KR101448353B1 - Method for analyzing the protease activity and the application thereof - Google Patents

Abstract

The present invention relates to a method for analyzing proteolytic enzyme activity, comprising the steps of: introducing a modified portion containing a carboxyl group at one end to the surface of metal nanoparticles to modify the surface of the metal nanoparticles to a carboxyl group; Preparing a connection portion into which polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by the protease to be analyzed; Aggregating the metal nanoparticles into which the carboxyl group has been introduced as an experimental group, in a buffer solution containing a divalent metal cation, an analyte to be analyzed and the connecting portion; Measuring the degree of aggregation of the metal nanoparticles; And comparing the measured degree of aggregation with the degree of aggregation of metal nanoparticles in a control group aggregated in a buffer solution containing no protease to be analyzed. The method for analyzing protease activity of the present invention can analyze the activity of protease in a simpler, less expensive and faster manner than the conventional technique, and is useful for screening protease activity inhibitor screening method and diagnosis of protease-mediated diseases Lt; / RTI >

Description

[0001] The present invention relates to a method for analyzing protease activity,

The present invention relates to a method for analyzing protease activity, and more particularly, to a method for analyzing the activity of proteolytic enzyme using the aggregation phenomenon of metal nanoparticles.

Protease is an enzyme that regulates protein activity or fate through hydrolysis of irreversible peptide bonds. Protease is a living organism that constitutes a protein that mediates a large number of life phenomena. It is a living organism such as protein catabolism, blood coagulation, cell growth and migration, protein activation, cell regulation and signal development, It plays a key role in pathological processes such as physiological phenomena and inflammation, cancer development and metastasis, and viral infection. Therefore, it is possible to develop an inhibitor that can diagnose diseases early or measure the activity of protease related enzymes by measuring the activity of the protease.

Recently, it has been newly found that protease plays a pivotal role in various human diseases such as cancer, dementia and AIDS. As an example, MMP (matrix metalloprotease) has been recognized as a factor that degrades the extracellular matrix in cells and the body in the past. However, in recent studies, it has been shown that they inhibit integrin signaling and pericellular matrix And it has been shown that it plays an important role in cancer growth such as new blood vessel formation, invasion of cancer cells, and metastasis.

In addition to the MMPs described above, it has been found that various types of proteases play a pivotal role in the pathogenesis of many diseases. Therefore, protease and its substrate proteins have attracted great interest as a main target of the development of new drugs. In particular, as new substrate proteins are discovered, the physiological functions of various proteolytic enzymes will be newly illuminated in the future, and new target protein proteases are expected to be discovered.

In the development of new drugs targeting such protease, it is very important to analyze the activity of the protease. Representative protease activity analysis methods currently used include peaks shift by spectroscopy using high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), or a phospholipid conjugated polypeptide substrate. And the like. However, these methods have a disadvantage in that they are not economical and time efficient to use in screening many drugs as in the development of a new drug because a multi-step measurement protocol is required.

Another method for analyzing the activity of proteolytic enzymes is to use a fluorogenic peptide or attach a pair of fluorescent substances or a fluorescent substance-quencher pair to a peptide substrate to generate FRET (Fluorescence Resonance Energy Transfer) There is a way to measure. However, since the two fluorescent materials can be rapidly photobleached by the initial external light energy, it is difficult to measure them for a long time, a fluorescence measurement equipment is separately required, and a suitable donor-receptor selection is not wide There is a problem.

Therefore, it is necessary to develop an additional technique for analyzing the activity of the proteolytic enzyme more simply, inexpensively and stably than the conventional analytical methods.

It is an object of the present invention to provide a method for analyzing the activity of proteolytic enzymes in a simpler, less expensive and stable manner.

According to an aspect of the present invention, there is provided a method for preparing a metal nanoparticle, comprising: introducing a modified portion containing a carboxyl group at one end to the surface of the metal nanoparticle to modify the surface of the metal nanoparticle to a carboxyl group; Preparing a connection portion into which polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by the protease to be analyzed; Aggregating the metal nanoparticles into which the carboxyl group has been introduced as an experimental group, in a buffer solution containing a divalent metal cation, an analyte to be analyzed and the connecting portion; Measuring the degree of aggregation of the metal nanoparticles; And comparing the measured degree of aggregation with the degree of aggregation of metal nanoparticles in a control group aggregated in a buffer solution containing no protease to be analyzed.

The method for analyzing protease activity of the present invention can analyze the activity of protease in a simpler, less expensive and faster manner than the conventional technique, and is useful for screening protease activity inhibitor screening method and diagnosis of protease-mediated diseases Lt; / RTI >

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1 is a schematic diagram for explaining the principle of a method for analyzing the activity of the protease of the present invention.
2 is a schematic view showing a structure in which a residue of a polyhistidine introduced at both ends of a polypeptide substrate is bonded to a carboxyl group present on the surface of metal nanoparticles through a divalent metal cation.
FIG. 3 is a graph showing the results of experiments in which the activity of proteolytic enzymes of the present invention was analyzed to confirm the structure of the polypeptide substrate and the coagulation conditions of the metal nanoparticles according to the presence or absence of the divalent metal cation.
(A) is a graph showing the results of measurement of E ₅₂₀ (absorbance at 520 nm);
(B) is a graph showing the ratio of the E ₅₂₀ value for the E ₇₀₀ value (E ₅₂₀ / E _700);
(C) is a photograph of the change in the color of the reactant as a result of aggregation reaction.
4 is a graph showing the results of a comparison of metal nanoparticle aggregation efficiencies of carboxyl groups and nitrilotriacetic acid (NTA) in a method for analyzing the activity of protease of the present invention.
(A) is a graph showing a result of measuring E ₅₂₀ ;
(B) is a graph showing E ₅₂₀ / E ₇₀₀ .
FIG. 5 is a graph showing E ₅₂₀ / E ₇₀₀ as a result of comparing the coagulation efficiency of metal nanoparticles according to the type of buffer solution and the ratio of the polypeptide substrate in the method for analyzing the activity of the protease of the present invention.
FIG. 6 is a graph showing the results of experiments comparing the coagulation efficiency of metal nanoparticles according to the kind of divalent metal cations in the method for analyzing the activity of the protease of the present invention,
(A) is a graph showing a result of measuring E ₅₂₀ ;
(B) is a graph showing E ₅₂₀ / E ₇₀₀ .
FIG. 7 is a graph showing the activity of MMP-7 according to the assay method of the present invention.
(A) is a graph showing a result of measuring E ₅₂₀ ;
(B) is a graph showing normalized E ₅₂₀ / E ₇₀₀ values according to the concentration of MMP-7.
8 is a view for explaining the principle of measuring the degree of agglomeration of metal nanoparticles using a silver-staining method.
FIG. 9 is a photograph of the result of measurement of the degree of agglomeration of metal nanoparticles by silver staining and observed with naked eyes.

Hereinafter, the present invention will be described in detail.

One. Analysis of protease activity

One aspect of the present invention provides a method for analyzing the activity of proteolytic enzymes.

The method for analyzing protease activity of the present invention comprises the steps of 1) introducing a modified moiety containing a carboxyl group at one end to the surface of metal nanoparticles to modify the surface of the metal nanoparticles with a carboxyl group; 2) preparing a junction in which polyhistidine is introduced at both ends of a polypeptide substrate which is specifically degraded by a protease to be analyzed; 3) As an experimental group, the step of aggregating the metal nanoparticles of step 1) in a buffer solution containing a divalent metal cation, a protease to be analyzed and a connecting portion of the step 2); 4) measuring the degree of aggregation of the aggregated metal nanoparticles in step 3); And 5) comparing the degree of aggregation measured in step 4) with the degree of aggregation of the metal nanoparticles of the control group aggregated in a buffer solution containing no protease to be analyzed.

1 is a schematic diagram for explaining the principle of the method for analyzing the activity of the protease.

Referring to FIG. 1 (A), the step (1) is a step of modifying the surface of the metal nanoparticles to a carboxyl group so that the metal nanoparticles can cohere with each other by the connecting portion.

The metal nanoparticles of the step 1) have a degree of aggregation depending on the degree of activity of the protease to be analyzed, and indirectly indicate the activity of the protease to be analyzed. Therefore, it is not particularly limited as long as it is a kind of metal which can be produced into nanoparticles and whose surface is easily modified. In particular, the metal nanoparticles may be composed of any one or two or more mixed metals selected from the group consisting of gold, silver, copper, platinum, palladium, nickel and iron.

The metal nanoparticles in the step 1) may have a diameter ranging from 2 nm to 50 nm, more preferably from 3 nm to 30 nm, such that optical characteristics such as absorbance can be changed by agglomeration.

A modified portion containing a carboxyl group is introduced into the surface of the metal nanoparticles in the step 1). The modifying unit modifies the surface of the metal nanoparticle so that the carboxyl group is exposed to the outside by bonding the other end of the metal nanoparticle to the surface of the metal nanoparticle not at one end where the carboxyl group exists. Therefore, it is preferable that the modifying unit is a polymer material having a carboxyl group at one end and a functional group capable of being easily bonded to the surface of metal nanoparticles such as a thiol group (-SH) at the other end. In particular, the modified part of the modified part may be HS- (polyethylene glycol) _n -COOH (wherein n is an integer of 1 to 20). In one embodiment of the present invention, it was confirmed that the metal nanoparticles modified with a carboxyl group were more efficiently aggregated than the metal nanoparticles modified with nitrilotriacetic acid (NTA (the first experimental group and the second experimental group in Fig. 4 Reference).

The modified portion of the step 1) may be introduced into the metal nanoparticles together with the modified density control portion to control the density of the carboxyl groups introduced into the surface of the metal nanoparticles. Therefore, the modified density control unit is preferably HS- (polyethylene glycol) _m -R (wherein m is an integer of 1 to 8, and R is an alkyl group having 1 to 5 carbon atoms), preferably 0.1 to 1 times In terms of molar ratio.

Referring to FIG. 1 (B), the step 2) is a step of manufacturing a connection part connecting and coagulating the metal nanoparticles of the step 1), and independently from the step 1) have.

The connecting portion of the step 2) has a polypeptide substrate which is specifically degraded by the protease to be analyzed and a polyhistidine which is introduced at both ends of the polypeptide substrate.

The polypeptide substrate constituting the linkage of the step 2) is a polypeptide sequence which is specifically degraded by the protease to be analyzed, and the sequence thereof may be changed depending on the protease to be analyzed. For example, when the analyte to be analyzed is MMP-14, the polypeptide substrate may be a polypeptide sequence comprising the polypeptide of SEQ ID NO: 9 (SGRIGFLRTAK), and when the analyte to be analyzed is thrombin, May be a polypeptide sequence comprising the polypeptide of SEQ ID NO: 15 (LVPRGS). Further examples of the types of the polypeptides contained in the polypeptide substrate are shown in Table 1 according to the type of protease to be analyzed. In addition, the sequence of the polypeptide substrate may be appropriately selected by those skilled in the art depending on the type of protease to be analyzed.

SEQ ID NO: Polypeptide sequence enzyme 3 GPIC (Et) F / FRLG Cathepsin D 4 GPLG / VRG MMP-2 5 PLG / LAARK 6 GVPLS / LTMG MMP-7 7 RPLA / LWRS 8 GPLGMRGL 9 SGRIGF / LRTAK MMP-14 10 GGLGQRCR / SANAILE uPA (urokinase plasminogen activator) 11 GWEHD / G Caspase-1 12 GDEVD / GSG Caspase-3 13 GVSQNT / PIVG HIV-1 protease 14 LVLA / SSSFGY HSV-1 protease 15 LVPR / GS Thrombin 16 HSSKL / Q PSA 17 KKKKVLP / IQLNAATDKGG Anthrax lethal factor endopeptidase 18 EVNL / DAGF B-Secretase 19 GI MMP-1, MMP-8, MMP-13 20 GL 21 ENLYFQ / S TEV protease

The polyhistidine constituting the connecting portion of the step 2) is a histidine multimer composed of 2 to 20 histidine, and is introduced through a peptide bond at the N-terminal and C-terminal of the polypeptide substrate, respectively. The polyhistidine introduced at both ends of the polypeptide substrate is bonded to carboxyl groups existing on the surface of the metal nanoparticles in step 1) via the bivalent metal cation with a structure as shown in FIG. 2, whereby the metal nanoparticles (See Fig. 2). In one embodiment of the present invention, it was confirmed that the metal nanoparticles did not agglomerate well when polyhistidine was not introduced at both ends (see the second experimental group and the fourth experimental group in Fig. 3).

Referring to FIG. 1C, step 3) is a step of coagulating the metal nanoparticles of step 1) with the connection of step 2).

In the step 3), the metal nanoparticles prepared in the step 1) are agglomerated in a buffer solution containing the divalent metal cations, the analyte to be analyzed and the linkage prepared in the step 2).

The divalent metal cation in the step 3) connects the polyhydroxystyrene of the connecting portion and the carboxyl group on the surface of the metal nanoparticle, and may be any one selected from the group consisting of Ni ²⁺ , Co ²⁺ , Zn ²⁺ and Cu ²⁺ Or two or more. In one embodiment of the present invention, it was confirmed that the metal nanoparticles were not agglomerated well when the bivalent metal cation was not present (see the third experimental group and the fourth experimental group in FIG. 3) Cobalt and zinc were found to coagulate the metal nanoparticles well (see FIG. 6).

The buffer solution of step 3) is preferably a solvent in which the agglomeration reaction of the metal nanoparticles occurs. Preferably, the buffer solution is a Tris buffer solution or a HEPES buffer solution, and the buffer solution is a neutral or weak base Tris buffer solution desirable. In one embodiment of the present invention, the flocculation efficiency of the metal nanoparticles was further improved in the environment of a neutral or weak basic buffer solution rather than a buffer solution containing acid or salt (see FIG. 5).

The aggregation in the step 3) may be performed by simultaneously injecting the divalent metal cations, the protease to be analyzed, the connecting portion and the metal nanoparticles into which the carboxyl group has been introduced into the buffer solution, and simultaneously reacting them. When the reaction is carried out as described above, the decomposition of the linking portion by the protease and the aggregation of the metal nanoparticles by the linking portion proceed simultaneously. Since the proteolytic enzyme is not accessible due to the steric hindrance caused by the metal nanoparticles, the linking part involved in aggregation of the metal nanoparticles before decomposition by the proteolytic enzyme is not decomposed. However, when the linking part is decomposed by the proteolytic enzyme before participating in the agglutination reaction of the metal nanoparticles, aggregation of the metal nanoparticles can not occur. Therefore, when the metal nanoparticles are agglutinated in the presence of the proteolytic enzyme as described above, the aggregation degree of the metal nanoparticles becomes lower than that of the agglutination reaction occurring in the absence of the reactive proteinase. In addition, the higher the activity of the protease, the lower the degree of aggregation of the metal nanoparticles. Therefore, the activity of the protease to be analyzed can be quantitatively and qualitatively analyzed by measuring the degree of aggregation of the metal nanoparticles as described above.

In addition, the step 3) may include: a) reacting the connecting portion with a protease to be analyzed in a buffer solution to decompose the polypeptide substrate in the connecting portion; And b) injecting and reacting the carboxyl group-introduced metal nanoparticle and the divalent metal cation into the buffer solution before the polypeptide substrate of step a) is completely decomposed. The step (a) is a step of causing a decomposition reaction of a linking part by the protease, and the step (b) is a step of causing a metal nanoparticle aggregation reaction by the linking part. When the metal nanoparticle aggregation reaction by the connecting portion in the step b) is first performed, the proteolytic enzyme can not approach due to the steric hindrance of the aggregated metal nanoparticles, so that the decomposition reaction of the connecting portion by the proteolytic enzyme occurs The activity of the proteolytic enzyme can not be measured and analyzed. Therefore, when the agglomeration reaction of the metal nanoparticles as in step b) is performed in the presence of the linking part decomposed by the proteolytic enzyme as in step a), compared with the agglutination reaction occurring in the absence of the reactive protease, The aggregation degree of the nanoparticles is lowered. In addition, the higher the activity of the protease, the lower the degree of aggregation of the metal nanoparticles. Therefore, the activity of the protease to be analyzed can be quantitatively and qualitatively analyzed by measuring the degree of aggregation of the metal nanoparticles as described above.

The step 4) is a step of measuring the degree of agglomeration of the metal nanoparticles aggregated in the step 3).

The measurement of the degree of cohesion in the step 4) may be performed by measuring the optical characteristics of the metal nanoparticles aggregated by the coagulation reaction in the step 3), or may be carried out by measuring the amount of the metal Or by measuring the amount of nanoparticles.

First, the measurement of the degree of aggregation of the step 4) can be performed by measuring the ratio of the absorbance at two wavelengths, which indicate the change of the absorbance by the aggregation in the step 3). As a result of the coagulation reaction in the step 3), when the metal nanoparticles are agglomerated to a large extent, redshift phenomenon of the wavelength exhibiting the maximum absorbance is exhibited and the maximum absorbance value is lowered.

Next, the degree of aggregation of the step 4) may be measured by measuring the amount of the metal nanoparticles which are free from agglomeration by the silver-staining method described in FIG. Referring to FIG. 8, as silver ions are reduced and adsorbed on the metal nanoparticles, the size of the metal nanoparticles increases and becomes dark gray. If fixer is added to this, color change due to silver halide will stop. According to this principle, the more non-aggregated metal nanoparticles are present, the more silver salt is formed and the solution becomes darker gray, and the less the metal nanoparticles are in solution, the more colorless the solution becomes. Therefore, the aggregation degree of the step 4) can be measured by staining the non-agglomerated metal nanoparticles by the silver-staining method and measuring the color change after the agglomeration of the step 3).

The step 5) is a step of analyzing the activity of the proteolytic enzyme using the degree of aggregation of the metal nanoparticles measured in the step 4), and the degree of aggregation measured in the step 4) ≪ / RTI >

The control in step 5) is performed in the same manner as in step 3), but is a result of the aggregation reaction of the metal nanoparticles without the protease to be analyzed. Since the control group does not contain a protease to be analyzed, the decomposition reaction of the connecting part does not occur, and all of the metal nanoparticles aggregate by the connecting part.

The degree of aggregation of the metal nanoparticles in the control group of the step 5) is carried out in the same manner as in the step 4).

By comparing the above step 5), it is possible to measure to what extent the degree of aggregation of the metal nanoparticles is reduced by the protease to be analyzed. For example, by comparing how much the maximum absorbance measured in step 4) is decreased or the maximum absorbance wavelength is long, it is possible to measure how much the aggregation degree of the metal nanoparticles is reduced by the analyte to be analyzed . Therefore, the enzymatic activity of the protease to be analyzed can be quantitatively and qualitatively analyzed by the comparison of the step 5).

In one embodiment of the present invention, gold nanoparticles having a surface modified with carboxy-PEG ₁₂ -thiol at a ratio of 1: 10 of methyl-PEG ₄ -thiol and having a radius of 5.3 ± 2.8 nm were dissolved in 20 mM Tris buffer The enzyme activity of the proteolytic enzyme MMP-7 was analyzed by coagulating with nickel cation and MMP-7 (see FIG. 7).

2. Screening method of protease inhibitors

Another aspect of the present invention provides a method for screening inhibitors of proteolytic enzymes.

The method of analyzing proteolytic enzyme activity of the present invention comprises the steps of: introducing a modified moiety having a carboxyl group at the terminal end thereof to the surface of metal nanoparticles to modify the surface of the metal nanoparticles with a carboxyl group; 2 ') to produce a linkage to which polyhistidine is introduced at both ends of a polypeptide substrate which is specifically degraded by the protease to be inhibited; 3 ') As the experimental group, the metal nanoparticles to which the carboxyl group of the step 1') is introduced are agglomerated in a buffer solution containing a divalent metal cation, an inhibitory substance protease, an inhibitor candidate substance and a connecting portion of the step 2 ' ; 4 ') measuring the aggregation degree of the aggregated metal nanoparticles in the step 3'); 5 ') comparing the degree of aggregation measured in step 4' with the degree of metal nanoparticle aggregation of a control group aggregated in a buffer solution not containing an inhibitor candidate material; And 6 ') determining that the inhibitor candidate contained in the test group exhibiting a higher degree of aggregation as compared with the control group is an enzyme activity inhibitor of the inhibitory protease.

The steps 1 '), 2') and 4 ') are the same as those described in the section " Method for Analyzing the Activity of Proteinase 1 " above. Therefore, this regard will be described only for the above-described detailed description of the source, using "1 activity assay of protease" item-specific configuration, hereinafter, be omitted and the method for screening inhibitors of the protease.

In the step 3 '), the metal nanoparticles prepared in the step 1') are agglomerated in a buffer solution containing a divalent metal cation, an inhibitory protease, an inhibitor candidate, and the linkage prepared in the step 2 ' do. The target is subjected to the above suppression effect by the protease inhibitor candidates since the additional step of adding 3) Unlike the inhibitor candidates in the "method 1 activity assay of protease". For example, if the added inhibitor candidate is in fact a substance that inhibits the enzymatic activity of the proteolytic enzyme, the decomposition reaction of the linkage by the proteolytic enzyme will be inhibited, and the metal nanoparticles will eventually flocculate. Conversely, if the added inhibitor candidate substance is a substance that does not actually affect the enzyme activity of the proteolytic enzyme, the degradation reaction of the linkage by the proteolytic enzyme is not inhibited, and the metal nanoparticles will not aggregate.

In the step 4 '), the degree of agglutination of the aggregated metal nanoparticles is measured in the step 3') as in the step 4) of " 1. Method for assaying protease activity ".

In the step 5 ') and the step 6'), the degree of coagulation of the metal nanoparticles measured in the step 4 ') is compared with the control group to screen the protease inhibitor.

The control in step 5 ') is performed in the same manner as in step 3'), but is a result of causing aggregation reaction of metal nanoparticles without inhibitor candidate material. Since the inhibitor candidate is not included in the control group, proteolytic enzymes are not affected by the enzyme activity, so that the degradation reaction of the linkage occurs and the aggregation reaction of the metal nanoparticles does not occur.

Based on the results of the above-mentioned step 5 '), it is considered that the test group exhibiting a higher level of aggregation than the control group is considered to be due to the inhibitor of protease, and the test group exhibiting higher degree of aggregation than the control group It is judged that the contained inhibitor candidate substance is an enzyme activity inhibitor of the inhibitory protease.

In the step 6 '), it is preferable that the degree of coagulation is as high as that having statistical significance by repeated experiment.

The inhibitor of proteolytic enzyme activity selected by the proteolytic enzyme inhibitor screening method can be used as a therapeutic agent for protease-mediated diseases. Therefore, the protease inhibitor screening method can be used for the screening of a therapeutic agent for protease-mediated diseases.

3. Methods for diagnosing protease-mediated diseases

Another aspect of the present invention provides a method for assaying protease activity for providing information necessary for diagnosis of protease-mediated diseases.

The protease activity assay method for providing information necessary for diagnosis of the protease-mediated disease comprises: introducing a modified moiety containing a carboxyl group at the 1 ' end of the metal nanoparticle to the surface of the metal nanoparticle, To a carboxy group; 2 ") a step of producing a junction in which polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by a protease to be analyzed; 3 ") grouping the metal nanoparticles into which the carboxyl group has been introduced, in a buffer solution containing a biological sample derived from the divalent metal cation, the linking portion, and the sample; 4 ") measuring the degree of aggregation of the metal nanoparticles; 5 ") comparing the measured degree of aggregation with the degree of metal nanoparticle aggregation of a control group aggregated in a buffer solution containing a biological sample derived from a normal subject; And 6 "). If the degree of aggregation of the experimental group is different from the degree of aggregation of the control group, it is determined that the subject has a risk of becoming or suffering from a protease-mediated disease.

The steps 1 ''), 2 '') and 4 '''are the same as those described in the item " 1. Analysis method of protease activity ". Therefore, a detailed description thereof will be omitted by referring to the description of the item " Method for Analyzing the Activity of Proteinase 1 & quot ;, and hereinafter, the protease activity for providing information necessary for diagnosis of the protease- Only the configuration specific to the analysis method is described.

The metal nanoparticles prepared in the step 1 " in the step 3 ") may be aggregated in a buffer solution containing a divalent metal cation, a biological sample derived from the sample, and the linkage prepared in the step 2 & do. The "1 activity assay of protease" Step 3) Unlike the biological sample of the agglutination reaction of the metallic nanoparticles derived from the subject because the biological sample of the subject-derived non-protease added in . ≪ / RTI > For example, if a biological sample derived from an added sample contains a proteolytic enzyme that is abnormally active in enzyme activity, a change in the decomposition reaction of the linking moiety due to the proteolytic enzyme will occur. As a result, the degree of aggregation of the metal nanoparticles Will also change.

The subject of step 3 ") is a subject that is suspected of having a protease-mediated disease or is in need of diagnosis of a risk of developing a protease-mediated disease, such as human, monkey, dog, goat, Vertebrae, and preferably mammals.

The biological sample of step 3 " is preferably a human-derived liquid sample, and the biological sample may be a blood sample, serum, plasma, saliva, sputum, lung sputum, multiple fluid, lymph fluid, body fluid, urine, cerebrospinal fluid, , Semen, vaginal fluid, amniotic fluid, amniotic fluid, synovial fluid, or tissue washing fluid, but is not limited thereto.

In the step 4 '', the degree of aggregation of the aggregated metal nanoparticles is measured in the step 3 '' as in the step 4) in " 1. Method for Analyzing the Activity of the Protease ."

In the above steps 5 '') and 6 ''), the degree of aggregation of the metal nanoparticles measured in the step 4 '' was compared with that of the control group, and it was judged that the subject had a risk of taking or taking a protease-mediated disease .

The control group of step 5 ") is carried out in the same manner as in step 3 " of the step 3 '', except that a biological sample derived from a normal individual, which is considered not to suffer protease-mediated disease, Is added to the metal nanoparticles to cause aggregation reaction of the metal nanoparticles. The control group, which contains biological samples derived from normal individuals, shows the enzymatic activity of the protease contained in a biological sample of an individual in the absence of protease-mediated disease.

The biological sample of step 5 " is preferably a human-derived liquid sample, and the biological sample may be a blood sample, serum, plasma, saliva, sputum, lung sputum, multiple fluid, lymph, fluid, urine, cerebrospinal fluid, , Semen, vaginal fluid, amniotic fluid, amniotic fluid, synovial fluid, or tissue washing fluid, but is not limited thereto.

Based on the result of the comparison in the step 5 "), the step 6 '' is considered to be that the experimental group exhibiting a higher or lower degree of aggregation than the control group contains a proteolytic enzyme having an abnormality in enzyme activity. Therefore, the subject provided with the biological sample included in the experimental group in which the degree of cohesion is different from that of the control group is considered to have a risk of or suffering from a protease-mediated disease.

It is preferable that the degree of coagulation is high or low in the step 6 '', which is high or low to the extent of having statistical significance by repeated experiments.

The protease-mediated diseases may be cancer, Alzheimer's disease, viral infectious diseases, bacterial infectious diseases, hemophilia, cardiovascular diseases and the like. In particular, the cancer may be a breast cancer, a liver cancer, a kidney cancer, a testicular cancer, a brain cancer, an ovarian cancer, a skin cancer, a lung cancer, a prostate cancer, a thyroid cancer, a pancreatic cancer, a cervical cancer, a colon cancer, a stomach cancer, a small bowel cancer, A squamous cell carcinoma, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a melanoma, a neuroblastoma or a retinoblastoma, and the like. In addition, the viral infection disease may be a disease infected by HIV (human immunodeficiency virus), HSV (Herpes Simplex Virus) or TEV (Tobacco Etch Virus). In addition, the bacterial infection disease may be a disease caused by Bacillus anthracis, and may be an anthrax.

Hereinafter, the present invention will be described in detail with reference to examples.

It is to be understood, however, that the following examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Analysis of Coagulation Conditions of Metal Nanoparticles

<1-1> Synthesis of metal nanoparticles

First, metal nanoparticles to be used for the measurement of protease activity were synthesized. Among various metals, gold (Au), which is easy to manufacture as nanoparticles, was used and nanoparticles were prepared according to the method known from the literature of Grabar et al. (Anal. Chem. (1995) 67, 735-743). More specifically, 100 μl of stock solution (300 μM) of HAuCl ₄ .3H ₂ O (Sigma-Aldrich, USA) was added to 100 ml of distilled water and continuously stirred to obtain a final concentration of 500 μM A working solution was made. To the working solution was added 2 ml of 30 mM sodium citrate (C ₆ H ₅ Na ₃ O ₇ .2H ₂ O) (Sigma-Aldrich, USA) (molar ratio of tetrachloromonohydrate: sodium citrate = 1: 2) And the mixture was stirred at a concentration of 800 μM. 100 μl of a stock solution of 300 mM sodium borohydride (NaBH ₄ ) (Sigma-Aldrich, USA) was rapidly added to the stirred solution and stirred to rapidly reduce and colloid gold ions, thereby preparing gold nanoparticles Respectively.

The gold nanoparticles prepared as described above were observed with UV-2550 (Shimadzu, Japan) and FE-SEM (Sirion, FEI, Netherlands). As a result, gold nanoparticles having a radius of 5.3 ± 2.8 nm Was produced.

<1-2> Surface modification of metal nanoparticles

<1-2-1> Surface modification of metal nanoparticles with carboxyl groups

To the gold nanoparticle solution prepared in Example <1-1>, methyl-PEG ₄ -thiol and carboxy-PEG ₁₂ -thiol in a ratio of 1:10 or 1: 1 were added to a final concentration of 300 μM, And reacted for 2 hours to modify the surface of the gold nanoparticles to a carboxyl group. The gold nanoparticles modified with carboxyl groups as described above were purified by centrifugation at 5000 g for 5 minutes using a Microcon YM-100 centrifugal filter unit (100 kDa MWCO).

<1-2-2> Surface modification of metal nanoparticles with nitrilotriacetic acid (NTA)

Example <1-1> in a 4 μM solution of gold nano-particles 25 ㎕ of concentration, 1 mM concentrations of the _{NTA-NH 2 (Nα, Nα} -Bis (carboxymethyl) -L-lysinehydrate) prepared in (Sigma-Aldrich, USA), 25 μl of a 2 μM EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide) (Thermo Scientific, USA) and distilled water to give a final volume of 100 μl, And the surface of the gold nanoparticles was modified with NTA. The gold nanoparticles modified with NTA as described above were purified by centrifugation at 8000 g for 10 minutes using Amicon ㄾ Ultra Centrifugal Filters (50 kDa) (Millipore, USA). The thus-prepared gold nanoparticles were washed with 20 mM Tris-HCl buffer (pH 7.4), resuspended, and stored at 4 ° C.

<1-3> Structure of polypeptide substrate and determination of coagulation conditions of metal nanoparticles according to presence or absence of divalent metal cation

In order to confirm the influence of the structure of the polypeptide substrate and the presence or absence of the divalent metal cation in the aggregation reaction of the surface-modified metal nanoparticles with the carboxyl group, the metal nanoparticles prepared in Example <1-2-1> The experimental group was designed as shown in Table 2 below.

In Table 2, SEQ ID NO: 1 having the sequence of HHHHHHGPLGMRGL is a polypeptide substrate having a structure in which histidine hexamer (HHHHHH) is linked to the N-terminus of GPLGMRGL (SEQ ID NO: 8), which is one of substrates of proteolytic enzyme MMP- And SEQ ID NO: 2 having the sequence of HHHHHHGPLGMRGLHHHHHH is a polypeptide substrate having a structure in which a histidine hexamer is linked to both N-terminal and C-terminal of GPLGMRGL to confirm the structure of the polypeptide substrate necessary for agglutination of the gold nanoparticles. Also, in Table 2, nickel chloride is injected to supply nickel divalent cations, which is to confirm the necessity of metal divalent cations for agglomeration of the gold nanoparticles.

As shown in Table 2, the solution having a final volume of 100 μl was vortexed, allowed to react at room temperature for 2 hours and allowed to stand for 2 hours. Then, the absorbance at 520 nm and 700 nm (E ₅₂₀ and E ₇₀₀ ) Was measured to confirm whether gold nanoparticles aggregated. When the gold nanoparticles aggregate, the absorbance at 520 nm (E ₅₂₀ ) is reduced and red shift occurs at the same time.

As a result, compared to the control group, the E ₅₂₀ values did not decrease in the first to third experimental groups and the ratio of the E ₅₂₀ values to the E ₇₀₀ values (E ₅₂₀ / E ₇₀₀ ) remained almost similar, whereas in the fourth experimental group The E ₅₂₀ value and the E ₅₂₀ / E ₇₀₀ value were significantly decreased (FIG. 3).

From the above results, it has been found that (1) the histidine polymer must be connected to both ends of the polypeptide substrate, and (2) the divalent metal cation is necessarily required for the surface-modified metal nanoparticles to be aggregated with the carboxyl group have.

<1-3> Comparison of Coagulation Efficiency of Metal Nanoparticles of Carboxyl Group and NTA

In order to compare the coagulation efficiencies of carboxyl-surface-modified metal nanoparticles and NTA-modified surface-modified metal nanoparticles, gold nanoparticles modified with a carboxyl group in the above Example <1-2-1> In Example < 1-2-2 >, gold nanoparticles modified with NTA were used to design an experimental group as shown in Table 3 below.

As shown in Table 3, the solution having a final volume of 100 μl was vortexed, reacted at room temperature for 2 hours and allowed to stand for 2 hours. Then, the absorbance at 520 nm and 700 nm (E ₅₂₀ and E ₇₀₀ ) Was measured to confirm whether gold nanoparticles aggregated.

As a result, the highest absorbance was observed at 520 nm in the first and second control groups in which neither the polypeptide substrate nor the divalent metal cation was added, and the ratio (E ₅₂₀ / E ₇₀₀ ) of the E ₅₂₀ value to the E ₇₀₀ value (Fig. 4). In the second experimental group that induced surface aggregation with gold nanoparticles modified with NTA, some redshift phenomenon was observed and the E ₅₂₀ / E ₇₀₀ value was decreased, leading to aggregation of gold nanoparticles (Fig. 4). However, it was observed that the red shift phenomenon appeared more remarkably in the carboxyl-surface-modified gold nanoparticles and the E ₅₂₀ / E ₇₀₀ value was further reduced (FIG. 4).

From the above results, it can be seen that, compared with the metal nanoparticles modified with NTA, metal nanoparticles whose surface was modified with a carboxyl group aggregate more efficiently in the presence of a polypeptide substrate having a histidine polymer connected at both ends thereof and divalent metal cations Able to know.

<1-4> Comparison of flocculation efficiency of metal nanoparticles according to the type of buffer solution and the ratio of polypeptide substrate

In order to confirm the influence of the kind of the buffer solution and the ratio of the polypeptide substrate on the aggregation of the metal nanoparticles, the metal nanoparticles prepared in Example <1-2-1> Respectively.

As shown in Table 4, the solution having a final volume of 100 μl was vortexed, reacted at room temperature for 2 hours and allowed to stand for 2 hours. Then, the absorbance at 520 nm and 700 nm (E ₅₂₀ and E ₇₀₀ ) Was measured to confirm whether gold nanoparticles aggregated.

As a result, the aggregation reaction of gold nanoparticles occurs efficiently at a mole ratio of 1:25 or more of the gold nanoparticles and the polypeptide substrate, and the aggregation reaction of the gold nanoparticles in the Tris buffer solution, particularly, the 20 mM Tris buffer solution (Fig. 5).

From the above results, the polypeptide substrate is added at a molar concentration of 25 times or more as compared with the metal nanoparticles, and the aggregation efficiency of the metal nanoparticles is further enhanced in a neutral or weakly basic buffer solution than in a buffer solution containing an acid or a salt .

<1-5> Comparison of Coagulation Efficiency of Metal Nanoparticles by Divalent Metal Cations

In order to confirm the influence of aggregation of metal nanoparticles on various kinds of divalent metal cations, an experiment group was designed using the metal nanoparticles prepared in Example <1-2-1> as shown in Table 5 below .

As shown in Table 5, the solution having a final volume of 100 μl was vortexed, allowed to react at room temperature for 2 hours and allowed to stand for 2 hours, and the absorbance at 520 nm and 700 nm (E ₅₂₀ and E ₇₀₀ ) Was measured to confirm whether gold nanoparticles aggregated.

As a result, regardless of the kind of the divalent metal cation, the aggregation efficiency of the gold nanoparticles was improved, but nickel, cobalt and zinc were found to be effective (FIG. 6).

Measurement of protease activity

<2-1> Measurement of protease activity

To determine the activity of proteolytic enzymes using agglutination of metal nanoparticles, an experimental group was designed using MMP-7 (Matrix Metalloprotase-7), one of the proteolytic enzymes, as shown in Table 6 below.

In Table 6, the gold nanoparticles, the polypeptide substrate, the nickel chloride, the Tris buffer solution and the MMP-7 were mixed together in a reaction tube at 37 ° C for 2 hours .

 As shown in Table 6, the second experimental group is a so-called '2-step method'. The peptide substrate and the MMP-7 were mixed in a reaction tube and reacted at 37 ° C for 2 hours. Then, the gold nanoparticles, And Tris buffer solution are added, and the reaction is continued for another 2 hours.

After reacting as described above, the reaction was allowed to stand for another 2 hours, and absorbance at 520 nm and 700 nm (E ₅₂₀ and E ₇₀₀ ) was measured with a UV-Vis spectroscope to confirm whether the gold nanoparticles aggregated.

As a result, the gold nanoparticles were not aggregated at all in the first control group except for the gold nanoparticles, and the highest absorbance at 520 nm was observed. In the second control group in which the polypeptide substrate and the nickel divalent cation were added, All were aggregated and the absorbance decreased remarkably at 520 ㎚. On the other hand, in the 1-7 experimental group and the second experimental group to which MMP-7 was added, the absorbance at 520 nm was somewhat reduced and a red shift phenomenon was observed, and the gold nanoparticles not aggregated due to the hydrolytic activity of MMP- (Fig. 7 (A)). Also, it was confirmed that the gold nanoparticles not aggregated in the 1-7 experimental group were more present than the second experimental group, and it was confirmed that the one-pot method was more effective in measuring the activity of the proteolytic enzyme.

 As a result of measuring the activity of proteolytic enzymes by treating MMP-7 at different concentrations (0 nM to 100 nM) according to the one-pot method as described above, the concentration of MMP-7 was 20 nM or less , The activity of MMP-7 was almost not exhibited. When the concentration of MMP-7 was 50 nM or more, the activity of MMP-7 became saturated (FIG. 7 (B)). From the above results, it can be seen that the method of measuring the protease activity of the present invention is applicable to a case where the concentration of the protease is 20 nM or more.

<2-2> Activity measurement by silver-staining method

In addition to the method of measuring the absorbance as in the embodiment <2-1>, the aggregation degree of the metal nanoparticles may be observed by observing the color change by staining the non-agglomerated metal nanoparticles with silver.

In order to confirm the application of the silver staining method as described above, the tubes subjected to the reaction in the first control group, the second control group and the first 1-7 test group of Example <2-1> were centrifuged at 6000 rpm for 20 minutes, After immersing the gold nanoparticles, 20 microliters of the supernatant containing unaggregated gold nanoparticles was obtained, and 20 microliters of the supernatant was added to the Silver Enhancement Kit ^(R) (Sigma-Aldrich, USA) The color change was observed after silver salt according to the manufacturer's manual.

As a result, dark gray was observed in the first control group in which gold nanoparticles were not agglomerated (FIG. 9A), and colorless in the second control group in which aggregation reaction occurred due to absence of MMP-7 (FIG. 9 B)). Pale gray was observed in the 1-7 experimental group in which de-aggregation was caused by MMP-7 (FIG. 9 (C)).

From the above results, it was confirmed that the silver staining method can be applied to measure the degree of aggregation of metal nanoparticles, and the activity of proteolytic enzyme can be measured using the silver staining method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

<110> Industry-University Cooperation Foundation Hanyang University <120> Method for analyzing the protease activity and the application          the <130> HY120041N <160> 21 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> MMP-7 substrate with 6His at N-termainal <400> 1 His His His His His His Gly Pro Leu Gly Met Arg Gly Leu   1 5 10 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> MMP-7 substrate with 6His at both N- and C-termainal <400> 2 His His His His His Gly Pro Leu Gly Met Arg Gly Leu His His   1 5 10 15 His His His His              20 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for Cathepsin D <220> <221> VARIANT <222> (4) &Lt; 223 > Xaa = Cys (Et) which is ethylcysteine <400> 3 Gly Pro Ile Xaa Phe Phe Arg Leu Gly   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 1st minimal substrate sequence for MMP-2 <400> 4 Gly Pro Leu Gly Val Arg Gly   1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2nd minimal substrate sequence for MMP-2 <400> 5 Pro Leu Gly Leu   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 1st minimal substrate sequence for MMP-7 <400> 6 Gly Val Pro Leu Ser Leu Thr Met Gly   1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 2nd minimal substrate sequence for MMP-7 <400> 7 Arg Pro Leu Ala Leu Trp Arg Ser   1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 3rd minimal substrate sequence for MMP-7 <400> 8 Gly Pro Leu Gly Met Arg Gly Leu   1 5 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for MMP-14 <400> 9 Ser Gly Arg Ile Gly Phe Leu Arg Thr Ala Lys   1 5 10 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for uPA <400> 10 Gly Gly Leu Gly Gln Arg Cys Arg Ser Ala Asn Ala Ile Leu Glu   1 5 10 15 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for Caspase-1 <400> 11 Gly Trp Glu His Asp Gly   1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for Caspase-3 <400> 12 Gly Asp Glu Val Asp Gly Ser Gly   1 5 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for HIV-1 protease <400> 13 Gly Val Ser Gln Asn Thr Pro Ile Val Gly   1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for HSV-1 protease <400> 14 Leu Val Leu Ala Ser Ser Ser Phe Gly Tyr   1 5 10 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for Thrombin <400> 15 Leu Val Pro Arg Gly Ser   1 5 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for PSA <400> 16 His Ser Ser Lys Leu Gln   1 5 <210> 17 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for Anthrax lethal factor          endopeptidase <400> 17 Lys Lys Lys Lys Val Leu Pro Ile Gln Leu Asn Ala Ala Thr Asp Lys   1 5 10 15 Gly Gly         <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for B-Secretase <400> 18 Glu Val Asn Leu Asp Ala Gly Phe   1 5 <210> 19 <211> 2 <212> PRT <213> Artificial Sequence <220> <223> 1st minimal substrate sequence for MMP-1 / MMP-8 / MMP-13 <400> 19 Gly Ile 166 <210> 20 <211> 2 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > 2nd minimal substrate sequence for MMP-1 / MMP-8 / MMP-13 <400> 20 Gly Leu 177 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> minimal substrate sequence for TEV protease <400> 21 Glu Asn Leu Tyr Phe Gln Ser  18 22

Claims (30)

Introducing a modified portion containing a carboxyl group into the surface of the metal nanoparticle to modify the surface of the metal nanoparticle to a carboxyl group;
Preparing a connection portion into which polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by the protease to be analyzed;
Aggregating the metal nanoparticles into which the carboxyl group has been introduced as an experimental group, in a buffer solution containing a divalent metal cation, an analyte to be analyzed and the connecting portion;
Measuring the degree of aggregation of the metal nanoparticles; And
And comparing the measured degree of aggregation with the degree of aggregation of metal nanoparticles in a control group aggregated in a buffer solution containing no protease to be analyzed. The method according to claim 1, wherein the metal nanoparticles are composed of any one or two or more mixed metals selected from the group consisting of gold, silver, copper, platinum, palladium, nickel and iron . The method according to claim 1, wherein the metal nanoparticles have a diameter of 3 nm to 30 nm. The method for analyzing the activity of a proteolytic enzyme according to claim 1, wherein the modified part is a high molecular substance having a carboxyl group at one end and a thiol group at the other end. The method of claim 4, wherein the modifying moiety is HS- (polyethylene glycol) _n -COOH,
Wherein n is an integer of 1 to 20. The method according to claim 1, wherein the modified part is introduced into the metal nanoparticle together with the modified density control part. The method of claim 6, wherein the modified density regulator is HS- (polyethylene glycol) _m- R,
M is an integer of 1 to 8,
Wherein R is an alkyl group having 1 to 5 carbon atoms. 2. The method according to claim 1, wherein the activity assay proteinase is selected from the group consisting of cathepsin, MMP (matrix metalloproteinase), caspase, secretase, urokinase ), HIV-1 protease, HSV-1 protease, TEV protease, Prostate Specific Antigen (PSA), endothelial factor endoproteinase Anthrax lethal factor endopeptidase, and thrombin. &Lt; Desc / Clms Page number 19 &gt; 2. The method according to claim 1, wherein the polypeptide substrate is a polypeptide sequence comprising a polypeptide represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 3 to 21 . The method according to claim 1, wherein the polyhistidine is composed of 2 to 20 histidines. The method of claim 1, wherein the aggregation of the metal nanoparticles
Wherein the analyzing step is carried out by injecting both the divalent metal cations, the protease to be analyzed, the connecting part and the metal nanoparticles into which the carboxyl group has been introduced into the buffer solution, and simultaneously reacting them. The method of claim 1, wherein the aggregation of the metal nanoparticles
Reacting the junction with a protease to be analyzed in a buffer solution to decompose the polypeptide substrate in the junction;
Characterized in that before the polypeptide substrate is completely decomposed, the metal nanoparticles to which the carboxyl group has been introduced are introduced into the buffer solution and the divalent metal cations are injected and reacted. The method according to claim 1, wherein the divalent metal cation is any one or two selected from the group consisting of Ni ²⁺ , Co ²⁺ , Zn ^2+, and Cu ²⁺ . 2. The method according to claim 1, wherein the buffer solution is a Tris buffer solution or a HEPES buffer solution. The method according to claim 1, wherein the measurement of the degree of cohesion is performed by measuring the ratio of absorbance at two wavelengths at which the absorbance changes by coagulation. 2. The method according to claim 1, wherein the measurement of the degree of coagulation is performed by staining the unagglutinated metal nanoparticles separated from the buffer solution by a silver-staining method and measuring a color change. Analysis of the activity of degrading enzymes. Introducing a modified portion containing a carboxyl group into the surface of the metal nanoparticle to modify the surface of the metal nanoparticle to a carboxyl group;
Producing a junction where polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by the inhibitory protease;
Aggregating the metal nanoparticles into which the carboxyl group has been introduced as an experimental group in a buffer solution containing a divalent metal cation, an inhibitory protease, an inhibitor candidate, and the connecting portion;
Measuring the degree of aggregation of the metal nanoparticles;
Comparing the measured degree of aggregation with a degree of metal nanoparticle aggregation of a control group aggregated in a buffer solution not containing an inhibitor candidate material; And
And determining that the inhibitor candidate contained in the test group exhibiting a high degree of aggregation as compared with the control group is an enzyme activity inhibitor of the inhibition subject protease. The metal nanoparticle according to claim 17, wherein the metal nanoparticles are composed of any one or two or more mixed metals selected from the group consisting of gold, silver, copper, platinum, palladium, nickel and iron and have a diameter of 3 nm to 30 nm Wherein the protease inhibitor is selected from the group consisting of: 18. The method of claim 17, wherein the modifying moiety is selected from the group consisting of HS- (polyethylene glycol) n-COOH (wherein n is an integer of 1 to 20)
Wherein the protein is introduced into the metal nanoparticle together with a modifying density regulator having a structure of HS- (polyethylene glycol) mR (wherein m is an integer of 1 to 8). 18. The method according to claim 17, wherein the polypeptide substrate is a polypeptide sequence comprising a polypeptide represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 3 to 21 . 18. The method for screening inhibitors of proteolytic enzymes according to claim 17, wherein the polyhistidine is composed of 2 to 20 histidine. 18. The method of claim 17, wherein the aggregation of the metal nanoparticles
And the step of injecting both of the divalent metal cations, the inhibitory protease, the connecting portion and the metal nanoparticles into which the carboxyl group has been introduced into the buffer solution, and simultaneously reacting them. The method for screening inhibitors of proteolytic enzymes according to claim 17, wherein the divalent metal cation is any one or more selected from the group consisting of Ni ^{2 +} , Co ^{2 +} , Zn ^{2 +,} and Cu ^{2 +} . 18. The method for screening inhibitors of proteolytic enzymes according to claim 17, wherein the measurement of the degree of aggregation is performed by measuring a maximum absorbance of the metal nanoparticles. Introducing a modified portion containing a carboxyl group into the surface of the metal nanoparticle to modify the surface of the metal nanoparticle to a carboxyl group;
Preparing a connection portion into which polyhistidine is introduced at both ends of a polypeptide substrate that is specifically degraded by the protease to be analyzed;
Aggregating the metal nanoparticles into which the carboxyl group has been introduced as an experimental group in a buffer solution containing a biological sample derived from a divalent metal cation, the linking portion, and a sample;
Measuring the degree of aggregation of the metal nanoparticles;
Comparing the measured degree of aggregation with a degree of metal nanoparticle aggregation of a control group aggregated in a buffer solution containing a biological sample derived from a normal individual; And
And determining that the subject has a risk of engulfing or taking a protease-mediated disease when the degree of coagulation of the experimental group is different from the degree of coagulation of the control group, information necessary for diagnosis of a protease-mediated disease Wherein the proteolytic enzyme activity analyzing method comprises the steps of: 26. The method of claim 25, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, lymph fluid, body fluid, urine, cerebrospinal fluid, spinal fluid, semen, vaginal fluid, amniotic fluid, Wherein the proteolytic enzyme activity assay is performed to provide information necessary for diagnosis of a protease-mediated disease. 26. The method according to claim 25, wherein the protease-mediated disease is any one selected from the group consisting of cancer, Alzheimer's disease, viral infectious disease and bacterial infectious disease. A method for analyzing protease activity. 27. The method of claim 27, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, renal cancer, testicular cancer, brain cancer, ovarian cancer, skin cancer, lung cancer, prostate cancer, thyroid cancer, pancreatic cancer, cervical cancer, colon cancer, gastric cancer, , Gastrointestinal cancer, genitourinary cancer, uterine cancer, head and neck cancer, coin cancer, small cell lung cancer, non-small cell lung cancer, Ebbis tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, melanoma, neuroblastoma and retinoblastoma Wherein the protease inhibitor is selected from the group consisting of a protease inhibitor and a protease inhibitor. 28. The method according to claim 27, wherein the viral infection disease is a disease infected by HIV (human immunodeficiency virus), HSV (Herpes Simplex Virus) or TEV (Tobacco Etch Virus) Proteinase activity assay to provide information. 28. The method according to claim 27, wherein the bacterial infection disease is a disease infected by Bacillus anthracis. 28. A method for analyzing protease activity for providing information necessary for diagnosis of protease-mediated diseases.

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