CN1852987A - Process for producing a-glycosylated dipeptide and method of assaying a-glycosylated dipeptide - Google Patents
Process for producing a-glycosylated dipeptide and method of assaying a-glycosylated dipeptide Download PDFInfo
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- CN1852987A CN1852987A CN 200480027120 CN200480027120A CN1852987A CN 1852987 A CN1852987 A CN 1852987A CN 200480027120 CN200480027120 CN 200480027120 CN 200480027120 A CN200480027120 A CN 200480027120A CN 1852987 A CN1852987 A CN 1852987A
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- glycated
- alpha
- proteolytic enzyme
- dipeptides
- peptide
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A process for producing an alpha-glycosylated dipeptide characterized in that a protease acts on a peptide having its N-terminus glycosylated or protein having its N-terminus glycosylated; and a method of assaying an alpha-glycosylated dipeptide characterized in that fructosyl-peptide oxidase acts on the alpha-glycosylated dipeptide obtained by the process so as to generate hydrogen peroxide and the thus generated hydrogen peroxide is measured. The provided process for producing an alpha-glycosylated dipeptide realizes simple, speedy and efficient production of an alpha-glycosylated dipeptide from a glycosylated protein or glycosylated peptide. The provided method of assaying an alpha-glycosylated dipeptide realizes short-time accurate quantitative determination of alpha-glycosylated dipeptide.
Description
Technical field
The present invention relates to prepare the method for alpha-glycated dipeptides and the method for measuring the amount of the alpha-glycated dipeptides that obtains by this preparation method.
Background of invention
Glycated protein is non-enzymatic glycated protein.Specifically, glycated protein produces as the result of the amino non-enzymatic covalent bonding on aldehyde radical on the sugared side (in other words on aldose (potential monose and derivative thereof with aldehyde radical) side) and the albumen side.In addition, such glycated protein is to form when (Amadori) resets in more than the Schiff's base that produces as reaction intermediate carries out Armagh.Therefore, glycated protein also be known as Armagh many in compound.
Glycated protein is present in body fluid for example body inner blood or biological specimen for example in the hair.The concentration that is present in the glycated protein in the blood depends primarily on for example concentration of glucose of the carbohydrate that is dissolved in the serum.Under the diabetes situation, the generation of glycated protein improves.And, be present in the concentration of the glycolated hemoglobin in the red corpuscle or the concentration of the glycosylated albumin in the serum, reflection average blood glucose level in the past in the regular hour.Therefore, the amount of measuring such glycated protein is very important for diagnosis or control of diabetes symptom.
Glycolated hemoglobin (hereinafter being abbreviated as HbA1c.) is a fructosyl albumen, described fructosyl albumen has by glucose and combines with the non-enzymatic of the-terminal amino acid of oxyphorase beta subunit forming the structure that Schiff's base produces, thereby causes by Armagh the fructose combination of resetting in many.Such HbA1c can individual month average glucose level of clinical reflection past 1-2.Therefore, HbA1c is the important indicator of control of diabetes, and needs rapid and precise measuring method thus.
At present, method as the amount of measuring HbA1c, IFCC Practical StandardMethods is (referring to people such as U., Clin.Chem.43,1944-1951 (1997)) a kind of measuring method is disclosed, described method comprises that the six peptide segments that will derive from the N-end of its β chain with HPLC are separated with endo-protease Glu-C hydrolysis (handling 18 hours in 37 ℃) HbA1c, and with the amount of for example capillary electrophoresis or mass spectrometric determination gains.Yet this method is problematic, because need special instrument and complicated program, and is uneconomic.
Therefore, enzymatic means has been proposed as the method for measuring the amount of HbA1c in the mode of simple program, low cost, pin-point accuracy.Such enzymatic means comprises with proteolytic enzyme the glycated protein sex change, makes Fructoamino-acid-oxidase act on the glycated amino acid of release, measures the amount of consequent hydrogen peroxide then.The oxidasic example that openly has been used for such enzymatic measuring method, comprise the bacteriogenic oxydase of corynebacterium (Corynebacterium) (referring to JP patent disclosure (Kokoku) 5-33997 B (1993) and JP patent disclosure (Kokoku) 6-65300 B (1994)), the oxydase (referring to JP patent disclosure (Kokai) 3-155780 A (1991)) that Aspergillus (Aspergillus) bacterial strain produces, gibberella belongs to the oxydase (referring to JP patent disclosure (Kokai) 7-289253A (1995)) that (Gibberella) bacterial strain produces, the oxydase (referring to JP patent disclosure (Kokai) 7-289253 A (1995) and JP patent disclosure (Kokai) 8-154672 A (1996)) that fusarium (Fusarium) bacterial strain produces, the oxydase (referring to JP patent disclosure (Kokai) 8-336386 A (1996)) that Penicillium (Penicillium) bacterial strain produces, and Ketoamine oxidase (referring to JP patent disclosure (Kokai) 5-192193 A (1993)).And following method (a)-(i) is therefore well-known already as an example, and wherein alpha-glycated amino acid (amino acid whose alpha-amino group is by saccharification) is released from the oxyphorase with saccharification-terminal amino acid:
Method (a), described method comprises 8M urea is added in the glycolated hemoglobin, mixture boiled 20 minutes with sex change, carry out trypsin treatment, measure the amount (referring to JP patent disclosure (Kokai) 8-336386 A (1996)) of gains then with the Fructoamino-acid-oxidase (FAOD) that derives from the Penicillium bacterial strain;
Method (b), described method comprises uses the protease treatment glycolated hemoglobin, measures the amount (referring to JP patent disclosure (Kokai) 10-33177 A (1998) and JP patent disclosure (Kokai) 10-33180 A (1998)) of gains then with the FAOD that derives from the Aspergillus bacterial strain;
Method (c), described method comprise the amount (disclosing 97/13872 pamphlet referring to international monopoly) of measuring glycolated hemoglobin with endo-protease and circumscribed proteolytic enzyme;
Method (d), described method comprise with serine carboxypeptidase comes enzymically treat to have the peptide or the albumen (referring to JP patent disclosure (Kokai) 2001-57897A) of the terminal Xie Ansuan of fructosyl N-;
Method (e), described method comprises with the 3rd leucic carboxyl side being handled from the terminal proteolytic enzyme of shearing of the β chain N-of HbA1c, with can with the histidyl-leucine from the fructosyl that produces valyl-histidyl--leucine excises the protease treatment gains that get off, measures the amount (referring to JP patent disclosure (Kokai) 2000-300294 A) of HbA1 c then;
Method (f), described method comprises uses the new enzyme that derives from corynebacterium and Rhodopseudomonas (Pseudomonas) glycated amino acid to be discharged (such endonuclease capable discharges from glycated protein and has the alpha-amino amino acid of saccharification), measures the amount (disclosing 00/50579 pamphlet referring to international monopoly) of gains then;
Method (g), described method comprises uses the new enzyme that derives from Sphingobacterium (Sphingobacterium), Sphingol single-cell genus (Sphingomonas), Comamonas (Comamonas), Mucor and Penicillium glycated amino acid to be discharged (such endonuclease capable discharges from glycated protein and has the alpha-amino amino acid of saccharification), measures the amount (disclosing 0/61732 pamphlet referring to international monopoly) of gains then;
Method (h), described method is included in tetrazole compound and exists down, comprise proteic sample with protease treatment, make thus obtained proteolysis gains and FAOX reaction, then the amount of rapid determination glycated protein (referring to the open 02/27012pamphlet of international monopoly); With
Method (i); described method comprises that deblocking aminopeptidase, two peptidyl aminopeptidases, leucine aminopeptidase, N-acyl amino acyl group-peptidohydrolase or hemicellulase are acted on contains terminal glycated peptide of N-or proteic testing liquid; discharge the terminal glycated amino acid of N-, measure the amount (referring to JP patent disclosure (Kokai) 2002-315600 A) of consequent glycated amino acid then.
Yet the experiment of carrying out according to present inventors does not promptly have by making various proteolytic enzyme act on the example that HbA1c discharges alpha-glycated amino acid yet.Specifically, various proteolytic enzyme can not cut into HbA1c the size less than alpha-glycated peptide.Almost there is not alpha-glycated amino acid to be cut by such proteolytic enzyme.As long as use above-mentioned Fructoamino-acid-oxidase, the amount of HbA1c just can not be measured with good sensitivity, and this has become final conclusion.As described above, measure for HbA1c, measure the measuring method that the good method of HbA1c relates to measure alpha-glycated peptide or preferably measures alpha-glycated dipeptides with hypersensitivity, described alpha-glycated peptide or alpha-glycated dipeptides are to act on such peptide or dipeptides as substrate with oxydase, discharge by protease treatment.The oxydase that acts on alpha-glycated dipeptides so has been disclosed JP patent disclosure (Kokai) 2001-95598 A and JP patent disclosure (Kokai) 2003-235585 A with the proteolytic enzyme that can excise glycosylated dipeptide.Yet,, just need have the active proteolytic enzyme of the higher alpha-glycated dipeptides of excision in order to realize that HbA1c measures to carry out fast than hypersensitivity.
Therefore, the purpose that the present invention will reach provides the method for the alpha-glycated dipeptides of preparation, by this method, alpha-glycated dipeptides (wherein the alpha-amino group of the-terminal amino acid of dipeptides is by the glycosylated dipeptide of saccharification) is discharged from glycated protein or glycated peptide effectively by a kind of protease treatment.Another purpose that the present invention will reach provides the method for the amount of measuring alpha-glycated dipeptides.Described method is by the amount of the alpha-glycated peptide that uses above-mentioned oxidase assay and discharge, can enough simple programs, with the height accurate way, measure the amount of glycated protein or glycated peptide at short notice.
The present invention is open
The result who achieves the above object as further investigation, present inventors find, by a kind of protease treatment, alpha-glycated dipeptides (wherein the alpha-amino group of the-terminal amino acid of dipeptides is by the glycosylated dipeptide of saccharification) can be discharged from glycated protein or glycated peptide effectively.Present inventors also find, by using the amount of the alpha-glycated peptide that above-mentioned oxidase assay discharges, and can highly accurate method, with simple program, measure glycated protein or glycated peptide at short notice.Like this, present inventors have just finished the present invention.
The invention provides following the present invention:
(1) method of the alpha-glycated dipeptides of preparation, described method comprise makes proteolytic enzyme act on terminal glycated peptide of N-or the terminal glycated protein of N-;
(2) according to the method for the alpha-glycated dipeptides of (1) preparation, the terminal glycated peptide of wherein said N-is fructosyl Val-His-Leu-Thr-Pro-Glu;
(3) according to the method for the alpha-glycated dipeptides of (1) preparation, the terminal glycated protein of wherein said N-is a glycolated hemoglobin;
(4) according to the method for the alpha-glycated dipeptides of (1), (2) or (3) preparation, wherein said proteolytic enzyme is that one or more are selected from following proteolytic enzyme: the proteolytic enzyme that is belonged to microorganisms such as (Streptomyces) by Aspergillus, bacillus (Bacillus), Rhizopus (Rhizopas), Tritirachium, glucose Coccus (Staphylococcus), chain enzyme bacteria, by the animal proteolytic enzyme that produces of pig and ox for example, and by the plant proteolytic enzyme that produces of papaya genus, Fructus Fici and pineapple for example;
(5) according to the method for (1), the alpha-glycated dipeptides of (2) or (3) preparation, wherein said proteolytic enzyme is that one or more are selected from following proteolytic enzyme: subtilisin, pronase, Dispase, neutral protease, Sumizyme MP, Proteinase K, papoid, ficin, bromeline, pancreatin, Glu-C and kethepsin;
(6) according to the method for the alpha-glycated dipeptides of (1) to (5) preparation, wherein said alpha-glycated dipeptides is a fVH; With
(7) measure the method for the amount of alpha-glycated dipeptides, described method comprises makes fructosyl peptide oxydase act on the alpha-glycated dipeptides that the preparation method according to (1) to (6) obtains, and measures the amount of the hydrogen peroxide that produces then.
To describe the present invention below.The present invention requires the right of priority of Japanese patent application 2003-326224 that submitted on September 18th, 2003 and the Japanese patent application 2003-421755 that submitted on November 19th, 2003, and comprises content and/or accompanying drawing in the specification sheets of above-mentioned patent application.
So long as by with albumen and aldose for example the non-enzymatic of glucose combine and produce, N-end glycated protein can be any albumen.
The example of the glycated protein that is produced by live body comprises glycosylated albumin and HbA1c.For example, the present invention can be used to aptly amount of measuring HbA1c etc.And, the terminal glycated peptide of N-among the present invention, not only comprise by with contained peptide in the sample and aldose for example the non-enzymatic of glucose combine the peptide that produces, and comprise the peptide that produces by enzymatic (for example proteolytic enzyme and peptase) or the terminal glycated protein of the above-mentioned N-of non-enzymatic (for example application of physical agitation and heating) cutting.Such glycated protein or glycated peptide also are present in food for example in fruit juice, candy, seasonings and the powdery food usually.As long as contain above-mentioned glycated protein or glycated peptide, the sample that the present invention contains glycated protein or glycated peptide can be any sample.The example of such sample can comprise the interior sample of body for example body fluid (for example food and saliva) and hair.The other example of such sample comprises top described food etc.These samples directly can be measured, perhaps be measured indirectly in that filtration, dialysis treatment etc. are back.And, for example, glycated protein that should measured quantity or glycated peptide can be carried out suitable concentrating, extraction, water, damping fluid etc. dilute then.
As long as can act on above-mentioned glycated protein or glycated peptide and discharge alpha-glycated dipeptides then, can be used in proteolytic enzyme of the present invention can be any enzyme.Can preferred proteolytic enzyme suitably be selected according to the type of glycated protein that is cut or glycated peptide.The such proteolytic enzyme or the example of peptase comprise Proteinase K, pronase, thermolysin, subtilisin, protaminase, pancreatin, kethepsin, carboxypeptidase, endo-protease Glu-C, papoid, ficin, bromeline and aminopeptidase.Especially in the present invention, the example that can discharge the proteolytic enzyme of alpha-glycated dipeptides effectively comprises: the proteolytic enzyme that derives from Aspergillus, for example " IP enzyme; AO proteolytic enzyme; peptase and molsin (all producing) " by KIKKOMAN CORPORATION, " protease A 5 (by KYOWAKASEI CO., LTD. produces) ", " umamizyme; protease A; proteolytic enzyme M and protease P (all producing) " by Amano Enzyme Inc., " sumizymeMP; sumizyme LP-20; sumizyme LPL and sumizyme AP (all producing) " and " proteolytic enzyme 6 (producing) " by Fluka by ShinNihon Chemical Co.Ltd.; Derive from the enzyme of Rhizopas, for example " peptase R (producing) " by Amano Enzyme Inc; Derive from the proteolytic enzyme of bacillus, for example " Dispase (producing) " by Roche, " subtilisin (producing) " by Boehringer Mannheim Corporation, " protease N (producing) " by Fluka, " proteolytic enzyme type VII (producing) " by Sigma-AldrichCorporation, " proteolytic enzyme (bacterium) (producing) " by Fluka, " protease N; proleather FG-F and proteolytic enzyme S (all producing) " by Amano Enzyme Inc., " proteolytic enzyme type X (producing) " by Sigma-Aldrich Corporation, " thermolysin (producing) " by DAIWA KASEI K.K., " pronase E (by Kaken Pharmaceutical Co.; Ltd. produces) " and " neutral protease (by TOYOBO., LTD. produces) "; Derive from the proteolytic enzyme that chain enzyme bacteria belongs to, for example " pronase (producing) " by Boehringer Mannheim Corporation, " proteolytic enzyme XIV type (producing) " and " Sumizyme MP (by TOYOBO., LTD. produces) " by Sigma-Aldrich Corporation; Derive from the proteolytic enzyme of Tritirachium, for example " Proteinase K (by Roche and Wako Pure ChemicalIndustries, Ltd. produces) "; Derive from the proteolytic enzyme of glucose Coccus, for example " Glu-C (producing) " by Boehringer MannheimCorporation; Derive from the proteolytic enzyme of plant, and for example " papoid (by Roche, Wako Pure Chemical Industries, Ltd., Sigma-AldrichCorporation, Amano Enzyme Inc. and ASAHI FOOD ﹠amp; HEALTHCARE, LTD. produce) "; " ficin (producing) "; " bromeline (producing) " and derive from the proteolytic enzyme of animal; for example " pancreatin (by Wako PureChemical Industries, Ltd. produces) " and " cathepsin B's (by Sigma-AldrichCorporation production) by Enzyme Inc. and Sigma-AldrichCorporation by Sigma-AldrichCorporation.The sample that contains these proteolytic enzyme is preferred especially the use.Above-mentioned proteolytic enzyme can be used separately, perhaps their two or more type combination be used.For example, for HbA1c, proved use endo-protease Glu-C can produce alpha-glycated six peptides (fructosyl Val-His-Leu-Thr-Pro-Glu) (people such as Kobold U., Clin.Chem.1997,43:1944-1951).Therefore, for preparing glycosylated dipeptide, be unusual effective means with Glu-C and the combination of above-mentioned proteolytic enzyme by HbA1c.
The treatment condition of sample can be any conditions, as long as proteolytic enzyme used herein can act on glycated protein under such condition, measure its amount, and alpha-glycated then dipeptides can be released at short notice and get final product.The content of selecting to depend on glycated protein in the sample substantially of the amount of proteolytic enzyme used herein, treatment condition etc.In example, the concentration adding of proteolytic enzyme (for example protease P of selling by Amano Enzyme Inc.) preferred 1mg/mL-20mg/mL with 0.5mg/mL-50mg/mL of Aspergillus bacterial strain will be derived from.And, also can add other proteolytic enzyme if desired.The pH that is used in protease treatment can be unadjusted pH.Perhaps, in order to reach the suitable pH of employed proteolytic enzyme effect, for example, can with suitable pH regulator agent for example hydrochloric acid, acetate, sulfuric acid, sodium hydroxide or potassium hydroxide with pH regulator to pH 2-pH 9 and preferred pH 3-pH 8.Handle and also can for example carry out between 20 ℃-50 ℃ in temperature range.According to employed enzyme, handle and also can carry out 45 ℃-70 ℃ comparatively high temps scope.Treatment time can be any time that is enough to make the glycated protein sex change.Specifically, processing can be carried out 1-180 minute and preferred 2-60 minute.Can directly use thus obtained treatment soln, perhaps if desired, carry out back indirect uses such as suitable heating, centrifugal, concentrated, dilution.
Subsequently, can measure the amount of the alpha-glycated dipeptides that obtains by aforesaid method.
As long as can measure the amount of alpha-glycated dipeptides, any method all is operable.With the height accurate way, with simple program, comprise method that makes oxydase act on alpha-glycated dipeptides and the method for using HPLC with the example of preferred method low-cost, that measure the amount of alpha-glycated dipeptides at short notice.
At first, description makes oxydase act on the method for alpha-glycated dipeptides.
Make oxydase act on above-mentioned alpha-glycated dipeptides, measure the amount of product that obtains by described effect or the product that is consumed then, make it possible to measure the amount of glycosylated dipeptide thus by enzymatic means.As oxydase, any enzyme all is operable, and condition is that it acts on alpha-glycated dipeptides produces hydrogen peroxide with catalysis reaction specifically.
The example of such enzyme comprise by be disclosed in JP patent disclosure (Kokai) 2001-95598 A by the fructosyl peptide oxydase of intestinal bacteria (Escherichia coli) DH5 α (pFP1) (FERM P-17576) preparation and be disclosed in the fructosyl peptide oxydase of JP patent disclosure (Kokai) 2003-235585 A.
Except above-mentioned example, the microorganism by the research occurring in nature or by study the enzyme of free animal or plant, can obtain to act on specifically alpha-glycated dipeptides produces the reaction of hydrogen peroxide with catalysis enzyme.And the such enzyme that obtains by research can obtain with gene recombination technology, and thus obtained recombinase also can be suitable is used.And, also can obtain such enzyme by modifying known Fructoamino-acid-oxidase etc.The example of known Fructoamino-acid-oxidase like this etc. comprises by the bacteriogenic oxydase of corynebacterium (JP patent disclosure (Kohyo) 5-33997 B (1993) and JP patent disclosure (Kohyo) 6-65300 B (1994)), oxydase (JP patent disclosure (Kokai) 3-155780 A (1991)) by the generation of Aspergillus bacterial strain, belong to the oxydase (JP patent disclosure (Kokai) 7-289253 A (1995)) that bacterial strain produces by gibberella, oxydase (JP patent disclosure (Kokai) 7-289253 A (1995) and JP patent disclosure (Kokai) 8-154672 A (1996)) by the generation of fusarium bacterial strain, by the oxydase (JP patent disclosure (Kokai) 8-336386 A (1996)) of Penicillium bacterial strain generation, and Ketoamine oxidase (JP patent disclosure (Kokai) 5-192193 A (1993)).
For by modifying the oxydase that acquisitions such as known Fructoamino-acid-oxidase act on alpha-glycated dipeptides, the microbial exposure that can produce above-mentioned known Fructoamino-acid-oxidase etc. is in ultraviolet ray, X ray, radiation etc.Perhaps, make such oxydase and mutagens for example ethyl methane sulfonate, N-methyl-N '-nitro-N-nitrosoguanidine or nitrous acid contact, with the processing that suddenlys change.The oxidasic microorganism that generation acts on alpha-glycated dipeptides is selected from thus obtained mutant microbial.Yet in general, the oxydase that acts on alpha-glycated dipeptides can be introduced for example acquisitions such as gene of above-mentioned known Fructoamino-acid-oxidase of gene (hereinafter being called wild type gene) by suddenling change.Any wild type gene can be used to introduce sudden change equally, condition is that described wild type gene is the wild type gene of above-mentioned Fructoamino-acid-oxidase or affiliated oxydase analogue, for example, described wild type gene can be by introducing the oxydase that sudden change obtains to act on alpha-glycated dipeptides.
By following method, can measure oxidasic the tiring of fructosyl peptide that acts on alpha-glycated dipeptides.Tiring so also can be measured by other method.
(1) preparation of reagent
Reagent 1 (R1): the 1.0kU peroxidase (hereinafter is abbreviated as POD, produce by KIKKOMAN CORPORATION) and 100mg 4-aminoantipyrene (hereinafter be abbreviated as 4AA, by Tokyo Kasei Kogyo Co., Ltd. produces) be dissolved in the 0.1M potassium phosphate buffer (pH 8.0).The gained formulations prepared from solutions is become the constant volume of 1L.
Reagent 2 (R2): 500mg TOOS (N-ethyl-N-(2-hydroxyl-3-sulfo group propyl group)-meta-aminotoluene is produced by DOJINDO LABORATORIES) is dissolved in the ion exchanged water.The gained formulations prepared from solutions is become the constant volume of 100mL.
Reagent 3 (R3): 1.25g fructosyl Val-His (MW416, its preparation will be described below) is dissolved in the ion exchanged water.The gained formulations prepared from solutions is become the constant volume of 10mL.
(2) measure
100 μ L R2 are added among the 2.7mL R1.Add in addition that 100 μ L contain the oxidasic enzyme solution of fructosyl peptide and with the solution thorough mixing, then 37 ℃ of preheatings 5 minutes.
Subsequently, add 100 μ L R3 and with the solution thorough mixing.With spectrophotometer (U-2000A, by Hitachi, Ltd. produces) measure optical density at 555nm change (37 ℃ with the R3 reaction before and react 5 minutes after difference between the optical density of mensuration).In addition, carry out the similar program of contrast solution, just add 100 μ L ion exchanged waters and replace 100 μ LR3.Be reflected in the optical density of the amount of the pigment that produces under the various concentration of hydrogen peroxide standardized solution of previous preparation by drafting, obtain graphic representation.Based on such graphic representation, obtain to change the amount of corresponding hydrogen peroxide with optical density.These numerical value are used as the activity unit in the enzyme solution.The amount that produced 1 μ mol hydrogen peroxide at 1 minute is confirmed as 1U.
By making above-mentioned fructosyl peptide oxydase act on the alpha-glycated peptide that discharges by protease treatment of the present invention, can measure the amount of alpha-glycated peptide in the sample.And, by measuring the amount of alpha-glycated peptide in the sample, can compare the efficient that proteolytic cleavage removes alpha-glycated peptide.The oxidasic amount of fructosyl peptide used herein depends on the amount of alpha-glycated peptide contained in the treatment soln.For example, can be with fructosyl peptide oxydase with 0.1U/mL-50U/mL, the ultimate density of preferred 1U/mL-10U/mL adds.Make the oxydase generation make employed pH of time spent and for example can be pH 3-pH 11, preferred pH 5-pH 9.Consider the oxidasic optimum pH of fructosyl peptide, preferably regulate pH to reach the pH that is suitable for measuring with damping fluid.Yet as long as pH can make such oxydase generation effect, pH is not limited to such pH.The method of regulating pH is not particularly limited.The example of such buffer reagent comprises N-[three (methylol) methyl] glycine, phosphoric acid salt, acetate, carbonate, three (hydroxymethyl)-aminomethane, borate, Citrate trianion, glutamic acid dimethyl ester, trishydroxymethyl glycine and HEPES.And, if desired, also can be with the pH regulator of the treatment soln after the protease treatment at above-mentioned pH.
For example, action time scope at 1-120 minute, between preferred 1-30 minute, and depend on the amount of the glycated peptide that is used as substrate.As long as be enough to make fructosyl peptide oxydase to act on such peptide, all be operable any time.For example, the operative temperature scope is between 20 ℃-45 ℃.Can select to be used for the temperature of general enzymatic reaction aptly.
Also can measure the amount of the hydrogen peroxide that produces by the oxidasic effect of fructosyl peptide with any method.The example of such method comprises the method for electrically that uses oxygen electrode and preferably uses peroxidase and the enzymatic means of suitable chromogenic substrate.For example, in the present invention, the simple and enzymatic means that the time is short of preferred service routine is measured.The example of reagent of amount of measuring hydrogen peroxide with enzymatic means by 5mM-500mM preferred 50mM-100mM buffer reagent (preferred pH 4-pH 10), 0.01 mM-50mM and preferred 0.1mM-20mM as the 4-aminoantipyrene of chromogenic substrate, 0.1U/mL-50U/mL and preferred 1U/mL-20U/mL peroxidase etc. are formed.
The example that is used for buffer reagent of the present invention comprises N-[three (methylol) methyl] glycine, phosphoric acid salt, acetate, carbonate, three (hydroxymethyl)-aminomethane, borate, Citrate trianion, glutamic acid dimethyl ester, trishydroxymethyl glycine and HEPES.The example of chromogenic substrate, except the 4-aminoantipyrene, comprise ADOS (N-ethyl-N-(2-hydroxyl-3-sulfo group propyl group)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxyl-3-sulfo group propyl group) aniline), 10-(carboxyl methyl-aminocarboxyl)-3, two (dimethylamino) thiodiphenylamine (DA-67) of 7-, N-(carboxyl methyl-aminocarboxyl)-4,4 '-two (dimethylamino) diphenylamine (DA-64).And, if desired, in the scope of not damaging the object of the invention, various additives be can suitably add, solubilizing agent, stablizer, tensio-active agent (for example triton X-100, bridge 35, Tween80 or cholate), reductive agent for example (for example dithiothreitol (DTT), mercaptoethanol or L-halfcystine), bovine serum albumin, carbohydrate (for example glycerine, lactose or sucrose) etc. comprised.
When the mensuration of the amount of carrying out hydrogen peroxide, generally speaking, preferably carry out producing the step of hydrogen peroxide simultaneously by oxidasic effect.In the present invention, for example, preferably with fructosyl peptide oxydase with 0.1U/mL-50U/mL, the amount of preferred 1U/mL-10U/mL is added in the reagent of amount of said determination hydrogen peroxide.
These reagent can the exsiccant forms or are used with dissolved state, and the form that the carrier on perhaps also can film for example floods the paper (for example scraps of paper that can flood) with reagent is used.Also can will be used for measuring the fixing repeated use then of enzyme of reagent by standard method.For example, be used to the temperature range measured between 20 ℃-45 ℃.Such temperature can be selected from the temperature that is used for general enzymatic reaction aptly.Measuring required time can be selected aptly according to various condition determinations.For example, the such time range that is used to measure can be at 0.1-60 minute, between preferred 1-10 minute.Color development degree (variable quantity of optical density) with spectrophotometric determination said determination reagent.With result and the contrast of standard absorption degree.So, the contained glycated peptide or the amount of glycated protein are just measured in the sample.Also general automatic analyser can be used for measuring.
Subsequently, will method that measure the amount of the glycated peptide that discharges by HPLC be described.
If desired, with treatment soln centrifuging or membrane filtration, suitably concentrate then and/or dilution after, the treatment soln that will contain the glycated peptide of release is used for HPLC directly or indirectly and measures.As long as can measure the amount of above-mentioned glycated peptide, the HPLC that uses among the present invention can be any HPLC.
The example of reversed-phase HPLC post used herein comprises CAPCEL-PAK C-18 (by Shiseido Co., Ltd. produces), TSKgel ODS80Ts (being produced by TOSOHCORPORATION) and Shodex RSpak RP18-415 (being produced by SHOWADENKO K.K.).Ion-exchange HPLC post used herein comprises TSKgelSP-2SW and TSKgel CM-2SW (being produced by TOSOH CORPORATION).
After protease treatment solution is adsorbed on such post, with target glycated peptide eluent wash-out.As long as be suitable in the present invention measuring, eluent can be any eluent.The example that is used for such eluent of reversed-phase column comprises the mixing solutions of mixing solutions, phosphate buffered saline buffer and acetonitrile of the acetonitrile that contains trifluoroacetic acid and water and the mixing solutions of ammonia soln and acetonitrile.The example that is used for such eluent of ion exchange column comprises the mixing solutions of phosphate buffered saline buffer and NaCl solution and the mixing solutions of acetate buffer and acetonitrile.By using such elutriant, can carry out segmentation or gradient elution.The example of preferred eluent comprises that 0.1%TFA (trifluoroacetic acid)/water is to the gradient elution agent of 0.1%TFA/30% acetonitrile etc.Being used for post of the present invention, eluent, elution requirement (for example flow velocity of elution process, eluent and temperature) etc. is appropriate combination.Therefore, preferably condition is carried out such setting, so that the peak of the elution peak of the alpha-glycated peptide of target and other component is far away as much as possible under the described conditions.
As long as can measure glycated peptide, can use any method to measure the glycated peptide of using the eluent wash-out.The example of such method used herein comprises the method for detection in the optical density of the wavelength of 210nm, 215nm etc., sampling, each is surveyed peak and then gains is carried out the method for mass spectroscopy with the peak of determining target molecular weight, the product of wash-out is carried out the method for thin-layer chromatographic analysis and the method for carrying out colorimetric analysis with ninhydrin method or sugared staining then by time sampling elutriated fraction.For example, when using the method for surveying optical density, calculate the elution peak area of the glycated peptide of surveying by monitor.The elution peak area of result and reference material is compared, can measure the amount of glycated peptide and glycated protein then.
Implement best mode of the present invention
By with reference to preparation embodiment and embodiment, the present invention is further described specifically.Yet scope of the present invention is not subjected to the restriction of these embodiment.
(preparation embodiment) prepares glycosylated dipeptide
Be used for alpha-glycated dipeptides of the present invention by following method preparation.
The commercially available dipeptides of 7.0g (27.6mmol) (valyl Histidine (Val-His), by BACHEM, Switzerland produces) is dissolved in the 14mL water.5.8mL acetate is added in this solution, and then in about 50 ℃ of dissolvings, that continues allows its clarification.Subsequently, add 120mL ethanol and mix with solution, add then 14g (77.8mmol) glucose and with the solution thorough mixing.
Subsequently, with solution the sealing container in 80 ℃ the heating 6 hours, in this process, solution is stirred once in a while.Reaction soln becomes brown in time.Reaction soln is sampled in time.Suitably after the dilution, solution is carried out reversed-phased high performace liquid chromatographic analysis, tlc analysis or mass spectroscopy.Detect the generation of target glycosylated dipeptide thus.Generally speaking, can be by 6-10 hour thermal treatment with the glycosylated dipeptide of productive rate acquisition preferably.Subsequently, collect reaction soln then with its concentrated 15-30 doubly with Rotary Evaporators.Enriched material is adsorbed onto with 99.5% ethanol equilibrated silicagel column (volume: 2000mL).With 99.5% washing with alcohol of post, so that remove for example unreacted glucose of miscellaneous component with two times of column volumes.Carry out wash-out with 85% ethanol of 90% ethanol of 95% ethanol of 3 times of column volumes, 3 times of column volumes, 3 times of column volumes and 80% ethanol of 3 times of column volumes in succession then.Each elutriated fraction is analyzed with tlc, reversed-phased high performace liquid chromatographic etc.Collection contains the fraction of the 95%-90% ethanol elution of target fructosyl Val-His.The product of collecting is concentrated and drying with Rotary Evaporators, obtain the partially purified product of about 3g thus.As the result of mass spectroscopy, find that the molecular weight of the product of purifying is MW 416, its molecular weight with fructosyl Val-His is consistent.And the structure of product is confirmed by nuclear magnetic resonance spectrometry analysis.By standard method, spent ion exchange resin improves purification degrees, with partially purified product absorption and take off suction.Gains are used for subsequently test.And, by with the similar approach of above-mentioned use Val, obtain the partially purified product of fructosyl Val.
Embodiment
(embodiment 1) discharges glycosylated dipeptide by saccharification six peptides
In order to screen the proteolytic enzyme that can effectively excise alpha-glycated dipeptides, make the listed proteolytic enzyme of table 1 act on alpha-glycated six peptides (fructosyl Val-His-Leu-Thr-Pro-Glu; By PEPTIDEINSTITUTE, INC. produces).Use fructosyl peptide oxydase or Fructoamino-acid-oxidase to measure the amount of consequent product.
＜preparation mmp reaction sample 〉
1.8mM alpha-glycated six peptides: 12 μ l
20mg/ml protein enzyme solution (when this concentration can not reach,, perhaps when proteolytic enzyme is liquid state, using same concentration): 8 μ l with this solution of high as far as possible prepared at concentrations
100mM potassium phosphate buffer pH 8.0 (pH is suitably changed according to optimum protein enzyme pH): 4 μ l
With the said components thorough mixing, make it then 37 ℃ of reactions 2 hours.Gains are 90 ℃ of heat treated 3 minutes, centrifugal then, obtain to be separated into the supernatant liquor of mmp reaction sample thus.In addition, replace substrate to carry out similar program, prepare dummy thus with distilled water.
＜be used for measuring the solution of the reaction of the glycosylated dipeptide of mmp reaction sample and glycated amino acid 〉
100mM potassium phosphate buffer pH 8.0
45mM 4AA
0.5mM TOOS
1U/ml POD (producing) by KIKKOMAN CORPORATION
0.1U/ml fructosyl peptide oxydase or Fructoamino-acid-oxidase
The above-mentioned reaction soln that 145 μ l are used for measuring the amount of glycosylated dipeptide and glycated amino acid is assigned to the hole of microtiter plate.Add the above-mentioned mmp reaction sample of 5 μ l and with this solution thorough mixing.With gains at 555nm (A
0) measure.Subsequently, 30 ℃ of incubations 20 minutes, then with gains at 555nm (A
1) measure.Replace the mmp reaction sample to carry out similar program with dummy, obtain A thus
0Blank and A
1Blank.Following formula is represented as the effect of the proteolytic enzyme that absorbs variation to alpha-glycated six peptides.
Δ A=(A
1-A
0)-(A
1Blank-A
0Blank)
In addition, four kinds of oxydase are used to measure in the above-mentioned reaction of amount of saccharification product below: as oxidasic FPOX-C of fructosyl peptide and FPOX-E (both are all produced by KIKKOMANCORPORATION); FAOX (producing) as Fructoamino-acid-oxidase by KIKKOMAN CORPORATION; And FLOD (producing) by Asahi KaseiCorporation.These oxydase are different aspect substrate specificity.Particularly, FPOX-C and FPOX-E act on fructosyl Val-His and fructosyl Val, and FAOX and FLOD only act on fructosyl Val.Therefore, can conclude that when fructosyl Val-His was cut by above-mentioned protease treatment, the optical density that will observe FPOX-C and FPOX-E changed.Can also conclude that if fructosyl Val is cut, the optical density that will observe FPOX-C, FPOX-E, FAOX and FLOD changes.
Table 1 display result (unit; MAbs)
| The proteolytic enzyme title | Origin | FPOX -C | FPOX -E | FAOX | FLOD |
| IP enzyme KIKKOMAN AO Proteinase K IKKOMAN peptase KIKKOMAN Molsin KIKKOMAN protease A 5 KYOWAKASEI Umamizyme Amano protease A Amano protease M Amano protease P Amano Sumizyme MP Shin Nihon Chemical Sumizyme LP-20 Shin Nihon Chemical Sumizyme LPL Shin Nihon Chemical Sumizyme AP Shin Nihon Chemical | Aspergillus | 38 63 65 5 21 37 78 85 126 142 71 8 5 | 51 46 50 8 14 20 51 63 89 105 52 6 5 | 1 0 1 1 0 0 0 0 2 0 0 0 2 | 2 0 0 1 0 0 0 4 1 0 1 0 2 |
| Proteolytic enzyme 6 Fluka | 119 | 87 | 0 | 0 | |
| Peptase R Amano Newlase F Amano | Rhizopas | 65 2 | 50 1 | 0 0 | 1 0 |
| Dispase Roche subtilopeptidase A Boehringer protease N Fluka protease type VII Sigma protease, the withered Fluka grass of bacterium bacillus protease protein enzyme N Amano Proleather FG-F Amano protease S Amano protease type X Sigma thermolysin DAIWA KASEI pronase E Kaken Pharmaceutical neutral proteinase TOYOBO | Bacillus | 63 10 114 12 41 63 4 129 73 73 31 132 | 32 6 82 10 33 44 4 87 53 51 11 105 | 1 1 0 2 1 0 0 0 0 2 1 0 | 2 0 0 2 2 0 0 0 1 2 3 0 |
| Pronase Boehringer proteolytic enzyme type XIV Sigma Sumizyme MP TOYOBO | Chain enzyme bacteria belongs to | 35 143 39 | 17 84 29 | 4 2 0 | 3 0 0 |
| Proteinase K Roche Proteinase K Wako | Tritirachium | 79 36 | 73 22 | 2 0 | 1 0 |
| AP-I Takara lysyl endopeptidase Wako | Achromobacter | 1 3 | 0 1 | 0 2 | 1 1 |
| Asp-N Takara | Pseudomonas | 0 | 0 | 0 | 0 |
| Pfu proteolytic enzyme Takara deblocking aminopeptidase Takara | Pyrococcus | 3 0 | 2 1 | 0 0 | 0 0 |
| PD enzyme KIKKOMAN | Penicillium | 1 | 2 | 1 | 1 |
| Aminopeptidase T Wako | Thermus | 0 | 0 | 2 | 0 |
| V8 proteolytic enzyme Takara V8 proteolytic enzyme Wako Glu-C Boehringer | The glucose Coccus | 1 3 4 | 2 0 2 | 1 0 3 | 2 0 1 |
| Papoid Roche papoid Wako papoid Sigma papoid W40 Amano papoid Asahi | Papaya belongs to | 90 51 52 49 55 | 69 30 27 21 27 | 3 0 0 1 2 | 1 0 1 0 0 |
| Ficin Sigma | Fructus Fici | 15 | 7 | 3 | 1 |
| Bromeline F Amano bromeline Sigma | Pineapple | 4 4 | 2 2 | 0 1 | 0 0 |
| The Sigma of Sigma cathepsin D of Sigma cathepsin C of pancreatin Wako cathepsin B elastoser Boehringer m-calpain Nacalai μ-calpain Nacalai trypsase Wako trypsase Sigma trypsase Takara alpha-chymotrypsin Sigma alpha-chymotrypsin Sigma pepsin Wako pepsin Sigma Aminopeptidase M Roche leucine amino peptidase Sigma Carboxypeptidase A Sigma protaminase Sigma N acyl amino acyl peptide Takara hydrolase | Pig pancreas ox spleen ox spleen ox spleen pig pancreas pig kidney swine erythrocyte pig pancreas pig pancreas pig pancreas pig pancreas pig pancreas pig pig pig pancreas pig pig pancreas pig pancreas pork liver | 28 21 0 2 1 1 1 2 2 5 1 0 1 0 1 3 0 3 0 | 17 16 1 1 1 1 1 2 1 1 0 2 2 0 1 3 0 3 0 | 0 1 2 0 1 1 1 1 0 0 1 2 2 0 1 1 0 1 0 | 1 1 1 1 0 0 0 2 0 1 2 1 1 0 1 1 0 2 1 |
When coming evaluating protein enzyme active with FAOX or FLOD by surveying the product that produces (surveying fructosyl Val), it approximately all is 0 that the optical density of all proteolytic enzyme situations of acquisition changes.This shows, is considered to have very weak excision fructosyl Val activity from the various proteolytic enzyme of glycated protein or glycated peptide excision fructosyl Val.Various proteolytic enzyme like this are leucine aminopeptidase(LAP)s, take off and seal aminopeptidase, N-acyl amino acyl group-peptidohydrolase and cathepsin C's (all being disclosed in JP patent disclosure (Kokai) 2002-315600 A); Aminopeptidase, carboxypeptidase, trypsinase, Quimotrase, subtilisin, Proteinase K, papoid, cathepsin B, stomach en-, thermolysin, lysyl endopeptidase, proleather and bromeline (all be disclosed in international monopoly and disclose 97/13872 reprint); And serine carboxypeptidase (being disclosed in JP patent disclosure (Kokai) 2001-57897 A).
On the contrary, when surveying, (survey fructosyl Val-His), under the situation of following enzyme, observe the intensive optical density and change: as IP enzyme, AO proteolytic enzyme, peptase, protease A 5, umamizyme, protease A, proteolytic enzyme M, protease P, sumizyme MP, sumizyme LP-20 and the proteolytic enzyme 6 of the enzyme that derives from Aspergillus with FPOX-C or FPOX-E; Peptase R as the enzyme that derives from Rhizopas; Dispase, subtilisin, protease N, proteolytic enzyme VII type, proteolytic enzyme (Bacterial), protease N, proteolytic enzyme X type, thermolysin, pronase E and neutral protease as the enzyme that derives from bacillus; Pronase, proteolytic enzyme XIV type and Sumizyme MP as the enzyme that derives from the chain enzyme bacteria genus; Proteinase K as the enzyme that derives from Tritirachium; Papoid and ficin as the enzyme that derives from plant; Pancreatin and cathepsin B with the enzyme that derives from animal.
Under the situation of following enzyme, observe more weak optical density and change: as molsin, sumizyme LPL and the sumizyme AP of the enzyme that derives from Aspergillus; Proleather FG-F as the enzyme that derives from bacillus; Glu-C as the enzyme that derives from the glucose Coccus; With as deriving from the bromeline of the enzyme of plant.As described above, prove that by above-mentioned protease treatment alpha-glycated dipeptides can be excised from alpha-glycated six peptides effectively.
(embodiment 2) proteolytic enzyme is with the activity of short reaction times excision glycosylated dipeptide
In order to screen the proteolytic enzyme that in the short reaction times, can excise alpha-glycated dipeptides effectively, carry out the test that does not change its various conditions similar to Example 1.Yet, the reaction times of proteolytic enzyme was foreshortened to 5 minutes by 2 hours.The amount of alpha-glycated dipeptides and alpha-glycated amino acid is measured in the reaction back.The result represents (being similar to embodiment 1) by following equation
Δ A=(A
1-A
0)-(A
1Blank-A
0Blank)
Simultaneously the result is summarized in table 2 (unit; MAbs).
Table 2
| The proteolytic enzyme title | The source | FPOX -C | FPOX -E | FAOX | FLOD |
| AO Proteinase K IKKOMAN peptase KIKKOMAN Molsin KIKKOMAN protease P Amano Sumizyme Shin Nihon MP Chemical | Aspergillus | 24 0 1 119 124 | 19 1 0 91 95 | 2 0 0 0 1 | 0 0 1 1 0 |
| Dispase Roche protease N Fluka proteolytic enzyme S Amano | Bacillus | 98 105 119 | 71 84 90 | 0 0 0 | 1 0 0 |
| Proteinase K Roche | Tritirachium | 26 | 20 | 2 | 2 |
| Papoid Roche | Papaya belongs to | 89 | 64 | 0 | 0 |
As with JP patent disclosure (Kokai) 2003-235585A in disclosed proteolytic enzyme (the AO proteolytic enzyme that derives from Aspergillus, peptase and molsin) relatively result, the proteolytic enzyme (protease P and sumizyme MP) that derives from Aspergillus shows than high about 5 times optical density variation under the AO proteolytic enzyme situation, derive from the proteolytic enzyme (Dispase of bacillus, protease N and proteolytic enzyme S) show that the optical density than high 4-5 under the AO proteolytic enzyme situation times changes, the proteolytic enzyme (Proteinase K) that derives from Tritirachium show almost with AO proteolytic enzyme situation under the optical density that equates change, and the proteolytic enzyme (papoid) that derives from plant shows than high about 4 times optical density variation under the AO proteolytic enzyme situation.Therefore, prove that the above-mentioned proteolytic enzyme of use can excise glycosylated dipeptide in the short period of time effectively.This shows that the amount of measuring glycated protein or glycated peptide in the short period of time with higher susceptibility is possible.
(embodiment 3) confirm the release of glycosylated dipeptide with HPLC
Above-mentioned alpha-glycated six peptides are dissolved in the water to prepare 5 mM solution.Add 0.01mL protein enzyme solution (papoid (producing), ficin (producing) or Dispase (producing)) and 0.09mL damping fluid (0.1M) and mix with each above-mentioned solution of 0.1mL by Roche by Sigma-Aldrich Corporation by Roche.Like this, carry out protease treatment.Said mixture was reacted 60 minutes at 37 ℃.Subsequently, suitably concentrated and dilution is carried out HPLC then and is measured with the solution of each processing.In order to carry out HPLC (reversed-phased high performace liquid chromatographic), use CAPCEL-PAK C-18 (by Shiseido Co., Ltd. produces).Gains are carried out gradient elution with 0.1%TFA (trifluoroacetic acid)/water to 0.1%TFA/30% acetonitrile as eluent.As reference material, use alpha-glycated dipeptides (fructosyl Val-His).As a result of, confirm that alpha-glycated dipeptides (fructosyl Val-His) is released by handling with each proteolytic enzyme (papoid, ficin or Dispase) in the treatment soln.
(embodiment 4) use the amount of proteolytic enzyme and oxidase assay saccharification six peptides
By following test, determine whether the proteolytic enzyme of screening among energy enough embodiment 1 and 2 and the amount of fructosyl peptide oxidase assay saccharification six peptides.
＜mmp reaction 〉
1.8mM alpha-glycated six peptides
3U/ml papoid (producing): 8 μ l by Roche
Water (to cumulative volume 24 μ l)
The amount of above-mentioned alpha-glycated six peptides that are used to react is respectively 0,1,2,3,4,5,6 and 7 μ l samples.Adding 8 μ l papoids and water to cumulative volume is 24 μ l.Allow solution 37 ℃ of reactions 10 minutes, heat-treated 5 minutes, carry out centrifugally then, obtain supernatant liquor thus as the mmp reaction sample at 90 ℃.In addition, replace substrate to carry out similar program, prepare dummy thus with distilled water.
＜be used for measuring the solution of the reaction of mmp reaction sample glycosylated dipeptide 〉
100mM potassium phosphate buffer pH 8.0
45mM 4AA
0.5mM TOOS
1U/mlPOD (producing) by KIKKOMAN CORPORATION
0.1U/ml fructosyl peptide oxydase, FPOX-C (producing) by KIKKOMANCORPORATION
The solution that 145 μ l are used for measuring the reaction of above-mentioned glycosylated dipeptide is assigned to the hole of microtiter plate.The above-mentioned mmp reaction sample of 5 μ l is added in each hole.Behind the thorough mixing, with gains at 555nm (A
0) measure.Subsequently, 30 ℃ of incubations 20 minutes, continue at 555nm (A
1) measure.In addition, replace the mmp reaction sample to carry out similar program, obtain A thus with dummy
0Blank and A
1Blank.When proteolytic enzyme is represented with the optical density variation the effect of alpha-glycated six peptides, obtain following formula.
Δ A=(A
1-A
0)-(A
1Blank-A
0Blank)
Fig. 1 is presented at the measurement result of the amount of alpha-glycated six peptides of each concentration.As shown in Figure 1, between the concentration of Δ A and alpha-glycated six peptides, there is linear relationship.Particularly, show by above-mentioned protease treatment and excise alpha-glycated dipeptides, can measure the amount of alpha-glycated six peptides at short notice with the height accurate way.
As mentioned above, show about known and handle alpha-glycated six peptides that obtain by HbA1c being carried out endo-protease Glu-C, by alpha-glycated six peptides are carried out protease treatment of the present invention and need not carry out electrocapillary phoresis or analytical reagent composition, make enzymatic more easily HbA1c measure and become possibility.
(embodiment 5) prepare alpha-glycated dipeptides by handling HbA1c with Glu-C and neutral protease
Whether can produce alpha-glycated dipeptides by following evidence by making Glu-C and neutral protease act on HbA1c.
＜mmp reaction 〉
14.4%HbA1c solution (by KYOWA MEDEX CO., LTD. produces): 44 μ l
0.5mg/ml Glu-C (by Wako Pure Chemical Industries, Ltd. produces): 36 μ l
150Mm ammonium acetate (pH 4.0): 8 μ l
Blended solution is incubated overnight at 37 ℃.Subsequently, 352 μ l neutral protease (2.4U/ml Dispases; Produce by Roche) be added in the solution, then with solution stirring.And, solution is incubated overnight at 37 ℃.Then with solution 92 ℃ of heat treated 5 minutes, afterwards with 12, centrifugal 5 minutes of 000rpm obtains the supernatant liquor as sample thus.In addition, replace Glu-C or Dispase to carry out similar program, prepare dummy thus with distilled water.
The reaction of contained alpha-glycated dipeptides in the＜mensuration mmp reaction sample 〉
Be prepared as follows the solution that is used for assaying reaction.In addition, FAOX among the use R1 and catalase are to remove the glycated amino acid that is entrained in the sample.
R1：
50mM POPSO damping fluid (pH 7.5) (producing) by DOJINDO LABORATORIES
5U/ml FAOX (producing) by KIKKOMAN CORPORATION
300U/ml catalase (producing) by KIKKOMAN CORPORATION
R2：
100mM Tris-HCl damping fluid (pH7.5) is by Nacalai Tesque, and Inc. produces)
0.1mM DA-64 (by Wako Pure ChemicalIndustries, Ltd. produces)
10mM Ca-EDTA (producing) by DOJINDO LABORATORIES
150U/ml POD (producing) by KIKKOMAN CORPORATION
0.15%NaN
3(by Wako Pure ChemicalIndustries, Ltd. produces)
40U/ml fructosyl peptide oxydase, FPOX-E (producing) by KIKKOMANCORPORATION
216 μ l R1 are added in the 30 μ l samples.React after 5 minutes, add 80 μ l R2 and mix with solution.Allow solution 37 ℃ of reactions 5 minutes.Be determined at the optical density (Δ Abs) (and the difference between the optical density of before the R2 reaction and reaction back mensuration) of the increase of 750 nm with Hitachi automatic analyser (model 7070).Like this, the optical density of increase is found to be 0.007.On the contrary, Δ Abs is 0 under the situation of dummy.In addition, even when FPOX-C (being produced by KIKKOMAN CORPORATION) also being obtained similar result during as fructosyl peptide oxydase.
Therefore, prove by producing alpha-glycated dipeptides with Glu-C and neutral protease processing HbA1c.Also proof can be measured the amount of the alpha-glycated dipeptides of generation with FPOX-E and-C simultaneously.
(embodiment 6) prepare alpha-glycated dipeptides by handling HbA1c with neutral protease
Make Glu-C and neutral protease act on HbA1c among the embodiment 5.In the present embodiment, upcheck with following experiment and make neutral protease act on HbA1c separately whether can to produce alpha-glycated dipeptides.
＜mmp reaction 〉
14.4%HbA1c solution (by KYOWA MEDEX CO., LTD. produces): 88 μ l
2.4U/ml neutral protease (Dispase; Produce by Roche): 352 μ l
Blended solution is incubated overnight at 37 ℃.Then with solution 92 ℃ of heat treated 5 minutes, then with 12, centrifugal 5 minutes of 000rpm obtains the supernatant (amount of used HbA1c is 2 times of institute's consumption among the embodiment 5 in this) as sample thus.In addition, replace neutral protease similarly to test, prepare dummy thus with distilled water.
The reaction of contained alpha-glycated dipeptides in the＜mensuration mmp reaction sample 〉
Used identical among used R1 and R2 and the embodiment 5 in this.
216 μ l R1 are added in the 30 μ l samples.Handle after 5 minutes, add 80 μ l R2 and mix with solution.Allow solution 37 ℃ of reactions 5 minutes.As a result, find that the optical density (Δ Abs) (and before the R2 reaction and the difference between the optical density of being measured after the reaction) in the increase that 750nm measures is 0.007.On the contrary, Δ Abs is 0 under the situation of dummy.The amount of used HbA1c is 2 times of institute's consumption among the embodiment 5 in this protease treatment.Yet, observe Δ Abs (=0.007) and equate with Δ Abs among the embodiment 5.And, even also observe similar result when being used as fructosyl peptide oxydase as FPOX-C (producing) by KIKKOMAN CORPORATION.Therefore, prove that handling HbA1c by independent use neutral protease can produce alpha-glycated dipeptides.Also proof can be surveyed the alpha-glycated dipeptides of generation with FPOX-E and-C simultaneously.
(embodiment 7) measure the amount of HbA1c with FPOX
HbA1c is contrasted (be used to measure the caliberator of the amount of factor HbA1c; By KYOWAMEDEX CO., LTD. produces) be dissolved in the solution of dilution of sample (specimen) (by KYOWAMEDEX CO., LTD. produces).Five kinds of HbA1c solution of preparation different concns (0.0%, 4.1%, 7.8%, 11.3% and 14.4%).Carry out following step with these solution.
＜mmp reaction 〉
Each HbA1c solution: 44 μ l
2.4U/ml neutral protease (Dispase; Produce by Roche): 176 μ l
Blended solution is incubated overnight at 37 ℃.Solution 92 ℃ of thermal treatments 5 minutes, with 12,000 rpm centrifugal 5 minutes then, is obtained the supernatant liquor as sample thus.In addition, replace neutral protease to carry out similar step, prepare dummy thus with distilled water.
The reaction of contained alpha-glycated dipeptides in the＜mensuration mmp reaction sample 〉
Used identical among used R1 and R2 and the embodiment 5 in this.
216 μ l R1 are added in 30 each sample of μ l.Handle after 5 minutes, add 80 μ l R2 and mix with solution.Allow this solution 37 ℃ of reactions 5 minutes.As a result of, in optical density (Δ Abs) that 750nm measure to increase (and before the R2 reaction and the difference between the optical density of being measured after the reaction).Fig. 2 show the concentration of HbA1c and the Δ Abs that obtains by this method between relation.Fig. 2 shows between the amount of hydrogen peroxide of HbA1c concentration and generation and has dependency.In addition, the Δ Abs that obtains by similar step in dummy always 0, is to replace neutral protease to be added in the HbA1c solution of different concns distilled water in dummy.
Whole publication, patents and patent applications that this paper quotes all quote in full does reference.
Industrial applicibility
According to the present invention, the method for preparing alpha-glycated dipeptides is provided, described method can prepare alpha-glycated dipeptides by glycated protein or glycated peptide simply, fast and efficiently. And, according to the present invention, providing the method for the amount of measuring alpha-glycated dipeptides, described method can be measured at short notice in highly accurate mode the amount of alpha-glycated dipeptides. Such assay method is especially effective aspect the mensuration of the amount of HbA1 c for example at the terminal glycated peptide of N-, albumen, protein subunit etc.
Brief description of the drawings
Fig. 1 has shown the result of the amount of measuring alpha-glycated six peptides.
Fig. 2 has shown the result who measures the amount of HbA1c.
Claims (7)
1. the method for preparing alpha-glycated dipeptides, described method comprise makes proteolytic enzyme act on terminal glycated peptide of N-or the terminal glycated protein of N-.
2. the method for the alpha-glycated dipeptides of preparation of claim 1, the terminal glycated peptide of wherein said N-is fructosyl Val-His-Leu-Thr-Pro-Glu.
3. the method for the alpha-glycated dipeptides of preparation of claim 1, the terminal glycated protein of wherein said N-is a glycolated hemoglobin.
4. the method for claim 1,2 or 3 the alpha-glycated dipeptides of preparation, wherein said proteolytic enzyme is that one or more are selected from following proteolytic enzyme: by the proteolytic enzyme of microorganisms such as Aspergillus, bacillus, Rhizopus, Tritirachium, glucose Coccus, chain enzyme bacteria genus, by the animal proteolytic enzyme that produces of pig and ox for example, and by the plant proteolytic enzyme that produces of papaya genus, Fructus Fici and pineapple for example.
5. the method for claim 1,2 or 3 the alpha-glycated dipeptides of preparation, wherein said proteolytic enzyme is that one or more are selected from following proteolytic enzyme: subtilisin, pronase, Dispase, neutral protease, Sumizyme MP, Proteinase K, papoid, ficin, bromeline, pancreatin, Glu-C and kethepsin.
6. the method for the alpha-glycated dipeptides of preparation of claim 1-5, wherein said alpha-glycated dipeptides is a fVH.
7. measure the method for the amount of alpha-glycated dipeptides, described method comprises makes fructosyl peptide oxydase act on the alpha-glycated dipeptides that obtains according to the preparation method of claim 1-6, measures the amount of the hydrogen peroxide that produces then.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105683390A (en) * | 2013-10-25 | 2016-06-15 | 龟甲万株式会社 | HbA1c measurement method using amadoriase acting on glycated peptides |
| CN113466384A (en) * | 2021-08-11 | 2021-10-01 | 昆明和合医学检验所有限公司 | Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood |
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| EP3270711B1 (en) * | 2015-03-19 | 2018-12-05 | Nestec S.A. | Sugar-dipeptide conjugates |
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| JP3949854B2 (en) * | 1999-10-01 | 2007-07-25 | キッコーマン株式会社 | Method for measuring glycated protein |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105683390A (en) * | 2013-10-25 | 2016-06-15 | 龟甲万株式会社 | HbA1c measurement method using amadoriase acting on glycated peptides |
| CN105683390B (en) * | 2013-10-25 | 2021-02-26 | 龟甲万株式会社 | HbA1c measurement method using amadoriase acting on glycosylated peptide |
| CN113466384A (en) * | 2021-08-11 | 2021-10-01 | 昆明和合医学检验所有限公司 | Liquid chromatography tandem mass spectrometry quantitative detection method for content of glycosylated hemoglobin in whole blood |
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