, human globulin component, and proteins other than the specific protein of interest, for example, human albumin and human hemoglobin, may be used. Is preferably free from proteins other than the specific protein of interest, and proteins other than the specific protein are preferably free from specific proteins.

For example, when the specific protein is a human globulin component and the proteins other than the specific protein are human albumin and human hemoglobin, the human globulin component preferably does not contain albumin and hemoglobin. Human hemoglobin preferably does not contain a human globulin component. The specific protein and the protein other than the specific protein may be used at any concentration.For example, when human albumin or human hemoglobin is used as the protein other than the specific protein, the prepared concentration of human albumin is 1%. ~ 200mg / ml is preferred, 10 ~ 100mg / ml
More preferably, the concentration is 35 to 55 mg / ml for a healthy human, and the prepared concentration of human hemoglobin is preferably 1 to 500 mg / ml, more preferably 10 to 200 mg / ml.

When the specific protein of interest is a globulin component, the prepared concentration of the human globulin component is 0.1 to 200 m
g / ml, preferably 0.5-100 mg / ml,
In the case of Fraction II and III, 5 to 25 mg / ml is most preferable, and in the case of chon Fraction IV, 1 to 15 mg / ml is most preferable. The human globulin component is a generic term for components other than albumin in serum proteins, and therefore the concentration may be appropriately changed according to the type of human globulin used since various types of production methods exist.

The substrate solution and the protease solution prepared as described above, preferably from 0.01 mU to 100 U (GA) / ml or
U (GH) / ml is mixed at a ratio of about 1: 0.01 to 1: 100 in the presence and absence of the candidate substance, preferably at a pH and temperature at which the protease can sufficiently act on the substrate solution. The reaction may be performed for minutes to 12 hours, more preferably for 5 minutes to 2 hours, to fragment the substrate solution.

In order to quantify the glycated amino acid or glycated peptide in the protease-treated substrate solution, the enzyme which subsequently or simultaneously acts on the at least glycated amino acid is preferably 0.01 to 000 U / ml, more preferably
The reaction is carried out at a concentration of 0.1 to 200 U / ml and at a pH and a temperature at which the enzyme acting on the saccharified amino acid can sufficiently react with the saccharified amino acids, preferably for 1 second to 12 hours, more preferably for 1 minute to 2 hours. The amount of the changed coenzyme, dissolved oxygen, hydrogen peroxide or other products may be measured directly or indirectly by the above method. The same operation is performed by using distilled water instead of the substrate solution, and subtracted from the measured value of the substrate solution as a reagent blank.

Finally, the quantitative values obtained from the respective candidate substances are compared, and when the candidate substances are added, the quantitative value of the specific protein is determined to be within the range in which human albumin or human hemoglobin can be quantified sufficiently. A candidate substance which is reduced to 50% to 75%, preferably 76 to 99%, and most preferably substantially ineffective to the quantitative value of hemoglobin is selected as a specific protein-selective protease inhibitor.

The specific protein selective protease inhibitor of the present invention can be provided in the form of a powder, granules, an aqueous solution or the like.

[0075]

Next, embodiments of the present invention will be described in detail, but the present invention is not limited thereto.

REFERENCE EXAMPLE 1 For the purpose of screening for a protease that does not act on the globulin component, glycated amino acids or glycated peptides produced by allowing the protease to act on albumin, the globulin component and hemoglobin were quantified using fructosamine oxidase.

<Substrate solution>1; HSA substrate solution (same as that used in the method for measuring the activity of protease on glycated protein) 2; G-II, III substrate solution, fructosamine value 48 μmol /
L (Globulins Human Cohn Fraction II, III; 16.9m
g / ml (manufactured by Sigma)] 3; G-IV substrate solution, fructosamine value 26 μmol / L [Gl
obulins Human Cohn Fraction IV; 6 mg / ml (Sigma)] 4; GI substrate solution, fructosamine value 77 μmol / L [globinin I; glovenin-I; immunoglobulin preparation (Takeda Pharmaceutical)] 5; Hb substrate Solution (same as that used in the method for measuring the activity of proteases on glycated proteins.) The fructosamine value of the substrate solution is determined by a fructosamine measurement kit (Auto Wako Fructosamine (manufactured by Wako Pure Chemical Industries, Ltd.)
Was measured.

<Preparation of Protease Reaction Sample> 200 μl of a substrate solution other than Hb, 100 mg / ml of a protease solution (if this concentration cannot be prepared, the concentration is as high as possible, and if the solution is the same, 40 μl) and 1 M Tris Mix 10 μl of buffer (pH 8) well, and react at 37 ° C for 30 minutes.
The solution was filtered through a membrane (Ultrafree MC; manufactured by Millipore), and the filtrate was used as a protease reaction sample. The same operation was performed using distilled water instead of the substrate to obtain a blank sample.

On the other hand, in the case of the Hb substrate solution, the substrate solution 150
μl, protease solution 200 mg / ml (if this concentration cannot be prepared, the concentration is as high as possible, and the solution is the same as it is) with 60 μl and 1 M Tris buffer (pH 8) 5 μl
l, mix well, and react at 37 ° C for 60 minutes.
(Fraction molecular weight: 10,000; manufactured by Millipore), and the filtrate was used as a protease reaction sample. The same operation was performed using distilled water instead of the substrate to obtain a blank sample.

<Measurement of glycated amino acid and glycated peptide in protease reaction sample><Composition of reaction solution> 50 mM Tris buffer pH 8.0 0.02% 4-AA (manufactured by Wako Pure Chemical Industries) 0.02% TOOS (Dojin Chemical Laboratory Co., Ltd.) 2U / ml FOD (made by Asahi Chemical Industry) 5U / ml POD (made by Sigma)

<Reaction procedure> The above reaction solution for measuring glycated amino acids
Dispense 300 μl into the cell, incubate at 37 ° C. for 3 minutes, and measure 555 nm (A ₀ ). Subsequently, 30 μl of a protease reaction sample was added, and the mixture was incubated at 37 ° C. for 5 minutes.
Measure 5 nm (A ₁ ). The A ₀ blank subjected to the same operation using a blank sample instead of the protease reaction samples, measuring the A ₁ blank. The effect of the protease on the glycated protein is represented by the following equation when represented by a change in absorbance. ΔA = (A ₁ −A ₀ ) − (A ₁ blank−A ₀ blank) Table 1 shows the action (ΔA) of representative proteases at pH 8 on albumin, globulin, and hemoglobin.

[0081]

[Table 1]

Since the activity of glycated proteins in the G-IV substrate solution was small or zero for all proteases, the measured values of the G-II and -III substrate solutions and the measured values of the GI substrate solution were almost the same. Therefore, Table 1 shows only the results of the GI substrate solution for the globulin component. As can be seen from Table 1, proteases derived from the genus Aspergillus and protease type XIV also act well on glycated globulin in globulin components. Furthermore, both endo-type and exo-type proteases acting on glycated albumin in albumin and glycated hemoglobin in hemoglobin showed an effect on glycated globulin in globulin components. Therefore, it was considered that when measuring glycated albumin in serum or plasma or glycated blood in whole blood and blood cells, the influence of the globulin component cannot be avoided only by selecting the protease. Similarly, it was thought that the effect of the specific protein could not be avoided by only selecting the protease.

[0083]

Example 1 <Screening of protease inhibitor selective for globulin component> Using the orientase 22BF (manufactured by Hankyu Bio-Industry Co.), which has a high effect on the HSA substrate solution, the various globulins were prepared based on the HSA substrate solution. Substances that reduce the protease action on the substrate solution were screened. Substrate solution (HSA substrate solution, G-
I substrate solution) 100 μl, orientase 22BF (100 mg / ml
About 9 mU (GA) / ml) 20 μl, 1 M Tris buffer (pH 8.0) 5
μl and 10 μl of a metal 0.10M to 0.01M, 10 mg / ml of protein A or protein G (manufactured by Sigma) and mixed well at 37 ° C. for 30 minutes to react with Ultrafree MC (fraction molecular weight 1).
And the filtrate is used as a protease reaction sample. The same operation is performed using distilled water in place of the substrate to obtain a blank sample, and a control sample to which distilled water is added in place of metal ion, protein A or protein G is used.

The metal solution to be added is KCl, NaCl, Na
F, NaN_Three, AgCl, LiCl, NH_FourCl, MgCl _Two, PbCl_Two, NiCl_Two, Mn
Cl_Two, SnCl_Two, GaCl_Two, GeCl_Two, CaCl_Two; 100mM, ZnCl_Two, CoC
l_Two, Na_FiveMoO_Four, FeSO_Four; 50mM, ZnSO_Four, Barium acetate, CuCl_Two,
 CuSO_Four, Fe_Two(SO_Four)_Three, AlCl_ThreeAt 10 mM (Wako Pure Chemical Industries, Ltd.)
Prepared.

<Reaction liquid> The same as in Reference example 1. <Reaction procedure> The same procedure as in Reference Example 1 was performed. Absorbance change ΔA (HSA) obtained from HSA substrate solution and
The ratio of absorbance change ΔA (GI) obtained from the GI substrate solution; ΔA (GI) / ΔA (HSA) X100 was compared in the presence and absence of various candidate substances (control sample). Table 2 shows the results.

[0086]

[Table 2]

As can be seen from Table 2, zinc, cobalt,
Lead, nickel, tin, gallium, copper, iron, aluminum ions, protein A and protein G have been confirmed to have the effect of inhibiting the protease action on glycated globulin in the globulin component, and these globulin component-selective protease inhibitors and proteases It has been clarified that proteins other than globulin can be mainly fragmented by using. It is also clear that other similar globulin component-selective protease inhibitors can be screened by using the same method. Furthermore, globulin component-selective protease inhibitors under different proteases and reaction conditions such as pH, temperature, etc. Screening was also possible.

Further, by using an Hb substrate solution instead of an HSA substrate solution, it is possible to screen for a globulin component-selective protease inhibitor which can be used in the measurement of glycated hemoglobin, and to use a specific target instead of the globulin component substrate solution. It was also found that the use of a protein substrate solution enables screening for a specific protein-selective inhibitor.

[0089]

Example 2 <Selective protease inhibitory effect of globulin component of lead ion><R-1> 10 mM Tris buffer pH 8.5 8 mM 4-AA (manufactured by Wako Pure Chemical Industries) 15 U / ml POD (manufactured by Sigma) 200 mg / ml protease; about 8 mU (GA) / ml 0.3 to 1.0 mM PbCl ₂ protease concentration is orientase 22BF; 0.3 mM (manufactured by Hankyu Bioindustry), protease-type-VIII; 0.4 mM, protease-type-XIV ; 1.0mM,
Protease-type-XXVII; 0.3 mM (all manufactured by Sigma) was used.

<R-2> 150 mM Tris buffer pH 8.5 12 mM TOOS (manufactured by Dojindo Laboratories, Japan) 24 U / ml FOD (manufactured by Asahi Kasei Corporation, Japan) <Substrate solution> Same substrate as in Reference Example 1 The solution was used. HS
Use A, G-II, III, G-IV, GI substrate solution.

<Operation> Transfer 270 μl of the above R-1 into a cell, add 9 μl of the substrate solution, react at 37 ° C. for 5 minutes, and react at 570 nm.
To measure the A _0. Followed by measuring the A ₁ of 570nm performs added 37 ° C. -5 minutes color development reaction R-2,90μl. Similarly, a blank was measured using distilled water instead of the sample, and Δ
A = (A ₁ −A ₀ ) − (A ₁ blank−A ₀ blank) was calculated. In addition, R-1 to which no lead ion was added was prepared and measured in the same manner as a control. Table 3 shows the results.

[0092]

[Table 3]

As can be seen from Table 3, orientase 22B
F, Protease-Type-VIII, Protease-Type-XIV, Protease-Type-XXVII All protease activities on glycated globulin in G-II, III, G-IV, GI substrate solutions in the presence of lead ions are reduced. On the other hand, the effect on glycated albumin in the HSA substrate solution was maintained. From this, it became clear that the globulin component-selective protease inhibitor screened in the present invention is effective regardless of the type of protease. It is also possible for various types of proteases to fragment albumin other than mainly globulin components by adding protease and protease inhibitor selective for globulin components to serum. It was revealed that glycated albumin can be measured without being affected by globulin components by measuring peptides using at least an enzyme acting on glycated amino acids. Further, it was found that the use of the present invention can also avoid the influence of globulin components when measuring glycated hemoglobin.

[0094]

Example 3 (Dilution linearity of saccharified albumin) <R-1> 10 mM Tris buffer pH 8.5 8 mM 4-AA (manufactured by Wako Pure Chemical Industries) 15 U / ml POD (manufactured by Sigma) 200 mg / ml orientase 22BF (manufactured by Hankyu Bio-Industry, Japan, 8 mU (GA) / ml) 0.3 mM AlCl3 <R-2> Same as in Example 2 <Substrate solution> The same substrate solution as in Reference example 1 was used. Use HSA, GI and G-IV substrate solutions.

<Operation> 0.0, 0.5 of HSA, G-IV and GI
, 1.0, 1.5, and 2.0-fold concentration samples were prepared and the dilution linearity was confirmed. The operation is the same as in Example 2. However, the HSA 1.0-fold concentration was measured 10 times and the CV value was calculated. The result is shown in FIG.
As can be seen from FIG. 1, no change in absorbance was obtained even when the concentrations of both the GI and G-IV substrate solutions were changed. On the other hand, HSA
It is clear that glycated albumin was quantified substantially without being affected by the globulin component, with the substrate solution having good linearity depending on the concentration. At HSA 1.0 times concentration
Good reproducibility was confirmed with a CV value of 1.2%, and it was clear that glycated albumin was measured selectively, with good sensitivity, and with good reproducibility in a reaction time of 10 minutes using the measurement system of the present invention. It became.

[0096]

Example 4 (Dilution linearity of glycated hemoglobin) <R-1> 77 mM Tris buffer pH 8.0 330 mg / ml protease type XIV (manufactured by Sigma,
1.3mU (Hb) / ml) 3.3mM AlCl 3 <R-2> Same as Example 1. <Substrate Solution> The same substrate solution as in Reference Example 1 was used. Use Hb, G-IV, GI substrate solution.

<Operation> 0.0, 0.5, Hb, G-IV and GI
Samples of 1.0, 1.5 and 2.0 times concentration were prepared and the linearity was confirmed. 65 μl of the above protease reaction reagent and substrate solution 150
μl was mixed well, reacted at 37 ° C. for 60 minutes, filtered through Ultrafree MC (fraction molecular weight: 10,000; manufactured by Millipore), and the filtrate was used as a protease reaction sample. The same operation was performed using distilled water instead of the substrate solution to obtain a blank sample. Next, 270 μl of R-2 was placed in the cell and the absorbance at 555 nm was measured.
To measure the A _0. Was further measured A ₁ of 555 nm subjected to 30μl added 37 ° C. -5 minutes coloring reaction protease reaction samples. Similarly, a blank sample is measured, and (A ₁ -A ₀ )-(A ₁
It was calculated blank -A ₀ blank). Further, a 1.0-fold concentration of Hb was measured ten times and the CV value was calculated. The result is shown in FIG.

As can be seen from FIG. 2, no change in absorbance was obtained even when the substrate concentration was changed for both GI and G-IV. On the other hand, it was clarified that glycated hemoglobin was quantified without substantially being affected by the globulin component, with favorable linearity being obtained depending on the concentration of Hb. In addition, at a Hb 1.0-fold concentration, a good reproducibility was confirmed with a CV value of 2.0%.
It was revealed that glycated hemoglobin was measured with good sensitivity and good reproducibility using the measurement system of the present invention.

[0099]

Example 5 (Linearity of Saccharified Albumin) <R-1> Same as Example 3. <R-2> Same as Example 3. <Substrate solution> Serum A) Diabetic patient GA% high serum value GA% = 32.9% Albumin concentration 4.3g / dl Serum B) Healthy person serum GA% = 16.4% Albumin concentration 4.1g / dl Serum A, B above 10: 0 , 8: 2, 6: 4, 4: 6, 2: 8, 0:10 to obtain a sample.

[0100] measuring the <operation> The R-1, A ₀ of 570 nm was added to the reaction 37 ° C. -5 min sample 27μl taken up in cell 810Myueru. Then R-2, was measured A ₁ of 570nm performs added 37 ° C. -5 minutes color development reaction 270Myueru. Similarly, a blank was measured using distilled water instead of the sample, and (A _1-
A ₀ ) − (A ₁ blank−A ₀ blank) was calculated. The results are shown in FIG.

As can be seen from FIG. 3, good linearity was obtained in samples having different saccharified albumin ratios while keeping the albumin concentration constant. Therefore, it was found that the glycated protein quantification method of the present invention can quantitatively detect actual glycated albumin in serum and plasma. In addition, since similar linearity was confirmed using a hemoglobin substrate solution prepared by lysing erythrocytes instead of serum, it was also confirmed that the method for quantifying glycated protein according to the present invention enables quantitative detection of glycated hemoglobin. Was.

[0102]

Example 6 (Correlation between saccharified albumin HPLC method and enzymatic method (the present invention)) <R-1> Same as Example 3. <R-2> Same as Example 3. <Substrate solution> 14 diabetic sera 25 healthy sera 25

<Operation> The operation is the same as in the second embodiment. The correlation between the enzyme method according to the present invention and a known HPLC method was confirmed using 14 sera of diabetic patients. The HPLC method was used to measure saccharified albumin (GAA-2000; manufactured by Kyoto Daiichi Kagaku).
The saccharified albumin ratio was measured at. The change in the absorbance obtained from the quantification method according to the present invention shows a very good correlation with the saccharified albumin ratio and the correlation coefficient r = 0.984, and it is clear that the quantification method according to the present invention accurately quantifies saccharified albumin. It became.

[Brief description of the drawings]

FIG. 1 shows the quantification curves and reproducibility of HSA, G-IV, and GI based on Example 3 of the present invention.

FIG. 2 is a quantitative curve and reproducibility of Hb, G-IV, and GI based on Example 4 of the present invention.

FIG. 3 is a quantification curve of glycated albumin based on Example 5 of the present invention.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA13 AA25 BA01 BA11 BA13 BB60 CA25 CA26 DA35 DA36 DA37 DA38 DA44 DA45 FA11 FB01 GC10 4B050 LL03 4B063 QA01 QA19 QQ79 QR03 QR16 QR41 QR48 QR50

Claims (24)

[Claims] 1. A method for selective protein fragmentation, comprising adding a protease and a protease inhibitor selective for a specific protein to a test solution to fragment mainly proteins other than the specific protein. 2. A method comprising adding a protease inhibitor selective for a protease and a globulin component to a test solution containing a globulin component and a protein other than the globulin component to fragment mainly the protein other than the globulin component. Method for selective fragmentation of a protein. 3. A method of adding a protease and a specific protein-selective protease inhibitor to a test solution, fragmenting only a specific protein, and quantifying glycated amino acids and / or glycated peptides generated from the protein. A method for quantifying a glycated protein, which is a feature. 4. A protease and a globulin component-selective protease inhibitor are added to a test solution containing a globulin component and a protein other than the globulin component to fragment mainly the protein other than the globulin component and produce the protein from the protein. A method for quantifying a glycated protein, comprising quantifying the glycated amino acid and / or glycated peptide. 5. The method according to claim 3, wherein the quantification of the glycated amino acid and / or glycated peptide produced is performed using an enzyme that acts on the glycated amino acid. 6. The method according to claim 5, wherein the enzyme acting on the glycated amino acid is fructosylamine oxidase. 7. The method according to claim 2, wherein the globulin component-selective protease inhibitor is a metal ion. 8. The method according to claim 7, wherein the metal ion is a transition metal, a group III or group IV metal ion alone or in combination. 9. The transition metal according to claim 8, wherein the metal ion of the group III or IV of the periodic table is a single ion of zinc, nickel, iron, copper, cobalt, aluminum, gallium, tin or lead or a combination thereof. Method. 10. The method according to claim 2, wherein the globulin component-selective protease inhibitor is protein G and / or protein A. 11. The method according to claim 3, wherein the protein is glycated albumin or glycated hemoglobin. 12. A composition for protein fragmentation comprising a protease and a protease inhibitor selective for a specific protein. 13. A protein fragmentation composition comprising a protease and a protease inhibitor that is selective for a globulin component. 14. A composition for quantifying a glycated protein, comprising a protease, a protease inhibitor selective for a specific protein, and an enzyme acting on a glycated amino acid. 15. A composition for quantifying a glycated protein, comprising a protease, a protease inhibitor selective for a globulin component, and an enzyme acting on a glycated amino acid. 16. The method according to claim 14, wherein the enzyme acting on the glycated amino acid is fructosylamine oxidase.
A composition as described. 17. The composition according to claim 13, wherein the protease inhibitor selective for globulin component is a metal ion. 18. The metal ion is a transition metal, Periodic Table III.
18. The composition according to claim 17, which is a metal ion of Group IV or Group IV alone or in combination. 19. The transition metal according to claim 18, wherein the metal ion of the group III or IV of the periodic table is zinc ion, nickel, iron, copper, cobalt, aluminum, gallium, tin or lead ion alone or a combination thereof. Composition. 20. The composition according to claim 13, wherein the globulin component-selective protease inhibitor is protein G and / or protein A. 21. The composition according to claim 14, wherein the protein is glycated albumin or glycated hemoglobin. 22. A globulin component-selective protease inhibitor. 23. The protease inhibitor according to claim 22, which is a metal ion. 24. Protein G and / or Protein A
23. The protease inhibitor according to claim 22, which is