JP2002315600A - Method for determining n-terminal saccharified protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating an N-terminal saccharified amino acid or peptide from a test solution containing an N-terminal saccharified peptide or protein, and measuring the separated amino acid or peptide, and to provide a measuring composition. SOLUTION: By acting deblocking aminopeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase, N-acylaminoacyl-peptide hydrolase or hemicellulase on a test solution containing an N-terminal saccharified peptide or protein, the N-terminal saccharified amino acid or peptide is separated, and the formed saccharified amino acid and/or saccharified peptide is determined. This method can perform the determination accurately, simply and speedily, and is useful for determining glycosylated hemoglobin in a blood sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for releasing a glycated amino acid or peptide at the N-terminus from a test solution containing a peptide or protein having a glycated N-terminus using a protease, And a composition used for this determination. According to the present invention, particularly, glycated hemoglobin in a blood sample can be accurately, simply and quickly quantified.

[0002]

2. Description of the Related Art Measurement of glycated protein is very important in diagnosing and managing diabetes. In particular, recent studies have shown that if glycated hemoglobin in blood is controlled to a value of 7% or less, the onset and progress of complications will occur. It has been proved that the risk rate of the disease can be significantly reduced, and is frequently used as an indispensable indicator in clinical practice.

[0003] As a method for quantifying glycated hemoglobin, an electrophoresis method, an ion exchange chromatography method, an affinity chromatography method, an immunization method and an enzyme method are generally known. However, the electrophoresis method and the chromatography method require expensive dedicated equipment, and the processing speed is slow, so that they are not suitable for a clinical test in which a large number of samples are processed. In addition, the immunization method has been rapidly spread in recent years because the analysis method is relatively simple and requires only a short time.However, since the antigen-antibody reaction is used, accuracy is not necessarily high in terms of reproducibility and influence of coexisting substances. There is a problem that can not be said.

[0004] As described above, an expensive dedicated apparatus is unnecessary, the processing speed is high, and the method for quantifying glycated hemoglobin with high accuracy, convenience, and low cost includes an enzymatic method. The definition of glycated hemoglobin is that the N-terminal amino acid of the hemoglobin β chain is glycated hemoglobin. Therefore, when measuring glycated hemoglobin using an enzyme, the N-terminal amino acid is converted from the glycated hemoglobin to the N-terminal glycated amino acid. Alternatively, a peptide containing an N-terminal glycated amino acid must be released and quantified. Generally, the glycation site of hemoglobin is about 60% α-amino group and about 60% ε-amino group.
40% saccharified, and N
Proteins whose ends are glycated and plugged are resistant to proteases. Examples of the release of an N-terminal glycated amino acid or a peptide containing an N-terminal glycated amino acid from hemoglobin in which the N-terminal amino acid is glycated include the following (a)
~ (H) are known.

(A) 8M urea is added to glycohemoglobin
After 20 minutes boiling denaturation, trypsinization, Penicillium (Penic
illium ) Fructosyl amino acid oxidase (FAO
Method of quantification in D) (JP-A-8-336386). (b) A method of treating glycohemoglobin with protease and quantifying it with FAOD derived from Aspergillus genus (JP-A-10-33177, JP-A-10-33180). (c) A method of performing protease treatment and detecting by FAOD (JP-A-5-192193). (d) A method for measuring glycated hemoglobin using endo-type and exo-type proteases (WO97 / 13872).

(E) A method in which a peptide or protein in which the N-terminal valine is fructosylated is treated with an enzyme using serine carboxypeptidase (JP-A-2001-57897). (f) a protease capable of cleaving the carboxyl group side of the third leucine from the N-terminus of the β-chain of hemoglobin A1c with a protease capable of cleaving the histidylleucine from the resulting fructosyl valyl histidyl leucine; A method for measuring hemoglobin A1c to be treated (JP-A-2000-300294). (g) a method of using a Corynebacterium (Corynebacterium) genus and Pseudomonas (Pseudomonas) from the genus novel enzymes of the α-amino group from a glycated protein to liberate is glycated amino acid, a glycated amino acid is liberated, to quantify this ( W
O00 / 50579). (h) Sphingobacterium that releases glycated amino acids from α-amino groups from glycated proteins
Genus, Sphingomonas (Sphingomonas) , Comamonas (C
omamonas) genus, Muko (Mucor) genus and Penicillium (Pen
a method of releasing glycated amino acids using a novel enzyme derived from the genus icillium ) and quantifying the same (WO00 / 61732).

In the above methods (a) to (d), since the ε and α amino groups are measured using FAOD which acts well on both saccharified saccharified amino acids, the α amino acid which is a definition of saccharified hemoglobin is used. It is not clear whether the group measures only glycated glycated amino acids.

Further, according to the experiments of the present inventors, (a)
The protease used in (d) is a synthetic substrate capable of confirming that the α-amino group acts on the glycated protein, for example, glycated valylparanitroanilide (hereinafter referred to as FValpN).
A). Therefore, the known method for measuring saccharified hemoglobin is not a saccharified α-amino group at the N-terminus which should be originally measured, but a saccharified ε.
It is considered that the amino group was measured.

On the other hand, by using the above methods (e) to (h), it is possible to measure the glycation site at the N-terminal of glycated hemoglobin. However, in the methods (e) and (f), a peptide containing a glycated amino acid at the N-terminus is cut out once with an endoprotease and then degraded to a glycated amino acid using carboxypeptidase. Protease is required.

The above (g) and (h) are for Corynebacterium
M(Corynebacterium) Genus, Pseudomonas(Pseudomona
sGenus, sphingobacterium(Sphingobacterium)
Genus, Sphingomonas(Sphingomonas)Genus, Comamonas(C
omamonas) Genus, Muko(Mucor) Genus and penicillium(Pen
icillium) Limited to enzymes from the genus. So far
Deblocking Aminop
eptidase), dipeptidyl aminopeptidase (Dipepti
dyl Aminopeptidase), leucine aminopeptidase
(Leucine Aminopeptidase), N-acylaminoacyl-
Peptide hydrolase (N-Acylaminoacyl-peptide h
ydrolase) or hemicellulase
To release the N-terminal glycated amino acid or glycated peptide.
And quantification of glycated peptide or glycated protein
No method has been reported.

[0011]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of solving the problems in quantifying such a peptide or a protein having a glycated N-terminal. That is, an object of the present invention is to provide a test solution containing a peptide or protein in which the N-terminus is glycated,
An object of the present invention is to provide a method for releasing a glycated amino acid or peptide at the N-terminus and quantifying the glycated amino acid or peptide, and a composition for the quantification. A further subject of the present invention is clinical biochemical testing,
In particular, it is an object of the present invention to provide a quantification method and a quantification composition useful for measuring glycated hemoglobin in blood in large quantities at low cost.

[0012]

Accordingly, the present inventors have synthesized FValpNA and the like in order to search for a protease that efficiently degrades the N-terminal of glycated hemoglobin. However, surprisingly, the protease which has been confirmed to have an effect on glycated hemoglobin could not confirm the activity of decomposing this synthetic substrate. Therefore, it was considered that the coloring by FAOD from glycated hemoglobin, which has been clarified so far, is not due to the glycated valine at the N-terminus but measures the sugar bound to the ε-amino group.

Thus, the present inventors have screened proteases from nature using a synthetic substrate such as FValpNA and the like. As a result, the protease used for deblocking of proteins in which the N-terminus of proteins or peptides is blocked, dipeptidylamino Peptidase (Dipeptid
yl Aminopeptidase), leucine aminopeptidase (L
eucine Aminopeptidase or hemicellulase (Hemi
celullase) was found to have the activity of degrading FValpNA.

However, proteases obtained from bacteria and fungi require complicated culturing and purification steps, and it is difficult to supply them in large quantities at low cost as reagents to clinical sites.
inopeptidase), dipeptidyl aminopeptidase (Di
peptidyl Aminopeptidase), leucine aminopeptidase (Leucine Aminopeptidase), N-acylaminoacyl-peptide hydrolase (N-Acylaminoacyl-peptid)
e hydrolase) or hemicellulase could be supplied in large quantities at low cost.

Further, the present inventors have found that glycated hemoglobin can be measured with good reproducibility, accuracy and convenience by using an enzyme that acts on glycated amino acids such as FAOD on these protease reaction solutions, thereby completing the present invention.

That is, according to the present invention, a test solution containing a peptide or protein having a glycated N-terminal is added to a deblocking aminopeptidase (Deblocking Aminopeptidase).
, Dipeptidyl aminopeptidase (Dipeptidyl Ami
nopeptidase), leucine aminopeptidase (Leucine
Aminopeptidase), N-Acylaminoacyl-peptide hydrolases
e) or hemicellulase (Hemicelullase),
The present invention relates to a method for releasing a glycated amino acid or a glycated peptide at the N-terminal and quantifying the same, and a composition used for the quantification. The present invention is a measurement method and a composition particularly useful for accurately, simply, rapidly and inexpensively quantifying glycated hemoglobin in a blood sample using such an enzyme.

Hereinafter, the structure and preferred embodiments of the present invention will be described in more detail. Any test liquid that can be used in the present invention may be used as long as it contains a peptide or protein whose N-terminal is glycated. Preferably a peptide containing the N-terminal of glycated hemoglobin or a test solution containing glycated hemoglobin, more preferably,
Examples include whole blood, hemolyzed whole blood, centrifuged red blood cells, washed red blood cells, and the like. A protease-treated solution obtained by fragmenting the separated red blood cells or glycated hemoglobin with a protease in advance can also be used as a preferable test solution.

The protease that can be used in the present invention is Deblocking Aminopepidase.
tidase), N-acylaminoacyl-peptide hydrolase (EC = 3.
4.19.1), dipeptidyl aminopeptidase (Dipeptid
yl aminopeptidase), leucine aminopeptidase (L
eucine Aminopeptidase, Hemicellulase
llase), any enzyme may be used.

Preferred examples of the enzyme include Deblocking Aminopeptidase, which is derived from the genus Pyrococcus.
, N-acylaminoacyl-peptide hydrolase (N-
Acylaminoacyl-peptide hydrolase), N-acylaminoacyl-peptide hydrolase from pig liver (N-Acylaminoacyl-peptide hydrolase; EC = 3.4.1
9.1), Dipeptidyl aminopeptidase
Aminopeptidase includes, for example, cathepsin C (Cathepsin C; EC = 3.4.14.1; from BoP) belonging to Dipeptidyl Aminopeptidase I (Dipeptidyl Aminopeptidase I).
vine Spleen; Sigma), dipeptidyl aminopeptidase II (Dipeptidyl Aminopeptidase II; EC =
3.4.14.2; located in various organ lysosomes), dipeptidyl aminopeptidase III (Dipeptidyl Aminopeptidase)
Angiotensinase belonging to III)
EC = 3.4.14.4; erythrocyte), dipeptidyl aminopeptidase IV (Dipeptidyl Aminopeptidase IV; EC = 3.
4.14.5; pig liver; Sigma, Leucine Aminopeptidase; EC = 3.4.11.
1; swine kidney, cytosol; Sigma), and hemicellulase (Hemicelullase) include, for example, hemicellulase "Amano" (manufactured by Amano Pharmaceutical Co., Ltd.), Sumiteam X (manufactured by Nippon Chemical Industry Co., Ltd.), and hemicellulase N (Tanabe Seiyaku) Co., Ltd.), cellulosin HC (manufactured by Hankyu Bio-Industry Co.), hemicellulase (manufactured by Tokyo Kasei) and the like, but the present invention is not limited to these enzymes.

Further, in the present invention, when the N-terminal glycated amino acid or glycated peptide is released, the above-mentioned protease which acts on the N-terminal glycated peptide or protein to release the N-terminal glycated amino acid or glycated peptide is used alone. Needless to say, another endoprotease may be allowed to act before or simultaneously with the reaction of the above protease. By doing so, the reaction rate for releasing the glycated amino acid or glycated peptide at the N-terminus can be increased.

The synthetic substrate used in the present invention for measuring the activity of a protease that acts on an N-terminal glycated peptide or protein and releases an N-terminal glycated amino acid or peptide is α-fructosyl amino acid derivative or α-fructosyl. Any substrate may be used as long as it is a synthetic substrate that is a peptide. α-fructosyl refers to an Amadori compound which is non-enzymatically generated by binding an α-amino group at the N-terminus of an amino acid or peptide to glucose.

When the synthetic substrate is a fructosyl amino acid derivative, a fructosyl valine derivative is most preferable. However, other amino acids may be used, and the C-terminus of the amino acid may be anilide, ester or the like for the purpose of distinguishing the substrate from the product. Must be protected in the form of The present inventors used paranitroanilide, which exhibits a yellow color when the protease is cleaved, for the purpose of facilitating protease screening, but any other protecting group may be used. When other protecting groups are used, for example, if the coloration can be induced only by the protease reaction, then the coloration may be used for the detection of protease, and if the coloration cannot be induced only by the protease reaction, the glycated amino acid generated is converted to FAOD. The protease may be detected by a known method after conversion into hydrogen peroxide.

When the synthetic substrate is a fructosyl peptide, the sequence is preferably the same as that of the glycated hemoglobin N-terminal. For example, fructosyl valyl histidine (Val-His)
From fructosyl valyl histidyl leucyl threonyl proline (Val-His-Leu-Thr-Pro) to fructosyl peptide of 2 to 5 residues or those obtained by derivatizing the peptide C-terminus thereof are most preferable. May be used.

The method of synthesizing the synthetic substrate is as follows: an amino acid derivative or peptide is synthesized by a known method, for example, the method of Hashib et al.
a H, J. Agric. Food Chem. 24: 70, 1976). Also, the synthesis can be performed using other methods. The derivatization of the amino acid or peptide at the C-terminus may be performed by a known method or a commercially available product may be used.

In the present invention, the activity of a protease that acts on a glycated peptide or protein at the N-terminus and releases a glycated amino acid or peptide at the N-terminus was measured by the following method. The activity was measured by FAOD (manufactured by Asahi Kasei Corporation) using FV
It utilizes the fact that it does not act on alpNA but acts on fructosyl valine (hereinafter abbreviated as FVal) generated by the action of protease.

<< Method of Measuring Protease Activity >><Composition of Reaction Solution> 6 mM FValpNA 15 mM Tris-HCl buffer pH7.5 (Wako Pure Chemical Industries) 2U / ml FAOD (Asahi Kasei Corporation) 2U / ml peroxidase ( Hereinafter abbreviated as POD, manufactured by Sigma) 0.01% 4-aminoantipyrine (hereinafter abbreviated as 4AA;
0.01% N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (hereinafter abbreviated as TOOS; manufactured by Wako Pure Chemical Industries)

Put 933 μL of the above reaction solution in a cuvette
Preheat at 37 ° C for 5 minutes. Add 67 μL of appropriately diluted protease-containing solution, stir to start the reaction,
The absorbance change ΔA per 10 minutes at 55 nm is measured. Further, a reaction solution in which distilled water was formulated in place of FValpNA as a substrate was prepared, and a similar operation was performed to measure a change in absorbance ΔA blank (hereinafter abbreviated as ΔAb). The amount of enzyme that produces 1 μM glycated amino acid per minute at 37 ° C. from the glycated amino acid derivative was defined as 1 U. The calculation formula is as the following formula 1.

[0028]

(Equation 1)

39.2: Millimolar extinction coefficient of 4AA-TOOS 2: Dye 1 in which 4-AA and TOOS are condensed from two molecules of hydrogen peroxide
Coefficient by which this produces a molecule 10: Reaction time (min) 1000: Total reaction volume (μL) 67: Enzyme solution volume (μL)

In the present invention, the method of measuring the released N-terminal glycated amino acid or peptide may be carried out by decolorizing the protein or the like using the reducing ability of the glycated amino acid or glycated peptide after deproteinization, and then quantifying by coloring the dye or the like. Also preferably, the protein may be quantified by means of chromatography, antigen-antibody reaction, enzymatic reaction or the like without deproteinization, and most preferably quantified using an enzyme acting on glycated amino acids or glycated peptides.

The enzyme acting on a glycated amino acid or a glycated peptide effectively acts on a glycated amino acid or a glycated peptide produced from a peptide or protein having a glycated N-terminal contained in a test solution by the action of the protease. Any enzyme may be used as long as it is an enzyme capable of measuring substantially glycated peptides and proteins at the N-terminus, but an enzyme whose α-amino group acts efficiently on a glycated amino acid or glycated peptide is preferable, Most preferred are enzymes in which the α-amino group specifically acts on glycated amino acids and the ε-amino group does not substantially act on glycated amino acids. Generally, an enzyme in which such an α-amino group specifically acts on a saccharified amino acid and an ε-amino group does not substantially act on a saccharified amino acid has poor stability. And an α-amino group may be measured using an enzyme that acts well on both saccharified amino acids, and a more stable ε-amino group may be measured using an enzyme that specifically acts on saccharified amino acids. Only the amino acid whose group is glycated is eliminated, and only the amino acid whose α amino group is glycated is measured using an enzyme which works well on both saccharified amino acids whose ε amino group and α amino group are highly saccharified. May be.

From the viewpoint of specificity, the most preferred example of an enzyme acting on glycated amino acids is an enzyme specific to glycated amino acids having an α-amino group which does not act on glycated amino acids such as fructosyl amino acid oxidase ( FAOD) (derived from the genus Corynebacterium (FERM P-8245)).
The stability is poor at a residual activity of 10% or less after a treatment for 10 minutes, which is not preferable for actual use. On the other hand an enzyme ε amino group and α-amino group acts well on both glycated amino acid is glycated as a highly stable enzyme Gibberella (Gibberella) genus or Aspergillus (Aspergillus) genus (e.g. IFO-636
5, -4242, -5710, etc.) from fructosamine oxidase, Candida (Candida) from genus fructosylamine de glycidyl gauze, Penicillium (Penicillium) genus (e.g. IFO-
4651, -6581, -7905, -5748, -7994, -4897, -533
7, etc.) from fructosyl amino acid degrading enzymes, Fusarium (Fusarium) genus (e.g. IFO-4468, -4471, -6384, -7
706, -9964, -9971, -31180, -9972 etc., oxidases acting on at least glycated amino acids such as ketoamine oxidases derived from the genus Acremonium or from the genus Debaryomyces , and more preferred. As an example, genetically modified fructosamine oxidase (manufactured by Asahi Kasei Kogyo Co., Ltd.), which has sufficient activity even in the presence of a protease, may be mentioned.

A method for measuring the activity of an enzyme acting on a glycated amino acid is described below. << Method for measuring the activity of enzymes acting on glycated amino acids >><Reactioncomposition> 50 mM Tris-HCl buffer pH7.5 0.03% 4-AA (Wako Pure Chemical Industries) 0.02% Phenol (Wako Pure Chemical Industries) ) 4.5 U / ml POD (manufactured by Sigma) 1.0 mM FVal or α-carbobenzoxy ε-D-fructosyl-L-lysine (synthesized and purified based on the method of Hashiba et al., Hashiba H, J. Agric. Food Chem. 24) : 70,1976, hereinafter abbreviated as FZLys.)

1 ml of the above reaction mixture was placed in a small test tube, and
Pre-warmed for 5 minutes, and then appropriately diluted enzyme solution 0.02 ml
And stirred to start the reaction. After exactly 10 minutes, the reaction was stopped by adding 2 ml of 0.5% SDS,
Measure the absorbance at 5 nm (As). The same operation is performed using 0.02 ml of distilled water instead of the enzyme solution as a blank, and the absorbance is measured (Ab). The enzyme activity is determined from the difference in absorbance (As-Ab) between the absorbance of the enzyme action (As) and the absorbance of the blind test (Ab). Separately, the relationship between the absorbance and the generated hydrogen peroxide is examined in advance using a standard solution of hydrogen peroxide. 37
The amount of enzyme that produces 1 μM hydrogen peroxide per minute
And the enzyme activity is calculated by the following formula (2).

[0035]

(Equation 2)

3.02: Total amount of reaction solution (ml) 0.02: Enzyme solution amount (ml) 10: Reaction time (min) 2: One molecule of dye formed by condensation of 4-AA and phenol from two molecules of hydrogen peroxide Coefficient 12.0: mmol absorption coefficient of 4AA-phenol

The composition of the reaction of the present invention for releasing an N-terminal glycated amino acid or glycated peptide from a test solution containing a N-terminal glycated peptide or protein is described below. The pH and the protease concentration may be determined so that the reaction proceeds efficiently in consideration of the optimal pH of the protease that releases the N-terminal glycated amino acid or glycated peptide from the test solution containing the protein.

For example, a deblocking aminopeptidase derived from the genus Pyrococcus (Deblocking
When Aminopeptidase (manufactured by Takara) is used, since the proteolytic activity is strong at around pH 6.5 to 9.0, the reaction pH can be selected from 6.5 to 9.0. The protease concentration may be 0.01 to 1000 as long as it can sufficiently decompose the protein in the test solution during the reaction time actually used.
U / ml is preferred, and 0.1 to 500 U / ml is more preferred.

With regard to the liquid composition of the reaction for measuring a glycated peptide or protein fragmented using an enzyme acting on a glycated amino acid, the reaction can be carried out efficiently in consideration of the optimum pH of the enzyme acting on the glycated amino acid to be used. The pH may be selected so as to proceed, and then the amount of enzyme acting on the glycated amino acid may be determined.

For example, in the case of recombinant fructosamine oxidase (manufactured by Asahi Kasei Kogyo), the region showing 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the reaction pH is 6.5 to 10.
10 can be selected. The enzyme addition concentration may be a concentration that can sufficiently measure glycated amino acids generated from 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 / ml. ml is more preferred, 0.5U-100U / m
l is most preferred.

From the above, the composition for quantifying a glycated protein in the present invention includes Deblocking Aminopeptidase, Dipeptidyl Aminopeptidase,
Leucine Aminopeptidas
e), those containing N-acylaminoacyl-peptide hydrolase or hemicellulase, or deblocking aminopeptidases (Deblocking Aminopeptidas)
e), dipeptidyl aminopeptidase (Dipeptidyl Ami
nopeptidase), leucine aminopeptidase (Leucine
Aminopeptidase), N-Acylaminoacyl-peptide hydrolase
Or it may be prepared as containing an enzyme acting on hemicellulase (Hemicelullase) and glycated amino acids,
For example, it can be provided as a liquid product, a frozen liquid product or a freeze-dried product.

Further, to the enzyme reaction composition for quantifying glycated protein according to the present invention, for example, a surfactant, a salt, a buffer, a pH adjuster, a preservative, and the like may be appropriately selected and added.

Examples of the surfactant include polyoxyethylene alkyl ethers (eg, polyoxyethylene (10) octyl phenyl ether (Triton X-100), polyoxyethylene (23) lauryl ether, etc.) and polyoxyethylene sorbitan fatty acid esters [For example, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40)
Etc.), polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbite fatty acid esters,
Polyoxyethylene alkylphenyl formaldehyde 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 esters, glycerin fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, N acyl amino acid salts, alkyl ether carboxylic acid salts, alkyl Phosphate, N acyl taurate, sulfonate, alkyl sulfate, betaine acetate amphoteric surfactant, imidazoline amphoteric surfactant, resin Derivatives (above Nikko Chemicals), ADEKATOL 720N, ADEKATOL B-795, ADEKATOL SO-120, ADEKANOL B-795 (both from Asahi Denka Kogyo), polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctylphenyl Ether, polypropylene glycol,
Polyvinyl alcohol, Triton X-305, Triton X-11
4, Triton X-405, Triton WR-1339 (all manufactured by Nacalai Tesque), and the like. These surfactants are available at 0.
It is preferably used at a concentration of from 01 to 10%, preferably from 0.05 to 5%.

Examples of the salts include lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride and the like. These salts are preferably used at a concentration of 1 mM to 5M, preferably 10 mM to 1M. Examples of the buffer include Tris-HCl buffer, glycine-NaOH buffer, phosphate buffer, Good's buffer and the like, and these are 10 mM to 2 M, preferably 20 mM.
It is preferred to use at a concentration of ~ 1M. Preservatives include, for example, sodium azide, from 0.01 to 10%, preferably from 0.05 to 10%.
-1% may be appropriately added.

In the method for quantifying a glycated protein according to the present invention, the enzyme action acting on a glycated amino acid is detected by, for example, using a dehydrogenase to determine the amount of change in a coenzyme, for example, using NAD as a coenzyme. The amount of change can be 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 with a colorimeter, or the amount of reduced Coenzymes include various diaphorases or phenazine methosulfate (hereinafter abbreviated as PMS), methoxy PMS,
Electron carriers such as dimethylaminobenzophenoxazinium chloride (Meldra Blue) and nitrotetrazolium, WST-1 and WST-8 (all manufactured by Dojindo Laboratories)
May be indirectly determined using a reducing color developing reagent such as various tetrazolium salts, or may be directly or indirectly measured by other known methods.

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

The amount of the above hydrogen peroxide may be determined by, for example, forming 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 coloring system of hydrogen peroxide is obtained by the oxidative condensation of a coupler such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (MBTH) with a chromogen such as phenol in the presence of peroxidase. A dyer-generating trinder reagent, a leuco-type reagent that directly oxidizes and develops a color in the presence of peroxidase, and the like can be used.

As the chromogen of the Trinder-type reagent, phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used. 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) (all manufactured by Dojindo Chemical Laboratories) and the like.

Specific examples of leuco reagents include o-dianisidine, o-tolidine, 3,3 diaminobenzidine,
3,5,5-tetramethylbenzidine (all from Dojindo Chemical Laboratories), N- (carboxymethylaminocarbonyl)-
4,4-bis (dimethylamino) biphenylamine (DA6
4), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67) (all manufactured by Wako Pure Chemical Industries, Ltd.) and the like.

Further, in the fluorescence method, compounds emitting fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, and diacetylfluorescin derivative, are used. In the chemiluminescence method, luminol and lucigenin are used as catalysts. , Isoluminol, pyrogallol and the like can be used.

Methods for producing aldehyde from alcohol using catalase or the like and quantifying the generated aldehyde include a method using the Huntch reaction, a method of coloring by condensation reaction with MBTH, a method of using aldehyde dehydrogenase, and the like. Is mentioned. Glucosone can be quantified using a known aldose reagent such as diphenylamine.

When hydrogen peroxide is measured 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. As the electrode measurement method, known methods such as amperometry, potentiometry, and coulometry can be used, and further, an electron carrier is interposed in the reaction between the oxidase or the substrate and the electrode,
The obtained oxidation and reduction currents or their electric quantities 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.

To quantify the glycated peptide or glycated protein in the test solution using the thus-prepared quantification composition of the present invention, 0.001 to 0.5 ml of the test solution is added to the quantification composition and the temperature is adjusted to 37 ° C. When reacting at a temperature and performing a rate assay, a few minutes to tens of minutes between two points after a certain time after the start of the reaction, for example, 1 minute after 3 minutes and 4 minutes, or 3 minutes after And the amount of changed coenzyme, dissolved oxygen, hydrogen peroxide or other products for 5 minutes after 8 minutes may be measured directly or indirectly by the above-mentioned method. The amount of the changed coenzyme, dissolved oxygen, hydrogen peroxide or other products after a certain period of time may be similarly measured. In this case, a known concentration of glycated amino acid,
The amount of glycated peptide and glycated protein in the test solution can be determined by comparing the change in absorbance and the like when measured using a peptide or protein.

[0055]

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 <Screening of protease that acts on N-terminal glycated peptide or protein to release glycated amino acid or glycated peptide at N-terminus> Screening consists of primary screening in which a large number of proteases are narrowed down and primary screening The screening was carried out in two stages of secondary screening in which fructosyl valine was purified from the synthetic substrate using fructosylamine oxidase.

(1) Primary screening <Reaction solution 1> 100 mM Tris-HCl buffer pH 8.5 2 mM FValpNA 200 μL of the above reaction solution 1 is placed in a 96-well plate, and 50 μL of a solution containing protease (higher concentration is preferable). ) Was added and reacted at room temperature for 10 minutes to overnight to select a yellow colored protease. Further, for the protease for which coloring was confirmed, take 900 μL of the above reaction solution in a cuvette, add 50 μL of a solution containing the protease, and change the absorbance at 500 nm Δ
A was measured. Further, a reaction solution in which distilled water was formulated in place of FValpNA as a substrate was prepared, and a similar operation was performed to measure a change in absorbance ΔA blank. The effect of the protease on the glycated amino acid derivative was calculated using a ΔA-ΔA blank.

(2) Secondary Screening The following activities were confirmed for proteases whose activity was recognized in the primary screening and proteases which had been reported to have an effect on glycated hemoglobin. <Reaction liquid 2> 6 mM FValpNA 15 mM Tris-HCl buffer pH7.5 (manufactured by Wako Pure Chemical Industries) 2U / ml FAOD (manufactured by Asahi Kasei) 2U / ml POD (manufactured by Sigma) 0.01% 4-AA (Wako Pure Chemical Industries) 0.01% TOOS (Wako Pure Chemical Industries)

The above reaction solution 2 was placed in a 933 μL cuvette.
Preheat at 37 ° C for 5 minutes. 67 μL of a solution containing an appropriately diluted protease is added, the reaction is started by stirring, and the absorbance change ΔA per 10 minutes at 555 nm is measured. Further, a reaction solution in which distilled water was formulated in place of FValpNA as a substrate was prepared, and a similar operation was performed to measure a change in absorbance ΔA blank. The effect of the protease on the glycated amino acid derivative was calculated using a ΔA-ΔA blank. Table 1 shows the results.

[0059]

[Table 1]

As shown in Table 1, surprisingly, no protease releasing activity from FValpNA was observed for proteases which have been said to be usable for measuring glycated hemoglobin.

On the other hand, Pyrococcus furiosas
ccus furiosus ) N-terminal blocked peptides such as deblocking aminopeptidase (Takara) and pig liver-derived N-acylaminoacyl-peptide hydrolase (Takara) And a protease which acts on proteins and releases N-terminal blocked amino acids, has a strong effect.

Similarly, a similar effect was confirmed for dipeptidyl peptidase which releases two amino acids from the N-terminus, for example, cathepsin C derived from bovine spleen and dipeptidyl aminopeptidase IV (Dipeptidyl aminopeptidase IV). This was thought to be because the protease recognized the glycated amino acid as two amino acids and released the glycated amino acid efficiently.

Among the aminopeptidases except for the special aminopeptidase described above, only leucine aminopeptidase had an activity of releasing fructosyl valine from the synthetic substrate, and no activity was confirmed from other aminopeptidases. It was presumed that the activity was confirmed only for leucine aminopeptidase because the size of the side chain of leucine was similar to that of the sugar bound to the N-terminal. Further, a similar activity was confirmed for hemicellulase.

[0064]

Example 2 <Reaction Curve for Synthetic Substrate> <Reaction Solution> Same as Reaction Solution 2 in Example 1.

<Operation> The reaction solution 2 shown in Example 1 was mixed with 9
Preheat in a 33μL cuvette at 37 ° C for 500 seconds.
Add 67 μL of an appropriately diluted protease-containing solution, stir to start the reaction, and measure the absorbance at 555 nm.
Further, a reaction solution in which distilled water was formulated in place of FValpNA as a substrate was prepared, and the change in absorbance was measured in the same manner. A water blank was subtracted from the absorbance obtained from FValpNA to calculate a reaction curve. The results are shown in FIG.

As can be seen from FIG. 1, deblocking aminopeptidase (manufactured by Takara), N-acylaminoacyl-peptide hydrolase (porcine liver-derived N-acylaminoacyl-peptide hydrolase;
Takara) and Petidylaminopeptidase IV (Dipe
ptidyl aminopeptidase IV) from FValpNA to FVal
It was observed that hydrogen peroxide was generated over time by FOD.

[0067]

Example 3 <Calibration curve using glycated hemoglobin as substrate><Reaction liquid 1> 10 mM Tris buffer pH 8.5 10 U / ml deblocking aminopeptidase (Takara, Japan)

<Reaction liquid 2> 150 mM Tris buffer pH 8.5 15 U / ml POD (manufactured by Sigma) 12 mM TOOS 8 mM 4-AA (manufactured by Wako Pure Chemical Industries) 2 U / ml FOD (manufactured by Asahi Chemical Industry) <Hb Substrate solution> Human hemoglobin was dissolved in distilled water to a concentration of 4 g / dl to obtain an Hb substrate solution. Hb substrate solution (4 g / d
Samples having a concentration of 0.0, 0.5, 1.0, 1.5 and 2.0 times of l) were used.

<Operation> Transfer 240 μl of the above reaction solution into a test tube and heat at 37 ° C. for 5 minutes. 8 μl of Hb substrate solution was added, and the reaction was started by stirring. After exactly 5 minutes, the protein was removed with trichloroacetic acid. After neutralization, 80 μl of reaction solution 2 was added to this solution.
Was added. The absorbance at 546 nm before and after the addition of the reaction solution 2 was measured, and the difference was defined as a change in absorbance. The same operation was performed using distilled water in place of the substrate to obtain a blank sample.

The sensitivity was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the Hb substrate solution. The Hb 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. 2, the Hb substrate solution obtained good linearity according to the concentration. In addition, good reproducibility was confirmed at a CV value of 2.5% at a Hb 1.0-fold concentration, indicating that glycated hemoglobin was measured selectively, with good sensitivity, and with good reproducibility using the measurement system of the present invention. It became clear.

[Brief description of the drawings]

FIG. 1 is a reaction curve when a synthetic substrate based on Example 2 of the present invention was used.

FIG. 2 is a calibration curve of glycated hemoglobin based on Example 3 of the present invention.

Claims (6)

[Claims] 1. A test solution containing a peptide or protein having a glycated N-terminus, a test solution containing deblocking aminopeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase, leucine aminopeptidase,
N-acylaminoacyl-peptide hydrolase (N-Acy
A method for quantifying an N-terminally glycated peptide or glycated protein, characterized by releasing a glycated amino acid or glycated peptide at the N-terminus by the action of laminoacyl-peptide hydrolase) or hemicellulase (Hemicelullase), and quantifying the glycated amino acid or glycated protein. 2. The method according to claim 1, wherein the amount of the released N-terminal glycated amino acid or glycated peptide is measured using an enzyme that acts on the glycated amino acid. 3. The method according to claim 1, wherein the N-terminal glycated peptide is an N-terminal fragment of glycated hemoglobin, or the glycated protein is glycated hemoglobin. 4. A deblocking aminopeptidase (De
blocking Aminopeptidase), Dipeptidyl Aminopeptidase, Leucine Aminopeptidase, N-Acylaminoacyl-Peptide Hydrolase (N-Acylamino)
acyl-peptide hydrolase) or hemicellulase (Hemicellulase)
A composition for quantifying a peptide or protein having an N-terminal glycated, comprising celullase). 5. Deblocking aminopeptidase (De
blocking Aminopeptidase, Dipeptidyl Aminopeptidase, Leucine Aminopeptidase, N-Acylaminoacyl-Peptide Hydrolase (N-Acylamino)
acyl-peptide hydrolase) or hemicellulase (Hemicellulase)
celullase) and an enzyme that acts on glycated amino acids, and a composition for quantifying an N-terminal glycated peptide or protein. 6. The quantification composition according to claim 4, wherein the N-terminal glycated peptide is a glycated hemoglobin N-terminal fragment, or the glycated protein is glycated hemoglobin.