WO2005111229A1 - Substrat pour la détection d'une activité enzymatique et procédé de détection d'une activité enzymatique avec celui-ci - Google Patents

Substrat pour la détection d'une activité enzymatique et procédé de détection d'une activité enzymatique avec celui-ci Download PDF

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Publication number
WO2005111229A1
WO2005111229A1 PCT/JP2005/001258 JP2005001258W WO2005111229A1 WO 2005111229 A1 WO2005111229 A1 WO 2005111229A1 JP 2005001258 W JP2005001258 W JP 2005001258W WO 2005111229 A1 WO2005111229 A1 WO 2005111229A1
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Prior art keywords
substrate
compound
fluorescent group
enzyme
enzyme activity
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PCT/JP2005/001258
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English (en)
Japanese (ja)
Inventor
Norikazu Nishino
Tamaki Kato
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Kitakyushu Foundation For The Advancement Of Industry, Science And Technology
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Priority claimed from JP2004144191A external-priority patent/JP2004357706A/ja
Application filed by Kitakyushu Foundation For The Advancement Of Industry, Science And Technology filed Critical Kitakyushu Foundation For The Advancement Of Industry, Science And Technology
Publication of WO2005111229A1 publication Critical patent/WO2005111229A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the present invention relates to a substrate for detecting an enzyme activity for detecting an enzyme activity and a method for detecting an enzyme activity using the same.
  • Patent Document 1 describes "a fluorescent probe in which both ends of a substrate peptide cleaved specifically by caspase, which is one of proteolytic enzymes, are modified with a fluorescent group that undergoes fluorescence resonance energy transfer.” ing.
  • Patent Document 1 JP-A-2000-316598
  • Patent Document 1 The technique disclosed in (Patent Document 1) is that, as a result of the substrate peptide of a fluorescent probe placed in a solution being cleaved by an enzyme, the fluorescent groups at both ends of the substrate peptide generate fluorescence resonance energy, and the fluorescence wavelength and fluorescence Since the intensity changes, the enzyme activity is detected by measuring the intensity.
  • a method for measuring enzyme activity there is a method in which a sample solution is injected into a cell having an opening formed in a microplate for fluorescence measurement, and high-speed measurement is performed by simultaneously measuring fluorescence on a plate reader. In this method, the opening area of the opening of the cell of the fluorescence measurement microplate needs to be formed large enough to inject the solution.
  • the present invention solves the above-mentioned conventional problems, and it is possible to detect enzyme activity only by measuring the fluorescence intensity or the like of a substrate using a change in the fluorescence intensity or the like as an index.
  • a substrate for enzyme activity detection that can detect enzyme activity even with a sample solution of (1) and can dramatically increase the degree of integration of the detection section without forming a cell or the like into which the sample solution is injected. With the goal.
  • the present invention can detect enzyme activity even in a small amount of a sample solution, can increase the detection sensitivity, can shorten the measurement time, can improve workability, and increase the measurement efficiency.
  • a method for detecting an enzyme activity wherein the enzyme activity of a sample solution containing a plurality of enzymes can be measured in a short time by using an enzyme activity detection substrate in which a fluorescent group is bonded to each of different types of peptides and the like.
  • a substrate for enzyme activity detection and a method for detecting enzyme activity using the same according to the present invention have the following configurations.
  • the substrate for enzyme activity detection according to claim 1 of the present invention comprises a substrate and a substrate directly bound to the substrate, or bound to the substrate via a first compound having one end fixed to the substrate. It has a configuration comprising: one fluorescent group; and a second compound bonded to the first fluorescent group with a peptide bond that is cleaved by an enzyme.
  • the first fluorescent group bonded to the substrate and the second fluorescent compound bonded to the first fluorescent group by a peptide bond that is cleaved by an enzyme the first fluorescent group is reacted with the enzyme to form a peptide bond.
  • the second compound is released. Since the fluorescence wavelength of the first fluorescent group from which the second compound has been released or the fluorescence intensity at a predetermined wavelength is different from that of the first fluorescent group in which the second compound is peptide-bonded, the enzyme activity is determined based on the change in the fluorescence intensity or the like. Can be detected.
  • the enzyme activity can be detected simply by contacting a very small amount of a sample solution containing the enzyme and measuring the fluorescence intensity of the substrate, and the enzyme activity can be detected.
  • the degree of integration of the detection unit can be dramatically increased.
  • Enzyme activity can be detected simply by contacting a very small amount of sample solution containing the enzyme, so that a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. It can be carried out.
  • the substrate may be a chlorinated honolem, a halogenated hydrocarbon such as dichloromethane, an ester such as ethyl acetate, N, N-dimethylformamide, or dimethyl used in a condensation reaction for forming a peptide bond.
  • Polar organic solvents such as sulfoxide, ethers such as dioxane and tetrahydrofuran, alcohols such as methanol and ethanol, and synthetic resins (polystyrene, etc.) insoluble in solvents such as pyridine, glass, etc.
  • a sheet formed in a plane or the like is used.
  • a commercially available carrier for solid phase organic synthesis such as Lantern Series (registered trademark) manufactured by Mimotobus Inc. can also be used.
  • amino acids As the first compound and the second compound, amino acids, peptides in which two or more amino acids are peptide-bonded, and the like are used.
  • the first compound and the second compound can be synthesized by immobilizing a C-terminal amino acid on a substrate and extending the peptide from the C-terminus using a conventional peptide synthesis method such as a solid phase method.
  • a sequential extension method in which the target amino acid sequence is sequentially extended from the C-terminal side to the N-terminal side
  • a fragment condensation method in which a plurality of short peptide fragments are synthesized and extended by coupling between the peptide fragments, etc. Can be used.
  • the peptide can be synthesized using a peptide synthesizer by introducing 91-fluorenylmethyloxycarbonyl (Fmoc) amino acid, t_butyloxycarbonyl (Boc) amino acid, or the like.
  • peptide bonds can be produced using a protease, or can be synthesized using genetic engineering.
  • a known method can be used, for example, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride.
  • DCC method, DCC_additive method, active ester method, carbonyldiimidazole method, redox method, method using Woodward reagent K, and the like Before conducting the condensation reaction, do not participate in the reaction in amino acids or peptides by known means. It can protect carboxyl groups, amino groups, etc., and can also activate carboxynole groups and amino groups involved in the reaction.
  • the first compound may be an amino acid or an amino acid sequence which is bound to the substrate by an amide bond, an ester bond, an ether bond, a thioether bond, a urethane bond or the like which is not cleaved by the enzyme, and which is not cleaved by the enzyme in the sample solution.
  • a compound such as a peptide is used.
  • the first fluorescent group those whose fluorescence wavelength or fluorescence intensity changes before and after the peptide bond between the first fluorescent group and the second compound is cleaved by the enzyme are used.
  • a peptide bond with the second compound is a non-fluorescent substance in a specific wavelength region, it emits fluorescence in the specific wavelength region when the peptide bond is cleaved to release the second compound.
  • Fluorescent groups for example, 4-methylcumaryl 7_amide (MCA), 7-amino_4_carboxymethylcoumarin (ACC), p-nitroalilide, ⁇ -naphthylamide, ⁇ -naphthyl ester and the like are preferably used.
  • an amino acid in which the peptide bond at the C-terminal side is selectively cleaved by the enzyme is used.
  • the second compound bonded to the first fluorescent group can be released to change the fluorescence intensity of the first fluorescent group in a specific wavelength region.
  • the second compound by forming the second compound with a peptide chain having a predetermined length (for example, about 15 mm) or more, a relatively long peptide chain is required for the cleavage action, rather than having high substrate specificity for each amino acid.
  • Enzymes such as elastase can also be detected, and the types of enzymes that can be detected can be increased.
  • enzymes include serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, kallikrein, perokinase, and elastase; aspartic proteases such as pepsin, cathepsin D, renin, and chymosin; Endopeptidase that cleaves internal peptide bonds of meta-oral proteases such as thermolysin and cystine proteases such as cathepsin B, H, L, and calpain; blood coagulation A system protease, a capture system protease, a hormone processing enzyme and the like are used.
  • serine proteases such as trypsin, chymotrypsin, thrombin, plasmin, kallikrein, perokinase, and elastase
  • aspartic proteases such as pepsin, cathepsin D, renin, and chymosin
  • the substrate for enzyme activity detection according to claim 2 of the present invention comprises a substrate, and the substrate is introduced into the side chain of a third compound having one end fixed to the substrate, via the third compound. Or a fifth compound bound to the third compound or a peptide bond that is cleaved by the enzyme with the third compound or the second fluorescent group bound to the substrate and bound directly to the substrate. And a third fluorescent group that binds to the fifth compound and has the second fluorescent group and fluorescence resonance energy transfer.
  • a second fluorescent group bound to the substrate by binding to the third or fourth compound, a fifth compound bound to the third or fourth compound by a peptide bond that is cleaved by an enzyme Since it has a second fluorescent group that binds to the fifth compound and a third fluorescent group that exhibits fluorescence resonance energy transfer, when the peptide bond is cleaved by the enzyme, the second fluorescent group and the third fluorescent group are dissociated. As the distance increases, the fluorescence resonance energy transfer does not occur, and the enzyme changes the spectrum from the fluorescence spectrum from the second fluorescent group (or the third fluorescent group) to the fluorescent spectrum from the third fluorescent group (or the second fluorescent group). It can be used as an activity measurement index, whereby the enzyme activity can be detected using changes in fluorescence intensity or the like as an index.
  • the fluorescence resonance energy transfer means that when two fluorescent compounds are present at a position close to each other in distance, the fluorescent spectrum of one of the two fluorescent compounds (called a donor) and the other (the donor) If the excitation spectrum overlaps with the excitation spectrum of the donor, the energy of the excitation wavelength of the donor is applied, and the fluorescence of the donor that should be observed is attenuated, and the fluorescence of the axceptor is observed instead.
  • the second fluorescent group and the third fluorescent group a combination of a donor and an acceptor in which fluorescence resonance energy transfer occurs can be used.
  • a third fluorescent group (or a second fluorescent group) which is an atomic group having an absorption band in a wavelength range overlapping with the fluorescent wavelength of the second fluorescent group (or the third fluorescent group) or the like is used.
  • Dnp dinitrophenyl
  • M ⁇ Ac 7-methoxycoumarin-1-yl
  • ABz anthraniloylbenzyl
  • Nma N-methylanthranilic acid
  • EDANS (5- (2'_aminoethyl) aminonaphthalene_1-sulfonic acid), tryptophan (Trp) and 5-dimethylamino-1-naphthalenesulfonic acid (Dns), carboxydichlorofluorescein (CDCF) ) And carboxymethyl rhodamine (CTMR), a combination of carboxydichlorofluorescein (CDCF) and carboxy X-rhodamine (CXR), a combination of lucifer yellow (LY) and carboxymethyl rhodamine (CTMR), and the like.
  • any of these donor receptors may be the second fluorescent group or the third fluorescent group. This is because if the second fluorescent group undergoes an outer change, it can be used as an indicator for measuring the enzyme activity.
  • tributophan or the like having a plurality of reaction points is used as the second fluorescent group, and the fourth fluorescent group is combined with the fluorescent resonance energy transfer.
  • Dns 5-dimethylamino-1-naphthalenesulfonic acid
  • the third compound and the fourth compound are the same as the first compound described in claim 1, and thus the description is omitted. Further, the fifth compound is the same as the second compound described in claim 1, and the description is omitted.
  • the length between the bonding portions of the second fluorescent group and the third fluorescent group bonded to the third compound or the fourth compound and the fifth compound, respectively is 100A or less. As the distance between the bond between the second fluorescent group and the third fluorescent group increases, the fluorescence resonance energy transfer tends to decrease and the change in the fluorescence intensity and the like tends to decrease. Is extremely small, and the sensitivity is lowered.
  • the substrate for enzyme activity detection according to claim 3 of the present invention comprises a substrate, a sixth compound having one end immobilized on the substrate, and a fourth fluorescent group introduced into the sixth compound.
  • the fourth firefly The seventh compound bound to the photogroup, the eighth compound bound to the seventh compound by a peptide bond cleaved by an enzyme, and the fourth fluorescent group bound to the eighth compound and fluorescence resonance energy transfer. And a fifth fluorescent group to be observed.
  • the distance between the substrate and the enzyme action point (peptide bond between the seventh and eighth compounds) is optimized, Enzyme can be allowed to act without being affected, enzyme activity can be detected more accurately, and detection sensitivity can be increased.In addition, it has the ability to cleave relatively long peptide chains, which does not have high substrate specificity for individual amino acids It is also possible to detect enzymes such as elastase, which are required for the above, and it is possible to increase the types of enzymes that can be detected.
  • tributophan Trp
  • Dns 5-dimethylamino_1_naphthalenesulfonic acid
  • the fourth fluorescent group requires a plurality of reaction points that bind to the sixth compound and the seventh compound.
  • the sixth compound is the same as the first compound described in claim 1, and therefore the description is omitted. Further, the seventh and eighth compounds are the same as the second compound described in claim 1, and thus the description is omitted.
  • the length between the bonding portions of the fourth fluorescent group and the fifth fluorescent group bonded to the seventh compound and the eighth compound, respectively, should be 100 A or less as described in claim 3. Is desirable.
  • the invention according to claim 4 of the present invention is the substrate for detecting an enzyme activity according to claim 2 or 3, wherein the fifth compound or the eighth compound is the fourth compound or the second compound.
  • the fourth compound is bonded to the second fluorescent group with a peptide bond cleaved by the enzyme, instead of being bonded to the conjugate by a peptide bond cleaved by the enzyme; or
  • the seventh compound has a structure in which the seventh compound is bonded to the fourth fluorescent group by a peptide bond cleaved by the enzyme.
  • the fourth compound or the seventh compound is bonded to the second fluorescent group or the fourth fluorescent group by a peptide bond that is cleaved by an enzyme, the fourth compound and the fifth compound are not included in the sample solution. Sequences can be made with amino acids that are not cleaved by the enzyme, and the degree of design freedom can be increased.
  • the amino acid of the fourth compound that binds to the second fluorescent group and the amino acid of the seventh compound that binds to the fourth fluorescent group are selectively cleaved at the C-terminal peptide bond by the enzyme. Things can be done.
  • the fourth and seventh compounds bound to the second and fourth fluorescent groups are cleaved by the enzyme and released, and the third and second fluorescent groups bound to the fifth compound,
  • the fluorescence intensity of the second and fourth fluorescent groups is changed in such a manner that no fluorescence resonance energy transfer occurs between the fifth and fourth fluorescent groups bound to the eighth compound. Can be.
  • the invention according to claim 5 of the present invention is the substrate for detecting enzyme activity according to any one of claims 1 to 4, wherein the second compound, the fifth compound, and the eighth The second fluorescent group introduced into the terminal group of the compound and / or the side chain of the third compound has an acetylated structure.
  • an acetyl ligating agent for acetylating the N-terminal of the second fluorescent group such as the terminal group of the peptide of the second compound or the third compound or the tributophan introduced into the side chain of the third compound, etc.
  • Acetic anhydride, N-hydroxysuccinimide acetate and the like can be used as an acetyl ligating agent for acetylating the N-terminal of the second fluorescent group.
  • the substrate for enzyme activity detection according to any one of claims 1 to 5 is contacted with a sample solution containing an enzyme to cause a reaction.
  • the enzyme activity can be detected simply by measuring the fluorescence intensity or the like of the substrate, so that the measurement time can be reduced. Workability can be increased and measurement efficiency can be increased.
  • Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution. It can be carried out.
  • the enzyme activity of a sample solution containing multiple enzymes can be determined. Measurement and analysis can be performed comprehensively in a short time, and measurement efficiency can be dramatically improved.
  • the sample solution containing the enzyme a solution adjusted to a pH at which the enzyme exhibits activity is used.
  • a buffer such as Tris_HCl, Hepes-K ⁇ H or the like can be added as a reaction buffer.
  • salts required for the expression of the enzyme activity and an activity protecting agent can be added.
  • the enzyme Since the fluorescent wavelength of the first fluorescent group from which the second compound has been released or the fluorescent intensity at a predetermined wavelength is different from that of the first fluorescent group in which the second compound is peptide-bonded, the enzyme is determined using the change in the fluorescent intensity as an index It is possible to provide a substrate for detecting enzyme activity which is excellent in versatility and capable of detecting an activity.
  • Enzyme activity can be detected simply by contacting a very small amount of sample solution containing the enzyme, so that a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a small amount of sample solution. It is possible to provide a substrate for enzyme activity detection which can be performed and has excellent operability.
  • the distance between the second fluorescent group and the third fluorescent group is increased, so that the fluorescence resonance energy transfer does not occur, and the transfer from the second fluorescent group (or the third fluorescent group) occurs.
  • the change in the spectrum from the fluorescence spectrum to the fluorescence spectrum from the third fluorescent group (or the second fluorescent group) can be used as an indicator of the enzyme activity.
  • a substrate for enzyme activity detection which is excellent in versatility and can be detected.
  • the enzyme can act without being affected by the substrate, the enzyme activity can be detected more accurately, and the detection sensitivity can be increased. Rather than having high substrate specificity for each amino acid, it is also possible to detect enzymes such as elastase that require a relatively long peptide chain for cleavage, thereby increasing the types of enzymes that can be detected.
  • a substrate for detecting enzyme activity can be provided.
  • exopeptidases such as aminopeptidase that acts on the N-terminal peptide bond can be significantly reduced, and even if these enzymes are contained in the sample solution, an endo-substrate with high substrate specificity can be used. It is possible to provide a substrate for detecting an enzyme activity, which is capable of accurately detecting the activity of an enzyme such as a peptidase and has excellent detection accuracy.
  • the enzyme activity can be detected simply by measuring the fluorescence intensity or the like of the substrate, so that the measurement time can be reduced. It is possible to provide a method for detecting an enzyme activity that can enhance workability and enhance measurement efficiency.
  • Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution.
  • a method for detecting enzyme activity that can be performed can be provided.
  • the enzyme activity of a sample solution containing multiple enzymes can be determined. It is possible to provide a method for detecting an enzyme activity, which can comprehensively measure and analyze in a short time and can dramatically improve the measurement efficiency.
  • FIG. 1 is a schematic diagram showing the principle of detecting enzyme activity of a substrate for detecting enzyme activity in Embodiment 1.
  • FIG. 2 is a schematic view showing the principle of detecting enzyme activity of a substrate for detecting enzyme activity in Embodiment 2.
  • FIG. 3 is a schematic diagram of a substrate for detecting enzyme activity in Embodiment 3.
  • FIG. 4 is a schematic diagram of an enzyme activity detection substrate according to a fourth embodiment.
  • FIG. 1 is a schematic diagram showing the principle of detecting enzyme activity of the substrate for detecting enzyme activity according to Embodiment 1 of the present invention.
  • 1 is a substrate for detecting the enzyme activity in Embodiment 1
  • 2 is a synthetic resin (polystyrene or the like) insoluble in solvents such as halogenated hydrocarbons and esters, glass, etc.
  • Substrate 3 formed into a curved surface, etc., 3 is directly connected to substrate 2 by peptide bonds, etc.
  • one of the fluorescent groups that changes in the fluorescence wavelength and the fluorescence intensity ie, 4-methylcoumaryl-7-amide ( (MCA) or the like
  • 4 is a second compound such as an amino acid or a peptide linked to the first fluorescent group 3 by a peptide bond cleaved by an enzyme 5 described below
  • 5 is the first fluorescent group 3 and the second fluorescent group.
  • An enzyme having a substrate specificity such as a serine protease that selectively cleaves a peptide bond with the conjugate 4
  • 6 is a fluorescent wavelength due to the selective release of the second compound 4 by the enzyme 5. Is the changed first fluorescent group.
  • the substrate 1 for enzyme activity detection configured as described above is prepared by fixing a C-terminal of an amino acid on the substrate 2 and elongating the peptide at the C-terminal, using a conventional peptide synthesis method such as a solid phase method.
  • a sequential extension method in which the desired amino acid sequence is sequentially extended from the C-terminal side to the N-terminal side
  • a fragment condensation method in which multiple short peptide fragments are synthesized and extended by coupling between peptide fragments, and a peptide synthesizer. It can be synthesized using a method such as Fmoc method, Boc method, etc.
  • Embodiment 1 The principle of detecting the enzyme activity of the substrate for enzyme activity detection of Embodiment 1 configured as described above will be described below.
  • the first fluorescent group 3 such as 4-methylcoumaryl-17-amide (MCA) of the enzyme activity detection substrate 1 shown in FIG. 1 (a) is a non-fluorescent substance and does not show fluorescence in a specific wavelength region.
  • MCA 4-methylcoumaryl-17-amide
  • the enzyme 5 such as a serine protease having substrate specificity is formed between the first fluorescent group 3 and the second compound 4. It selectively cleaves peptide bonds (see Figure 1 (b)).
  • the first fluorescent group 6 from which the second conjugated product 4 is released becomes a fluorescent substance such as 7-amino-methyl coumarin (AMC), and the fluorescent wavelength or the fluorescent intensity in the specific wavelength region is the same as that of the second compound 4. Since it is different from the peptide-bonded first fluorescent group 3, the enzyme activity can be detected using the change in fluorescence intensity or the like as an index (see FIG. 1 (c)).
  • AMC 7-amino-methyl coumarin
  • a fluorescent group is bonded to the substrate, only a very small amount of a sample solution containing the enzyme is brought into contact with the substrate as a detection unit, and the fluorescence intensity of the substrate after a predetermined time is measured.
  • a cell into which a sample solution is injected onto a substrate can detect enzyme activity depending on the amount of enzyme, the type of enzyme, etc., using the change in fluorescence intensity or the like corresponding to the number of molecules modified by the action of the enzyme as an index. Since the detection unit can be miniaturized without the necessity of forming such elements, the degree of integration of the detection unit on the substrate can be dramatically increased.
  • Enzyme activity can be detected only by contacting a very small amount of a sample solution containing an enzyme.Therefore, a large amount of sample solution is not required for measurement, and enzyme activity can be detected even with a very small amount of sample solution. It can be carried out.
  • the first fluorescent group 3 is directly bonded to the substrate 2
  • the first compound such as an amino acid or a peptide bonded to the substrate 2
  • the first fluorescent group 3 is bonded to the substrate 2 via the first compound.
  • the second compound 4 is bound to the first fluorescent group 3 by a peptide bond cleaved by the enzyme 5, as described in the first embodiment. This makes it possible to optimize the distance between the substrate 2 and the enzyme action point (the peptide bond between the first fluorescent group 3 and the second compound 4), thereby allowing the enzyme to act without being affected by the substrate. The effect is obtained that the activity can be detected more accurately.
  • the first compound is synthesized with a peptide or the like having a sequence that is not cleaved by the enzyme in the sample solution.
  • the first fluorescent group 3 bonded to the substrate 2 via the first compound is released from the substrate by the action of the enzyme, and before and after contacting the enzyme.
  • the first fluorescent group 3 in which the fluorescence intensity or the like has changed drifts in the sample solution, and the relationship between the binding location of the first fluorescent group 3 to the substrate 2 and the change in the fluorescence intensity or the like at that location is unclear.
  • FIG. 2 is a schematic diagram illustrating the principle of detecting enzyme activity of the substrate for detecting enzyme activity according to Embodiment 2 of the present invention. Note that the same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.
  • reference numeral 10 denotes a substrate for detecting an enzyme activity in Embodiment 2
  • 11 denotes a third compound such as an amino acid or peptide having one end immobilized on the substrate 2
  • 12 denotes a side chain of the third conjugate 11. Introduced third fluorescent group 14 described below and fluorescence resonance energy transfer can be seen.
  • Second fluorescent groups such as (7-methoxycoumarin_4_yl) acetyl (M ⁇ Ac) and tryptophan (Trp), and 13 is the third fluorescent group
  • Fifth compound such as amino acid and peptide linked by compound 11 and peptide bond cleaved by enzyme 5, and 14 is dinitrophenyl (Dnp), 5-dimethylamino-1-naphthalenesulfonic acid (Dns ) Is a third fluorescent group.
  • the second fluorescent group 12 and the third fluorescent group 14 are bonded to each other at a distance (100 A or less) at which fluorescence resonance energy transfer is observed.
  • Reference numeral 15 denotes a second fluorescent group whose fluorescence wavelength and the like have been changed by the selective release of the fifth conjugate 13 to which the third fluorescent group 14 is bonded by the enzyme 5.
  • the fluorescence of the second fluorescent group 12 and the third fluorescent group 14 of the enzyme activity detection substrate 10 shown in FIG. 2A are bonded to each other at a distance where fluorescence resonance energy transfer can be seen, the fluorescence of the second fluorescent group 12
  • the vector and the excitation spectrum of the third fluorescent group 14 overlap, and when the energy of the excitation wavelength of the second fluorescent group 12 is applied, the fluorescence of the second fluorescent group 12 that should be observed is attenuated, and The fluorescence of the third fluorescent group 14 is observed.
  • the substrate for detecting an enzyme activity in the second embodiment is configured, the following operations can be obtained by adding the functions described in the first embodiment.
  • the fluorescent wavelength of the second fluorescent group is Since it is possible to set the area, it is possible to use a visible light detection device such as a commercially available CCD camera to perform measurement, which is excellent in versatility.
  • FIG. 3 is a schematic view of a substrate for detecting enzyme activity according to Embodiment 3 of the present invention. Note that the same components as those of the second embodiment are denoted by the same reference numerals, and description thereof is omitted.
  • reference numeral 20 denotes a substrate for detecting an enzyme activity in Embodiment 3
  • reference numeral 21 denotes a side chain or terminal of a second fluorescent group 12 such as tributophan ( ⁇ ) having a plurality of reaction points directly bonded to the substrate 2.
  • This is the fourth compound such as amino acids and peptides introduced into the product.
  • the fifth compound 13 is bonded to the fourth compound 21 by a peptide bond that is cleaved by the enzyme.
  • the difference between the substrate for enzyme activity detection in the third embodiment and the second embodiment is that the second fluorescent group 12 directly bonded to the substrate 2 is bonded to the fourth compound 21.
  • the principle of detecting the enzyme activity on the enzyme activity detecting substrate according to the third embodiment configured as described above is the same as that described in the second embodiment, and a description thereof will be omitted.
  • the substrate for detecting enzyme activity in Embodiment 3 is configured. Therefore, after the operation described in Embodiment 2 is performed and the second fluorescent group is immobilized on the substrate, the normal operation is performed. It can be synthesized simply by elongating the fourth compound or the fifth compound using the peptide synthesis method, so that the operability of synthesizing the substrate for enzyme activity detection is excellent and the product yield can be increased. Is obtained.
  • the compound 21 may have an amino acid sequence that is not cleaved by an enzyme, so that the second fluorescent group 12 and the fourth conjugate 21 are bound by a peptide bond that is cleaved by the enzyme.
  • an effect is obtained that the degree of freedom of the amino acid sequence constituting the fourth compound and the fifth compound can be increased and the design can be facilitated.
  • FIG. 4 is a schematic view of a substrate for detecting an enzyme activity according to Embodiment 4 of the present invention. Note that the same components as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.
  • 30 is a substrate for detecting enzyme activity in Embodiment 4
  • 31 is a sixth compound such as an amino acid or peptide having one end bonded to the substrate 2
  • 32 is one reaction site bonded to the sixth conjugate 31.
  • a fourth fluorescent group such as tributophan ( ⁇ ⁇ ⁇ ), 33 is a seventh compound such as an amino acid or a peptide to which another reactive site of the fourth fluorescent group 32 is bonded
  • 34 is a seventh fluorescent compound 33 and an enzyme.
  • the eighth compound 35 such as an amino acid or a peptide, bound by a peptide bond is bound by the eighth bond 34, and fluorescence resonance energy transfer is observed with the fourth fluorescent group 32.
  • It is a fifth fluorescent group such as naphthalenesulfonic acid (Dns).
  • the principle of detecting the enzyme activity in the enzyme activity detecting substrate according to the fourth embodiment configured as described above is the same as that described in the second embodiment, and a description thereof will be omitted.
  • the substrate for detecting an enzyme activity in Embodiment 4 is configured, and thus the following operation is obtained by adding the function described in Embodiment 2 to the substrate.
  • the substrate 2 and the enzyme action point (the peptide bond between the seventh and eighth conjugates 33 and 34)
  • the enzyme can act without being affected by the substrate 2, the enzyme activity can be detected more accurately, the detection sensitivity can be increased, and the substrate specificity for each amino acid can be improved. It is also possible to detect enzymes such as elastase that require a relatively long peptide chain for the cleavage action, which can increase the number of detectable enzymes.
  • the eighth conjugated product 34 is bonded to the seventh conjugated product 33 by a peptide bond that is cleaved by an enzyme.
  • the 34th compound and the seventh compound 33 have an amino acid sequence that is not cleaved by the enzyme, so that the fourth fluorescent group 32 and the seventh compound 33 are bound by a peptide bond cleaved by the enzyme.
  • an effect is obtained that the degree of freedom of the amino acid sequence constituting the seventh compound and the eighth compound can be increased and the design can be facilitated.
  • Example 1 the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded spherical substrate as a substrate for detecting the enzyme activity.
  • the method will be described.
  • DMF (10 ml) was flown into the Fmoc-Aca-PEGA resin (0.05 mmol / g, 0.5 g) in the vessel with the cock open, and solvent replacement and washing were performed.
  • the cock was closed, 20% piberidine / DMF was added, and the mixture was reacted for 30 minutes to remove Fmoc. Thereafter, the cock was opened to remove 20% pyridine ZDMF, and then washed with DMF (10 ml).
  • Ala-Ala-Pro-Lys (Boc) -ACC_Aca-Aca-PEGA resin to which Ala-Ala-Pro-Lys was bound was obtained. After that, close the cock and dilute DIEA (2.5 mmol, 0.435 ml) and acetic anhydride (1.25 mmol, 0.117 ml) in DMF (2 ml), add them, and allow them to react for 1 hour. Acesila-Ala_Ala_Pro-Lys (Boc) -ACC-Aca_Aca_PEGA resin was obtained.
  • Gael A and Gael B are the type of enzyme.
  • Gael A (or Gael B) and Gael C is the presence or absence of the enzyme, and Gael A (or Gael B, C) and Gael.
  • the difference from D is the presence or absence of a substrate for enzyme activity detection.
  • the enzyme activity detection substrate of Example 1 was about 315-fold in the well A with trypsin and in the well B with chymotrypsin. It was confirmed that the difference was about 10 times. As a result, the substrate for enzyme activity detection of Example 1 It was shown that the enzyme activity can be detected based on the amount of the enzyme and the type of the enzyme. Further, comparing the fluorescence values of the wells A and B, the enzyme activity detection substrate of Example 1 shows that the fluorescence value of trypsin is about 30 times larger than that of chymotrypsin. Force S confirmed.
  • Example 1 This indicates that the enzyme activity detection substrate of Example 1 has specificity depending on the enzyme, and that qualitative analysis of the enzyme having activity is possible.
  • a sample solution containing the same type of enzyme was brought into contact with the same type of enzyme activity detection substrate and the fluorescence value was measured after a predetermined time, the change in the fluorescence value was modified by the action of the enzyme. It was speculated that quantitative analysis of the enzyme was possible because it corresponds to the number of molecules.
  • Example 2 an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for enzyme activity detection.
  • the method will be described.
  • a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape.
  • One lantern as a substrate (D-series manufactured by Mimotops Co., introduction rate 18 mmol / unit) was placed in a screw tube, and the mixture was stirred with 20% piperidine ZDCM (1 mL) for 30 minutes to remove the Fmoc group.
  • the plate was washed three times with DMF (1 ml) and three times with DCM (1 ml), and Pmoc_Aca_Aca_lantern having one end of the first compound (Aca-Aca) immobilized on a substrate (lantern) was obtained.
  • the Boc group of the Lys side chain was removed using 25% TFA / DCM (lml) to obtain the desired substrate for detecting the enzyme activity (Ac-Ala-Ala-Pro-Lys-ACC_Aca-Aca_lantern) of Example 2.
  • the Boc group of the Lys side chain was removed using 25% TFA / DCM (lml) to obtain the desired substrate for detecting the enzyme activity (Ac-Ala-Ala-Pro-Lys-ACC_Aca-Aca_lantern) of Example 2.
  • the fluorescence values before and after the reaction were measured.
  • the fluorescence value was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 460 nm using a WALLAC ARVOTM SX 1420 multilabel counter (manufactured by PerkinElmer).
  • the initial fluorescence value was 39,000
  • the fluorescence value after 18 hours of reaction was 145,000
  • the enzyme having activity can be detected also in the enzyme activity detecting substrate of Example 2 using lantern as the substrate.
  • Example 3 an enzyme activity was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting enzyme activity.
  • the method will be described.
  • a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape.
  • One lantern as a substrate (D_series, introduced by Mimotops Co., 18 mmol / unit) was placed in a screw tube, and the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to cut out the Fmoc group.
  • the measurement was performed using a WALLAC ARVOTM SX 1420 multilabel counter (PerkinElmer) to measure the fluorescence value before adding the enzyme and the fluorescence value 30 minutes after adding the enzyme at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. And asked for the difference.
  • chymotrypsin has the specificity of selectively cleaving peptide bonds mainly at the C-terminal side of aromatic amino acid residues
  • trypsin is mainly at the C-terminal side of lysine. It is presumed that it was expressed because it has the specificity of selectively cleaving peptide bonds.
  • Example 4 In Example 4, the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting the enzyme activity. Hereinafter, the method will be described.
  • a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape.
  • One lantern (D_series, Mimotops Co., 18 ⁇ mol I) was used as the substrate in the screw tube, and the mixture was stirred with 20% piperidine ZDCM (1 ml) for 30 minutes to remove the Fmoc group. .
  • Fmoc_Pro_OH and Fmoc-Ala-OH are introduced into Fmoc-Lys (Boc) -Lys (Ac-Trp (Boc)-)-Aca-Aca-lantern to extend the peptide by repeating the same operation as in Example 3.
  • Dns_Cl Fmoc_Pro_OH and Fmoc-Ala-OH
  • the lantern was washed three times with DMF (1 ml) and three times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then, the mixture was washed three times with DCM (1 ml), five times with H ⁇ (1 ml), and three times with DCM (1 ml), and dried under reduced pressure to allow the third and fifth compounds to bind (Ala -Ala-Pro_Lys-Lys-Aca-Aca) provided with Trp as a second fluorescent group in the side chain and Dns as a third fluorescent group at the end Dns for enzyme activity detection of Example 4 of interest -Ala-Ala-Pro-Lys-Lys (Ac-l rp—no Aca— Aca—lantern obtained.
  • the fluorescence values before and after the reaction were measured.
  • the fluorescence value was measured using an FP-6600 fluorescence spectrophotometer (manufactured by Jasco) at an excitation wavelength of 280 nm and a fluorescence wavelength of 350 nm.
  • the initial fluorescence value was 37
  • the fluorescence value after 18 hours of reaction was 567
  • Fmoc group was removed using piperidine ZDCM, and washed with DCM and DMF.
  • Fmoc-Lys (Boc) _OH, HATL ;, HOAt and DIEA were dissolved in DMF and reacted. Then, it was washed with DMF DCM to obtain Fmoc-Lys (Boc) -TYp (Boc) -lantern.
  • Fmoc_Lys (Boc) _Trp (the same procedure as in Example 3 in which Fmoc_Pro_uH and Fmoc_Ala_OH are introduced into Boc lantern) is repeated to elongate the peptide, and Dns-Cl is further reacted to obtain Dns-Ala-Ala- Pro—Lys (Boc) _T ⁇ (Boc) -lantern was obtained.
  • the lantern was washed with DMF and DCM, and reacted with TFAZDCM to remove the Boc group. After that, the resultant was washed with DCM, H0, and DCM, and dried under reduced pressure.
  • Example 5 The substrate for enzyme activity detection of Example 5 which is provided with Trp as a second fluorescent group at the terminal of (Ala_Ala_Pro-Lys) to which the compound is bound and Dns at the terminal as a third fluorescent group
  • Example 5 When the enzyme activity detection ability of the substrate for enzyme activity detection of Example 5 was measured in the same manner as in Example 4, the detection ability of the substrate for enzyme activity detection of Example 5 was almost the same as that of Example 4. Was done. This indicates that the enzyme having activity can be detected also in the enzyme activity detection substrate of Example 5, and the substrate is bound to the substrate by using a fluorescent group such as tributophan having a plurality of reaction points. It has been clarified that the degree of freedom in designing an amino acid sequence of a peptide or the like can be further increased.
  • Fmoc-Trp (Boc) _Aca_Aca_lantern in which the sixth compound (Aca_Aca) was bound to the substrate (lantern) was protected.
  • Fmoc-Lys (Boc) _OH, Fmoc-Pro_OH, and Fmoc_Ala-OH were introduced to extend the peptide by repeating the same operation as in Example 3, and further reacted with Dns-Cl to obtain Dns-Ala-Ala-OH.
  • Example 7 the activity of the enzyme was measured by synthesizing a peptidyl fluorescent group-bonded flat substrate as a substrate for detecting the enzyme activity.
  • the method will be described.
  • a carrier made of a multi-plate synthetic resin for peptide synthesis (lanthanum series (registered trademark) manufactured by Mimotobus Co., Ltd.) was cut off by only one plate and made into a planar shape.
  • One lantern as a substrate (D-series manufactured by Mimotops, 18 ⁇ mol I introduction rate) was placed in a screw tube, and the mixture was stirred with 20% piperidine / DCM (1 mL) for 30 minutes to remove the Fmoc group. .
  • the lantern was washed three times with DMF (1 ml) and three times with DCM (1 ml), and reacted with 25% TFA / DCM for 30 minutes to remove the Boc group. Then 3 times with DCM (1 ml) and 5 times with H 0 (1 ml)
  • Example 7 After washing three times with DCM (1 ml) and drying under reduced pressure, the third compound and the fifth compound were combined (Ala-Ala-Pro-Phe-Lys-Aca-Aca).
  • the substrate for enzyme activity detection of Example 7 having TYp as a second fluorescent group on the side chain and Dns as a third fluorescent group at the end.
  • the enzyme was reacted with 50 ⁇ g of enzyme.
  • Chymotrypsin B Chymotrypsin
  • Fluorescence values were obtained using an FP-6600 fluorescence spectrophotometer (manufactured by Jasco) at an excitation wavelength of 280 nm and a fluorescence wavelength of 350 nm. The fluorescence value before the enzyme was removed and the fluorescence value 20 minutes after the enzyme was removed. And the difference was determined.
  • the ability to detect the activity for the type of enzyme can be changed by changing the type of the amino acid sequence of the peptide or the like to which the fluorescent group is bound.
  • an example was shown in which the result changes depending on a single amino acid residue in the sequence, such as chymotrypsin or trypsin. Since the amino acid sequence can be freely designed over the entire length of the sequence, it is possible to obtain an enzyme activity detection substrate that can express infinite substrate specificity and detect a wide variety of enzymes by designing the amino acid sequence. It was revealed.
  • the present invention relates to a substrate for detecting an enzyme activity for detecting an enzyme activity, and an enzyme activity using the same.
  • the enzyme activity can be detected simply by measuring the fluorescence intensity of the substrate using the change in the fluorescence intensity etc. as an index, and the operability is excellent, and the enzyme activity is detected even with a small amount of sample solution.
  • the enzyme activity detection substrate to which the enzyme is bound it is possible to provide an enzyme activity detection method that can measure the enzyme activity of a sample solution containing a plurality of enzymes in a short time.

Abstract

Substrat pour la détection d'une activité enzymatique capable de détecter toute activité enzymatique en mesurant seulement l'intensité de la fluorescence, etc. d'un substrat en utilisant tout changement d'intensité de la fluorescence, etc. comme indice et qui excelle en termes d'aptitude à l'exploitation, lequel substrat pour la détection d'une activité enzymatique permet la détection d'une activité enzymatique même lorsque la quantité de solution d'analyte est infime et ne nécessite pas la formation de cellules pour l'incorporation de la solution d'analyte, etc. permettant de cette manière de réaliser un renforcement impressionnant de l'intégrité d'une partie de détection. Il est fourni un substrat pour la détection d'une activité enzymatique, comprenant un substrat, un premier groupe fluorescent directement lié au substrat ou lié au substrat via un premier composé ayant une de ses extrémités fixée au substrat et un second composé lié au premier groupe fluorescent au moyen d'une liaison peptidique clivée par des enzymes.
PCT/JP2005/001258 2004-05-13 2005-01-28 Substrat pour la détection d'une activité enzymatique et procédé de détection d'une activité enzymatique avec celui-ci WO2005111229A1 (fr)

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JP2004-144191 2004-05-13
JP2004144191A JP2004357706A (ja) 2003-05-13 2004-05-13 酵素活性検出用基板及びそれを用いた酵素活性の検出方法

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0246280A (ja) * 1988-06-20 1990-02-15 Becton Dickinson & Co 蛍光および反応速度を増強するための器具ならびにその使用法
JP2003503013A (ja) * 1999-05-05 2003-01-28 オーロラ バイオサイエンシズ コーポレーション 光学的プローブおよびアッセイ
JP2004504854A (ja) * 2000-05-03 2004-02-19 エクスプレッシブ　コンストラクツ，インコーポレイテッド 細菌汚染検出装置および使用方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0246280A (ja) * 1988-06-20 1990-02-15 Becton Dickinson & Co 蛍光および反応速度を増強するための器具ならびにその使用法
JP2003503013A (ja) * 1999-05-05 2003-01-28 オーロラ バイオサイエンシズ コーポレーション 光学的プローブおよびアッセイ
JP2004504854A (ja) * 2000-05-03 2004-02-19 エクスプレッシブ　コンストラクツ，インコーポレイテッド 細菌汚染検出装置および使用方法

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