CN109750082B - Fluorescent polypeptide substrate for detecting human gelatinase MMP-2 and application thereof - Google Patents
Fluorescent polypeptide substrate for detecting human gelatinase MMP-2 and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent polypeptide substrate for detecting human gelatinase MMP-2. The fluorescent polypeptide substrate comprises an amino acid sequence shown as Peptide II, wherein valine at the 1 st position of the amino acid sequence is combined with fluorescent group 5-carboxyfluorescein, and lysine at the 11 th position is combined with fluorescence quenching group 5-carboxytetramethylrhodamine. The fluorescent polypeptide substrate reacts with human MMP-2, the enzymatic reaction kinetic constant Km is 315 mu M, and Kcat/Km: 2565M‑1·S‑1(ii) a Human MMP-9 at an enzymatic activity concentration of less than 6. mu.M is hardly reactive with the fluorescent polypeptide substrate of the present invention. The fluorescent polypeptide substrate for detecting the activity of the human MMP-2 does not react with human gelatinase MMP-9 of different families, and has certain specificity. The method for detecting the human MMP-2 and screening the human MMP-2 inhibitor is simple and convenient to operate, is quick and has good application prospect.
Description
Technical Field
The invention relates to a fluorescent polypeptide substrate containing a specific sequence or motif and capable of detecting human gelatinase-2 (MMP-2) and an activity detection method thereof, belonging to the technical field of biology.
Technical Field
Matrix Metalloproteinases (MMPs) can almost degrade various protein components of extracellular matrix, and are a Zn-dependent class2+The endopeptidase family is mainly derived from various tissues and cells such as brain tissue, endothelial cells, fibroblasts and smooth muscle cells[1]. MMPs can be classified into six major classes, i.e., gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, MMP-8, MMP-13 and MMP-18), stromelysins (MMP-3, MMP-10 and MMP-11), stromelysins (MMP-7 and MMP-26), membrane-type matrix metalloproteinases (MT-MMPs; (MMP-14 and MMP-15, etc.) and other secreted MMPs (MMP-19 and MMP-21, etc.), according to their substrate specificity, sequence similarity and domain composition[2]. MMPs are involved not only in physiological processes such as tissue remodeling, angiogenesis, wound healing and embryogenesis and development, but also in processes such as hypertension, preeclampsia, acute myocardial infarction, aneurysm formation, venous expansion, malignancyPlays a key role in the pathophysiological process of diseases such as tumor[3,4]. By detecting matrix metalloprotease, the pathogenic mechanism of the disease can be deeply understood, which is helpful for early diagnosis and treatment of the disease, so the research and development of matrix metalloprotease detection technology are important.
Gelatinases, including MMP-2 and MMP-9, primarily degrade gelatin (denatured collagen) and type iv collagen, and cleave precursors of MMPs to activate them, and also degrade bioactive factors that regulate the inflammatory response of atherosclerosis, such as: growth factors, cytokines, tumor necrosis factor-beta 1, and the like. A large number of researches show that MMP-2 and MMP-9 are involved in the physiological and pathological processes of embryo implantation, vascular smooth muscle cell proliferation, extracellular matrix degradation, thrombotic diseases, heart failure, malignant tumors and the like[4-15]. However, despite their structural similarity, the pathophysiological conditions involved in certain diseases are not exactly the same. For example, in heart failure, MMP-2 appears to be more dependent on neurohumoral regulation than MMP-9, whereas MMP-9 is closely related to the etiology of ischemic heart failure and the myocardial ischemia process, and plays a selectively inhibitory role in the myocardial remodeling process after myocardial infarction[16,17]. Through discriminant analysis, the circulating MMP-2 can be used as a biomarker for predicting the cardiovascular events in the early stage of heart failure of the new heart failure patients, and has higher prognostic value; while several studies have clearly demonstrated the prognostic role of circulating MMP-9 on the progression of heart failure or cardiovascular death events following myocardial infarction[18-21]. Therefore, the deep research on the pathogenesis of MMP-2 and MMP-9 is expected to realize the effective diagnosis and treatment of related diseases.
The current common enzyme activity detection methods for MMPs mainly comprise: the protease activity is detected by methods such as fluorescent probe technology, high performance liquid chromatography, isotope determination method, spectrophotometry, electrochemical method and the like. The fluorescent probe technology can realize the potential of real-time, quantitative and visual detection, the sensitivity is generally higher than that of other types by a plurality of orders of magnitude, and meanwhile, the synthesis and purification method of the fluorescent polypeptide is simple and convenient, the influence of temperature, ionic strength, pH and the like on experimental results can be avoided, so that the fluorescent probe technology is widely used in the fields of biological structure research, chemical analysis and the like, and particularly has obvious effect in related research of the protease.
Basic principle of detection of MMPs Activity by fluorescent substrates: a fluorescent group and a quencher are respectively combined at two ends of a Fluorescence Resonance Energy Transfer (FRET) peptide, and in the complete FRET peptide, the fluorescent group is quenched by the quencher, so that energy can not be detected within the excitation wavelength of the fluorescent group; after MMPs are cut into two independent fragments, the fluorescence of the fluorescent group is recovered, and the fluorescence intensity can be monitored under specific excitation/emission (Ex/Em) wavelength, so that the aim of detecting the activity of the protease is fulfilled[22]。
However, there is substrate similarity between families of MMPs. A sensitive substrate and a detection method which have high specificity, can detect the activity of MMP-2 and can distinguish MMP-2 and MMP-9 belonging to gelatinase are established, and are important for developing the function research of the gelatinase.
Reference documents:
[1]Galis ZS,Khatri JJ.Matrix metalloproteinases in vascular remodeling and atherogenesis:the good,the bad,and the ugly[J].Circulation research.2002,90(3):251-62.
[2]Visse R,Nagase H.Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure,function,and biochemistry[J].Circulation research.2003,92(8):827-39.
[3]Dabek J,Kulach A,Gasior Z.The role of matrix metalloproteinases in acute coronary syndromes[J].European journal of internal medicine.2007,18(6):463-6.
[4]Wang X,Khalil RA.Matrix Metalloproteinases,Vascular Remodeling,and Vascular Disease[J].Advances in pharmacology(San Diego,Calif).2018,81:241-330.
[5]Bergers G,Brekken R,McMahon G,Vu TH,Itoh T,Tamaki K,et al.Matrix metalloproteinase-9triggers the angiogenic switch during carcinogenesis[J].Nature cell biology.2000,2(10):737-44.
[6]Sheu BC,Hsu SM,Ho HN,Lien HC,Huang SC,Lin RH.A novel role of metalloproteinase in cancer-mediated immunosuppression[J].Cancer research.2001,61(1):237-42.
[7]Dong Z,Kumar R,Yang X,Fidler IJ.Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma[J].Cell.1997,88(6):801-10.
[8]O'Reilly MS,Wiederschain D,Stetler-Stevenson WG,Folkman J,Moses MA.Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance[J].The Journal of biological chemistry.1999,274(41):29568-71.
[9]Shi Y,Su C,Hu H,Yan H,Li W,Chen G,et al.Serum MMP-2as a potential predictive marker for papillary thyroid carcinoma[J].PloS one.2018,13(6):e0198896.
[10]Hannocks MJ,Zhang X,Gerwien H,Chashchina A,Burmeister M,Korpos E,et al.The gelatinases,MMP-2and MMP-9,as fine tuners of neuroinflammatory processes[J].Matrix biology:journal of the International Society for Matrix Biology.2017.
[11]Kurzepa J,Kurzepa J,Golab P,Czerska S,Bielewicz J.The significance of matrix metalloproteinase(MMP)-2and MMP-9in the ischemic stroke[J].The International journal of neuroscience.2014,124(10):707-16.
[12]Shevchenko AV,Golovanova OV,Konenkov VI,Tolkacheva OM,Maksimov VN,Voevoda MI,et al.[Analysis of the gene polymorphism of matrix metalloproteinase-2and-9in patients with coronary heart disease][J].Terapevticheskii arkhiv.2010,82(1):31-4.
[13]Wu H,Shou X,Liang L,Wang C,Yao X,Cheng G.Correlation between plasma angiopoietin-1,angiopoietin-2and matrix metalloproteinase-2in coronary heart disease[J].Archives of medical science:AMS.2016,12(6):1214-9.
[14]Kobusiak-Prokopowicz M,Krzysztofik J,Kaaz K,Jolda-Mydlowska B,Mysiak A.MMP-2and TIMP-2in Patients with Heart Failure and Chronic Kidney Disease[J].Open medicine(Warsaw,Poland).2018,13:237-46.
[15]Xu L,Chen Y,Ji Y,Yang S.Influencing factors of NT-proBNP level inheart failure patients with different cardiacfunctions and correlation with prognosis[J].Experimental and therapeutic medicine.2018,15(6):5275-80.
[16]Creemers EE,Cleutjens JP,Smits JF,Daemen MJ.Matrix metalloproteinase inhibition after myocardial infarction:a new approach to prevent heart failure？[J].Circulation research.2001,89(3):201-10.
[17]Sanchis L,Andrea R,Falces C,Llopis J,Morales-Ruiz M,Lopez-Sobrino T,et al.Prognosis of new-onset heart failure outpatients and collagen biomarkers[J].European journal of clinical investigation.2015,45(8):842-9.
[18]Bajraktari G,Miccoli M,Buralli S,Fontanive P,Elezi S,Metelli MR,et al.Plasma metalloproteinase-9and restrictive filling patternas major predictors of outcome in patients with ischemic cardiomyopathy[J].European journal of internal medicine.2012,23(7):616-20.
[19]Dini FL,Buralli S,Bajraktari G,Elezi S,Duranti E,Metelli MR,et al.Plasma matrix metalloproteinase-9better predicts outcome than N-terminal protype-B natriuretic peptide in patients with systolic heart failure and a high prevalence of coronary artery disease[J].Biomedicine&pharmacotherapy＝Biomedecine&pharmacotherapie.2010,64(5):339-42.
[20]Wagner DR,Delagardelle C,Ernens I,Rouy D,Vaillant M,Beissel J.Matrix metalloproteinase-9is a marker of heart failure after acute myocardial infarction[J].Journal of cardiac failure.2006,12(1):66-72.
[21]Kelly D,Khan SQ,Thompson M,Cockerill G,Ng LL,Samani N,et al.Plasma tissue inhibitor of metalloproteinase-1and matrix metalloproteinase-9:novel indicators of left ventricular remodelling and prognosis after acute myocardial infarction[J].European heart journal.2008,29(17):2116-24.
[22]Stryer L.Fluorescence energy transfer as a spectroscopic ruler[J].Annual review of biochemistry.1978,47:819-46.
disclosure of Invention
In view of the above, the present invention aims to provide a fluorescent polypeptide substrate with high specificity, which can detect human gelatinase MMP-2.
The invention adopts the following technical scheme: a fluorescent polypeptide substrate for detecting human MMP-2 is synthesized by using a chemical synthesis method, wherein the substrate comprises an amino acid sequence shown as Peptide II, the molecular weight is 1906Da, and the amino group of the 1 st valine at the N end of the amino acid sequence is combined with a fluorescent group, namely 5-carboxyfluorescein (5-FAM), and the amino group of the 11 th lysine is combined with a fluorescence quenching group, namely 5-carboxytetramethylrhodamine (5-TAMRA).
When irradiated by laser light, the fluorescent group in the fluorescent polypeptide substrate can emit energy to detect the fluorescence intensity. When the fluorescent polypeptide substrate is irradiated by laser with a certain wavelength, energy emitted by the fluorescent group is quenched by the quenching group, and the energy cannot be detected within the excitation wavelength of the fluorescent group; when the fluorescent polypeptide substrate is cleaved by the protease, the energy emission of the fluorophore is restored and detected.
The fluorescent polypeptide substrate can react with human MMP-2 with the enzyme activity concentration of 0.15-6 mu M, the enzymatic reaction kinetic constant Km is 315 mu M, and Kcat/Km: 2565M-1·S-1(ii) a However, human MMP-9, which had an enzymatic activity concentration of less than 6. mu.M, reacted little with the fluorescent polypeptide substrate of the present invention. It is demonstrated that the fluorescent polypeptide substrate of the present invention can be used for detecting the activity of human MMP-2 and can distinguish between MMP-2 and MMP-9 belonging to human gelatinase.
The fluorescent polypeptide substrate can be used for detecting the activity of human MMP-2 and screening human MMP-2 inhibitors.
The fluorescent polypeptide substrate can be used for detecting the activity of MMP-2 in human serum, the serum to be detected after APMA activation is taken to be mixed with excessive fluorescent polypeptide substrate in reaction buffer solution, a 100 mu l reaction system is established on a black 96-pore plate, the fluorescence intensity is detected at 37 ℃ under the excitation wavelength and emission wavelength (Ex/Em) corresponding to a fluorescence group combined by the fluorescent polypeptide substrate, a standard curve is established, the activity of the MMP-2 enzyme is calculated, and the activity of the MMP-2 in different serum samples can be compared by expressing the speed mu mol/L.s.mg of protease cracking substrate with unit mass.
In an embodiment, the fluorescent clique to which the fluorescent polypeptide substrate of the invention binds corresponds to an excitation wavelength of 485nm and an emission wavelength of 538 nm. The reaction buffer comprises: 50mM Tris, pH7.4, 20mM CaCl2，200mM NaCl，0.005％Brij-35。
The invention also provides a method for screening the human MMP-2 inhibitor, which comprises the steps of mixing a sample to be tested with the activated human MMP-2 in a reaction buffer solution, incubating at 37 ℃, adding a proper amount of the fluorescent polypeptide substrate, establishing a 100 mu l reaction system on a black 96-pore plate, and generating a fluorescence value of the fluorescent polypeptide substrate at the specific Ex/Em at 37 ℃. If the activity of the human MMP-2 enzyme is inhibited, the fluorescent peptide chain substrate cannot be enzymolyzed, the fluorescent group connected with the 1 st isoleucine at the N end is quenched by the quenching group connected with the 11 th lysine site, so that the fluorescence intensity is weakened or even zero, the sample to be detected is considered to be the inhibitor of the human MMP-2, and the Ki value of the inhibitor can be calculated.
The invention also provides a kit for detecting MMP-2 enzyme, which comprises the fluorescent polypeptide substrate. The reaction buffer solution in the kit is 50mM Tris with pH of 7.5 and 10mM CaCl2，150mM NaCl，0.05％Brij-35。
The kit provided by the invention has the advantages of good stability, long storage time and convenience in MMP-2 activity detection, and can be applied to detection of MMP-2 activity in serum.
The invention chemically synthesizes a fluorescent polypeptide substrate Peptide II according to the characteristics of human gelatinase MMP-2, and the Peptide II has definite specificity and pertinence. Human MMP-9 having an enzymatic activity concentration of less than 6. mu.M reacted with the fluorescent polypeptide substrate of the present invention with little reaction. It is demonstrated that the fluorescent polypeptide substrate of the present invention can be used for detecting the activity of human MMP-2 and can distinguish between MMP-2 and MMP-9 belonging to human gelatinase. Can provide a new idea for the MMP-2 activity change research and mechanism research in cardiovascular diseases; meanwhile, a more convenient approach is provided for the targeted MMP-2 medicament treatment research.
Drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 is a HPLC analysis of a fluorescent polypeptide substrate of the present invention.
FIG. 2 is a mass spectrometric analysis of a fluorescent polypeptide substrate of the present invention.
FIG. 3 is an enzymatic reaction of a fluorescent polypeptide substrate of the present invention with human MMP-2. The fluorescent polypeptide substrate is a fluorescent polypeptide substrate Peptide II shown in the invention, the substrate concentration is 50 mu M, and the enzymatic activity concentration of human MMP-2 is 0 mu M and 3 mu M. The abscissa is time (min) and the ordinate is relative fluorescence intensity (. DELTA.RFU).
FIG. 4 is a graph showing the enzymatic reaction of a fluorescent polypeptide substrate of the present invention with human MMP-2 at various concentrations of enzymatic activity. The fluorescent polypeptide substrate is Peptide II, the substrate concentration is 50 μ M, and the human MMP-2 concentration is 0 μ M, 0.15 μ M, 0.3 μ M, 0.6 μ M, 1.2 μ M, 3 μ M, 6 μ M respectively. The horizontal axis represents MMP-2 enzyme activity concentration (. mu.M), and the vertical axis represents relative fluorescence intensity (. DELTA.RFU)
FIG. 5 is a graph showing the enzymatic reaction of a fluorescent polypeptide substrate of the present invention with human MMP-2 and human MMP-9 at different enzyme activity concentrations. The fluorescent polypeptide substrate is Peptide II, the substrate concentration is 50 mu M, and the enzyme activity concentrations of MMP-2 and MMP-9 are respectively 0.6 mu M, 3 mu M and 6 mu M. The abscissa is time (min) and the ordinate is relative fluorescence intensity (. DELTA.RFU).
Detailed Description
The embodiment of the invention discloses a detection method for detecting the activity of human gelatinase MMP-2.
Example (b): the fluorescent polypeptide substrate is chemically synthesized by Beijing Saibutsu bioengineering company, recombinant human MMP-2 and recombinant human MMP-9 are purchased from American R & D Systems company, APMA and a fluorescent substrate XIV for MMP-2 and MMP-9 activity titration are purchased from American Anaspec company, and GM6001 (broad-spectrum MMPs inhibitor) is purchased from American MCE company.
1. Synthesis and storage of fluorescent polypeptides
Chemically synthesizing a fluorescent polypeptide substrate, wherein the sequence is shown as Peptide II, synthesizing the fluorescent polypeptide substrate, purifying by high pressure liquid chromatography, and detecting the molecular weight of the fluorescent polypeptide substrate by mass spectrometry, as shown in figure 1 and figure 2. The fluorescent polypeptide substrate is stored in a refrigerator at the temperature of-20 ℃ in a freeze-dried powder form. DMSO dissolves the fluorescent polypeptide substrate freeze-dried powder of the invention to prepare 1mM solution, and the solution is stored at-20 ℃.
2. Enzymatic reaction of gelatinases with the fluorescent polypeptide substrates of the invention
2.1 kinetic parameters Km, Kcat of the enzymatic reaction of the fluorescent polypeptide substrates according to the invention
50 mu l of human gelatinase MMP-2 and human gelatinase MMP-9 enzyme solutions with the same enzyme activity concentration or equal volume of reaction buffer solution and the fluorescent polypeptide substrate are respectively incubated for 30min at 37 ℃, then 50 mu l of Peptide II working solution is added into a black opaque 96-well plate to establish a 100 mu l reaction system, the final enzyme concentration is 3 mu M, and the Peptide II working solution comprises 50mM Tris and 10mM CaCl2150mM NaCl, 0.05% Brij-35, at a final concentration of 50. mu.M; after enzyme and substrate are mixed uniformly, the mixture is immediately put into a fluorescence enzyme labeling instrument, the fluorescence intensity is read by an Ex/Em-485 nm/538nm filter at 37 ℃, the reading is carried out once every 30s for 1000 times in total, then dynaFit4 software is used for solving the kinetic parameters Km and Kcat of the enzymatic reaction of human MMP-2 to the fluorescent polypeptide substrate Peptide II, and the result is shown in figure 3.
2.2 comparison of the sensitivity and specificity of the fluorescent polypeptide substrate of the invention for detecting human gelatinase
Respectively adding human MMP-2 with different enzyme activity concentrations of 0.15 μ M, 0.3 μ M, 0.6 μ M, 1.2 μ M, 3 μ M and 6 μ M; MMP-9 concentrations are respectively 0.6 mu M, 3 mu M and 6 mu M, 50 mu l of enzyme solution or equal volume of reaction buffer solution and the fluorescent polypeptide substrate are respectively incubated for 30min at 37 ℃, then 50 mu l of the fluorescent polypeptide substrate working solution is added into a black opaque 96-well plate to establish a 100 mu l reaction system, the final concentration of the fluorescent polypeptide substrate working solution is 50 mu M, the enzyme and the substrate are mixed uniformly and then immediately placed into a fluorescent enzyme labeling instrument, the fluorescence intensity is read by an Ex/Em 485nm/538nm filter at 37 ℃, the fluorescence intensity is read once every 30s and the total reading is 1000 times, and the result is shown in figures 4 and 5.
As can be seen from the results of FIG. 3, the enzymatic reaction kinetics constant Km for the reaction of the fluorescent polypeptide substrate of the present invention with human MMP-2 is 315. mu.M, Kcat/Km: 2565M-1·S-1(ii) a FIG. 4 illustrates that low concentrations (150nM) of human MMP-2 can be detected with the fluorescent polypeptide substrates of the present invention; FIG. 5 shows that human MMP-9 hardly reacts with the fluorescent polypeptide substrate of the present invention at an enzyme activity concentration of 6 μ M or less, indicating that the fluorescent polypeptide substrate of the present invention has a certain specificity when reacting with gelatinase, and MMP-2 and MMP-9 can be distinguished at an enzyme activity concentration of 6 μ M or less.
3. Application of fluorescent polypeptide substrate
3.1 determination of MMP-2 enzyme Activity in human serum
The fluorescent polypeptide substrate can be used for detecting the activity of MMP-2 in human serum, the serum to be detected after APMA activation is taken to be mixed with excessive fluorescent polypeptide substrate in a reaction buffer solution, a 100 mu l reaction system is established on a black 96-pore plate, the excitation wavelength and the emission wavelength are 485nm/538nm under the excitation wavelength and the emission wavelength (Ex/Em) corresponding to a fluorescence group combined by the fluorescent polypeptide substrate at 37 ℃, the fluorescence intensity is detected, a standard curve is established, the activity of the MMP-2 enzyme is calculated, and the activity of the MMP-2 in different serum samples can be compared by expressing the speed mu mol/L.s.mg of protease cracking substrate per unit mass.
3.2 screening of human MMP-2 inhibitors
Mixing a sample to be tested with the activated human MMP-2 in a reaction buffer solution, incubating for 1 hour at 37 ℃, adding the fluorescent polypeptide substrate (the final concentration is 50 mu M), establishing a 100 mu l reaction system on a black 96-well plate, and reading the fluorescence intensity at 37 ℃ by an Ex/Em-485 nm/538nm filter. If the activity of the human MMP-2 enzyme is inhibited, the fluorescent peptide chain substrate cannot be enzymolyzed, and a fluorescent group connected with the 1 st isoleucine at the N end is quenched by a quenching group connected with the 11 th lysine site, so that the fluorescence intensity is weakened or even zero, and the sample to be detected is considered to be the inhibitor of the human MMP-2.
Then, after the activated MMP-2 and a sample solution to be detected diluted in equal proportion or a reaction buffer solution with the same volume are incubated for 1 hour at 37 ℃, then a working solution of the fluorescent polypeptide substrate (the final concentration is 50 mu M) is respectively added to establish a 100 mu l reaction system, the reaction system is put into a fluorescence microplate reader, the fluorescence intensity is read by using an optical filter with Ex/Em being 485nm/538nm at 37 ℃, and the Ki value of the inhibitor is calculated by using Dynafit4 software.
The above description, while indicating embodiments of the present invention, is intended to be illustrative, not exhaustive, and not limited to the embodiments shown. Many variations and modifications will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
SEQUENCE LISTING
<110> first Hospital affiliated to Kunming medical university
<120> a fluorescent polypeptide substrate for detecting human gelatinase MMP-2
<130> 2019-3-8
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213> Artificial sequence
<400> 1
Val Gly Tyr Ala Ile Gly Thr Gln Gln Ala Lys
1 5 10
Claims (7)
1. A fluorescent polypeptide substrate for detecting human gelatinase MMP-2, which is characterized in that: the amino acid sequence of the fluorescent polypeptide substrate is shown as SEQ ID NO. 1, the molecular weight is 1906Da, and valine at the 1 st site and lysine at the 11 th site of the amino acid sequence are respectively combined with a fluorescent group and a fluorescence quenching group.
2. The fluorescent polypeptide substrate for detecting human gelatinase MMP-2 according to claim 1, wherein: the combined fluorophore and fluorescence quenching group are a fluorophore 5-carboxyfluorescein combined with the amino group of valine at position 1 and a fluorescence quenching group 5-carboxytetramethylrhodamine combined with the amino group of lysine at position 11.
3. According toThe fluorescent polypeptide substrate for detecting human gelatinase MMP-2 according to claim 1, wherein: 50mM Tris, 10mM CaCl at pH 7.52150mM NaCl, 0.05% Brij-35 at 37 ℃, the MMP-2 catalyzes the enzymatic reaction kinetic constant Km of the reaction of the fluorescent polypeptide substrate to be 315 mu M, Kcat/Km: 2565M-1·S-1 。
4. The fluorescent polypeptide substrate for detecting human gelatinase MMP-2 according to claim 1, wherein: 50mM Tris, 10mM CaCl at pH 7.52Under a reaction system of 150mM NaCl, 0.05% Brij-35 and a temperature of 37 ℃, the reaction of the fluorescent polypeptide substrate and human MMP-9 with the enzyme activity concentration of less than 6 mu M approaches zero.
5. A fluorescent polypeptide substrate kit for detecting the activity of human gelatinase MMP-2 is characterized in that: comprising the fluorescent polypeptide substrate of claim 1.
6. The fluorescent polypeptide substrate kit for detecting the activity of human gelatinase MMP-2 according to claim 5, wherein: the reaction buffer corresponding to the kit is 50mM Tris, 10mM CaCl with pH 7.52，150mM NaCl，0.05% Brij-35。
7. The fluorescent polypeptide substrate kit for detecting the activity of human gelatinase MMP-2 according to claim 5, wherein: the reaction corresponding to the kit was carried out at 37 ℃.
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| Matrix Metalloproteinase-9 Reduces Islet Amyloid Formation by Degrading Islet Amyloid Polypeptide;KathrynAston-Mourney等;《Molecular Bases of Disease》;20130201;第288卷(第5期);第3553-3559页 * |
| 基质金属蛋白酶MMP-2/9分子探针对临床结肠癌组织的检测;余祖红等;《科学通报》;20171229;第63卷(第3期);第318-326页 * |
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