CN104792979B - Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 - Google Patents
Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 Download PDFInfo
- Publication number
- CN104792979B CN104792979B CN201410450501.XA CN201410450501A CN104792979B CN 104792979 B CN104792979 B CN 104792979B CN 201410450501 A CN201410450501 A CN 201410450501A CN 104792979 B CN104792979 B CN 104792979B
- Authority
- CN
- China
- Prior art keywords
- mmp
- peptide substrates
- fluorescent peptide
- fluorescent
- detection human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 (MMP-12) and a detection method. The fluorescent polypeptide substrate comprises an amino acid sequence represented by Peptide I, wherein isoleucine at the first site of the amino acid sequence is combined with a fluorescent group of 5-carboxyfluorescein, and lysine at the twelfth site of the amino acid sequence is combined with a fluorescence quenching group of 5-carboxytetramethylrhodamine. The kinetic constant Km of an enzymatic reaction between the fluorescent polypeptide substrate and MMP-12 is 35muM, and a ratio of Kcat to Km is 35428M＜-1＞.s＜-1＞. The ratio of Kcat to Km is 6.4 times as large as that of an enzymatic reaction between MMP-9 and the fluorescent polypeptide substrate, and MMP-1 and MMP-3 hardly react with the fluorescent polypeptide substrate. According to the invention, the method for detecting activity of the MMP-12 in human serum is simple to operate and fast, and has a certain specificity and a bright application prospect.
Description
Technical field
The present invention relates to a kind of fluorescence of the detection MMP-12 (mmp-12) containing particular sequence or motif
Peptide substrate and its activity test method, belong to biological technical field.
Background technology
Matrix metalloproteinase (matrix metalloproteinases, mmps) is a class dependence calcium ion containing zinc
Endopeptidase family, can Multiple components in degradation of cell epimatrix and basement membrane, having the following characteristics that 1. can degradation of cell
Epimatrix and the composition of basement membrane；2. secreted with zymogen forms, there is after activation biologic activity；3. rely on calcium ion and maintain it
Stability；4. can be suppressed by endogenous tissue type tissue inhibitor of metalloproteinase.Mmps derives from Various Tissues and cell, such as
Endotheliocyte, smooth muscle cell, fibroblast and cerebral tissue[1-3].Mmps not only participates in organizer under normal physiological conditions
The generation of official's form, cell migration and angiogenesis etc., also play an important role in pathologic remodeling process, such as class wind
Wet arthritis[4]The invasion and attack of tumor and transfer[5]And chronic obstructive pulmonary disease[6], cardiovascular disease[7]Deng.
The people that is otherwise known as is huge bites for MMP-12 (matrix metalloproteinases-12, mmp-12)
Cell elastase, 1975 by werb etc.[8]Find in mouse peritoneal macrophages first, shapiro in 1993 etc.[9]
In the pulmonary alveolar macrophage of smoker, successful clone goes out the cdna sequence of mmp-12.Mmp-12 is divided with the zymogen forms of 54-kda
Secrete, after secretion, self cleavage falls aminoterminal sequence, generate the activity form of 45-kda, then the sequence life cutting off carboxyl terminal
Ripe 22-kda activity form[9,10], the substrate of mp-12 is elastoser, Collagen Type VI, Fn Fiberonectin, layer glue
Even albumen, vitronectin, Dan Baiduotang proteoglycan PG, chondroitin sulfate, myelin basic protein and alpha1-antitrypsin.In vivo
, with plasminogen as substrate, plasminogen is formed after being hydrolyzed for mmp-12, mmp-3, mmp-9, mmp-7
The fragment of class angiostatin[11]；Mmp-12 can also activate other mmps, such as mmp-2 and and mmp-3, amplify and accelerate
The degraded of albumen[12].
Research finds that mmp-12 is closely related with development with the generation of multiple diseases, such as chronic obstructive pulmonary disease[6], hat
Cardiopathia plaque rupture[13], dissection of aorta[14], abdominal aortic aneurysm[15], wound deep venous thrombosis[16]Deng.But due to mmps
Between complicated interaction mechanism and research method restriction, its concrete mechanism of action illustrates not yet.Hence set up one accurately
Efficient mmp-12 activity test method seems particularly significant in understanding the function of mmp-12 and the research of dependent interaction mechanism.
The main method of detection proteinase activity has spectrophotography, fluorescence method, isotopic measurement, electrochemistry side at present
Method etc..Sensitivity often several orders of magnitude high than spectrophotography sent out due to fluorescence, fluorescence intensity is also and exciting light
Light source relevant；Fluorescent polypeptide synthesizes, purification process is easier and can avoid ionic strength, ph, temperature in experimentation
Deng the impact to experimental result for the many factors, it is used more and more in enzymology.
The fluorogenic substrate principle of detection mmps activity is to be coupled fluorescence radiation base respectively at the protease substrate two ends of synthesis
Group and quenching group, the intensity that can produce fluorescence by detection substrate after being cut detects the feelings of protease cleavage reaction
Condition[17, 18].In no protease, because fluorescent core and quencher are mutually close to each other, this complete peptide chain does not have endogenic
Fluorescence activity, when this peptide chain of albumen enzymatic breaking, can not be quenched agent again by detached fluorescent core and effectively be quenched and show fluorescence
Activity, you can measure the fluorescent value that protease cracking substrate produces in specific ex/em.
Report multiple fluorescent peptide substrates that can be hydrolyzed by mmp-12 at present, but specificity is low, by mmp-12 water
With other mmps, hydrolysis, such as the mmp-12 fluorescence bottom synthesized by U.S. anaspec company can also occur while solution
Thing can be simultaneously by mmp-1,2,3,8 and 13 enzyme action.Contain multiple mmps in clinical serum sample, the function of mmp-12 be with multiple
Family member and tissue depressant be common to be adjusted and completes, thus since using in these fluorescent peptide substrates detection serum
The activity of mmp-12 lessens the reliability of detection.It is not suitable for Clinical detection mmp-12 enzymatic activity.
Content of the invention
In view of this, it is an object of the invention to provide a species specificity high can detect mmp-12 activity fluorescent polypeptide
Substrate and detection method.
For realizing the above-mentioned purpose of the present invention, the technical solution used in the present invention is as follows:
Using chemosynthesis method synthesize a kind of fluorescent peptide substrates of detection mmp-12 enzymatic activity, this substrate include as
Aminoacid sequence shown in peptide, and the relying of the amino of isoleucine at the 1st, its aminoacid sequence n end and the 12nd
The amino of propylhomoserin is respectively in connection with having fluorophor and fluorescent quenching group.
The application of fluorescein(e) dye widely, including fluorescence microscope, Flow Cytometry and immunofluorescence analysis etc..
The fluorophor of aminoacid sequence shown in peptide for the described combination and fluorescent quenching group are: with the 1st different bright ammonia
The fluorophor 5 CF 5(6)-Carboxyfluorescein (5-fam) that the amino of acid combines, the fluorescence being combined with the amino of the lysine of the 12nd is sudden
Go out group 5 carboxyl tetramethylrhodamin (5-tamra).The 1st, fluorescent peptide substrates of the present invention its aminoacid sequence n end
Isoleucine and the 12nd Lysine binding have fluorophor and fluorescent quenching group, in fluorescent peptide substrates when laser irradiates
Fluorophor emitted energy, in order to fluoroscopic examination.The fluorophor that can use and fluorescent quenching group are a lot of in the industry
, including fluorescein 1 amino naphthalenes 8 carboxylic acid (edans), CF 5(6)-Carboxyfluorescein (fam).But it is preferably, of the present invention
Fluorophor is 5 one CF 5(6)-Carboxyfluorescein (5-fam), and fluorescent quenching group is 5 one carboxyl tetramethylrhodamins (5-tamra).
When certain wavelength laser irradiates this fluorescent peptide substrates, the 1st, n end isoleucine and the 12nd lysine glimmering
Light group emitted energy simultaneously, but this emitted energy again by site each other close to and absorbed each other it is impossible to launch energy
Amount.And work as after this fluorescent peptide substrates digested by mmp-12, because isoleucine and lysine are separated from each other, so that energy is sent out
Penetrate and be detected.
The fluorescent peptide substrates that the present invention provides can be hydrolyzed by mmp-12, the enzyme of mmp-12 catalytic fluorometry peptide substrate reaction
Promote kinetics constant km: 35 μm, kcatFor 1.24s-1, kcat/kmFor 35428m-1.s-1.Wherein mmp-9 enzyme action institute of the present invention
State fluorescent peptide substrates kcat/km6.4 times, mmp-1,3 almost reactionless with fluorescent peptide substrates of the present invention.Explanation
Fluorescent peptide substrates of the present invention have certain specificity with mmps when reacting.
Present invention also offers a kind of method detecting mmp-12 enzymatic activity in serum, take the test serum after apma activation
Mix in reaction buffer with excessive above-mentioned fluorescent peptide substrates, and the reactant of 100 μ l is set up on black 96 orifice plate
System, at 37 DEG C under the described fluorescent peptide substrates corresponding excitation wavelength of combined with fluorescent group of institute and launch wavelength, detects fluorescence
Intensity, calculates proteinase activity from standard curve, with the speed μm ol/l.s.mg table of unit mass protease cracking substrate
Show, relatively the mmp-12 activity of different serum samples.
Detection method of the present invention is many containing specific test plasma sample and fluorescence using the irradiation of certain wavelength laser
Peptide substrates, if in plasma sample mmp-12 enzymatic activity very low it is impossible to enzymolysis fluorescence peptide substrates, be connected to the 1st, n end different bright
The fluorophor of propylhomoserin is connected the quencher quenching of the lysine sites of the 12nd, when this peptide chain of albumen enzymatic breaking, quilt
Detached fluorescent core can not be quenched agent again and effectively be quenched and show fluorescence activity, you can measure protease cracking substrate in spy
Determine the fluorescent value of ex/em generation.Concentration in normal plasma for the mmp-12 is unknown, therefore first measures blood using the method for elisa
The concentration of middle clearly mmp-12, in institute's test sample basis, mmp-12 concentration is about 4ng/ml.In order to ensure the accuracy of testing result, this
Add excessive fluorescent peptide substrates in the method for invention described detection mmp-12 activity, make mmp-12 in plasma sample fully anti-
Should.In Examples below 1, the consumption of described fluorescent peptide substrates is 18 μm of ol, is far longer than mmp-12 content in serum.
The method of detection mmp-12 activity of the present invention is easy and simple to handle, quickly, has certain specificity.
In Examples below 1, the fluorophor that fluorescent peptide substrates of the present invention are combined is fluorescein -5- horse
Carry out amide, described emission wavelength is 485nm, and launch wavelength is 538nm.Described reaction buffer includes: 50mm tris,
Ph7.4,20 mm cacl2, 200mm nacl, 0.005%brij-35.
The present invention also provides a kind of test kit of detection mmp-12 enzyme, including above-mentioned fluorescent peptide substrates.Described reagent
Reaction buffer in box includes 50mm tris, ph7.4,20 mm cacl2, 200mm nacl, 0.005%brij-35.
Stabilization of kit of the present invention is good, and the holding time is long, and detection mmp-12 activity is convenient, can be applicable to detect
Mmp-12 activity in serum.
Brief description
Fig. 1 is the hplc analysis chart of fluorescent peptide substrates of the present invention.
Fig. 2 is the mass spectral analyses figure of fluorescent peptide substrates of the present invention.
Fig. 3 is the lineweaver-burk (a) that fluorescent peptide substrates of the present invention are reacted with mmp-12
With michaelis-menton (b) scatterplot.
Fig. 4 is the reaction broken line with mmp-1,3,9,12 for the fluorescent peptide substrates of detection mmp-12 activity of the present invention
Figure.Fluorescent peptide substrates are the fluorescent peptide substrates shown in the present invention, 0.5 μm of concentration of substrate, and mmp-1,3,9,12 concentration is
15nm.Abscissa is time min, and vertical coordinate is Relative fluorescence units rfu.
The scatterplot that Fig. 5 is reacted with mmp-12 in serum for fluorescent peptide substrates described in present example 1, wherein 1, No. 2
The tremulous pulse serum of but normal coronary angiogram patient taken from by sample, 3, No. 4 samples take from coronarography confirm to have narrow
The tremulous pulse serum of patients with stable angina pectoris, fluorescent peptide substrates are the fluorescent peptide substrates shown in the present invention, and concentration of substrate is
18 μm, abscissa is time min, and vertical coordinate is Relative fluorescence units rfu.The numerical value of each of chart point takes and repeats reality twice
The meansigma methodss (mean ± sd) tested.
Specific embodiment
The embodiment of the invention discloses a kind of fluorescent peptide substrates of detection mmp-12 activity and detection method.This area skill
Art personnel can use for reference present disclosure, is suitably modified technological parameter, but specifically, all similar replacements and
Change apparent to those skilled in the art, they are considered as including in the present invention.For a further understanding of
The present invention, with reference to example, the present invention is described in detail, but is not intended to limit the invention to embodiment.
Embodiment: fluorescent peptide substrates (chemosynthesis of Beijing SBS Genetech bio-engineering corporation) of the present invention, mmp-
1st, 12 be purchased from r ＆ d systems company of the U.S., mmp-9,3 be purchased from raybiotech company of the U.S., apma be purchased from the U.S.
Anaspec company, 5-fam is purchased from sigma company of the U.S., and in detection serum, the elisa test kit of mmp-12 is purchased from eiaab (force
Han Yiaibo Science and Technology Ltd.).
1st, the synthesis of fluorescent polypeptide and storage.
Chemosynthesis fluorescent peptide substrates, sequence is the fluorescent peptide substrates horizontal high voltage liquid phase of synthesis shown in peptide
Chromatograph and mass spectral analyses are to detect purity and the quality (Fig. 1, Fig. 2) of fluorescent peptide substrates.Fluorescent peptide substrates are with lyophilized powder shape
Formula is stored in -20 DEG C of refrigerators.
2. the enzyme kineticss parameter of mmp-12 catalytic fluorometry peptide substrate calculates.
Mmp-12 proenzyme adds 1mm apma, incubates 2 hours, diluted enzymatic solution with reaction buffer after activation at 37 DEG C
For 15nm.Dmso dissolves fluorescent peptide substrates lyophilized powder and is made into the solution of 1mm, and reaction buffer dilutes the dmso substrate of 1mm
Solution is 10～320 μm of substrate working solution.
Enzymatic solution and substrate working solution are respectively placed in 37 DEG C of incubation 15min, in the in the hole of 96 orifice plates of black non transparent
It is separately added into the substrate working solution 50 μ l of variable concentrations and isopyknic reaction buffer or enzymatic solution sets up 100 μ l reactants
System, enzyme-to-substrate is immediately placed in fluorescence microplate reader after mixing, and uses the optical filter of ex/em=485nm/538nm to read fluorescence at 37 DEG C
Intensity, is spaced 1min, reads 120 times.
5-fam Specification Curve of Increasing: because fluorescent peptide substrates itself can absorb certain fluorescent value it is therefore desirable to reality
In testing, the substrate working solution of used variable concentrations does 5-fam standard curve to correct because being absorbed by fluorescent peptide substrates respectively
And the fluorescent value being quenched.With reaction buffer, 5-fam is diluted to 0.03 ~ 1.25 μm of work in 96 orifice plates of black non transparent
Make liquid, every hole 50 μ l, then add isopyknic substrate working solution in hole, ex/em=485nm/ is used on fluorescence microplate reader
The optical filter of 538nm reads fluorescence intensity, draws standard curve.Using identical method, 5 ~ 160 μm of substrate working solution is divided
Do not do standard curve.According to standard curve, rfu is converted to the molar concentration of the product of enzyme catalysis fluorescent peptide substrate generation.
The kinetic parameter k of the enzymatic reaction of fluorescent peptide substratesm、kcatIt is according to enzyme and variable concentrations substrate (10～80
μm) response curve and double-reciprocal plot method calculate.(design conditions: in the first few minutes of reaction, product generates
Rate or substrate conversion efficiency should be less than 10%).
Shown in Fig. 3 result, a: double-reciprocal plot show that y intercept is 1/vmax=0.01122 s/ μm, x-axis intercept is -1/
km=-0.02835l/μm.B: when fluorescent peptide substrates concentration is about 120 μm, maximum reaction velocity in enzyme-to-substrate, reaches
Concentration of substrate during maximum reaction velocity half is km, kmIt is about 30 μm.Therefore mmp-12 hydrolyzes fluorescent polypeptide of the present invention
The k of substratemFor km: 35 μm, kcatFor 1.24s-1, kcat/kmFor 35428m-1.s-1.
3rd, the specificity analyses of enzyme catalysis fluorescent peptide substrate reaction.
At 37 DEG C, activate mmp-1,3,9,12 respectively with 1mm apma, concrete soak time is shown in Table 1.
Table 1 mmps soak time table
.
With reaction buffer, the mmps after activating is diluted to 30nm, dilution 1mm fluorescent peptide substrates are 1 μm of substrate work
Make liquid, after being respectively placed in 37 DEG C of incubation 15min, add 50 μ l enzymatic solution and isopyknic substrate working solution in black 96 orifice plate,
Set up the reaction system of 100 μ l, enzyme-to-substrate puts into fluorescence microplate reader, with the optical filter of ex/em=485nm/538nm after mixing
Read fluorescence intensity, every 5min reads once, reads 12 times altogether.
From Fig. 4 result, through the reaction of 60 minutes, with respect to the fluorescent peptide substrates in the mmp-12 enzyme action present invention
Efficiency, the k of mmp-12 enzyme action fluorescent peptide substrates of the present inventioncat/kmIt is mmp-9 enzyme action substrate of the present invention kcat/km
6.4 times, mmp-1,3 almost reactionless with fluorescent peptide substrates of the present invention.Fluorescent polypeptide of the present invention is described
Substrate has certain specificity with mmps when reacting.
4th, example 1: the detection of human serum mmp-12 activity.
Take the tremulous pulse blood serum sample of 4 parts of different patients, 1, No. 2 is but normal coronary angiogram patient, 3, No. 4 is crown dynamic
Arteries and veins radiography confirms there is narrow patients with stable angina pectoris.First use mmp-12 concentration in elisa method detection serum, in black not
The minimum serum sample of 50 μ l mmp-12 concentration is added in 96 transparent orifice plates, then with reaction buffer by remaining 3 parts of serum samples
This dilution makes mmp-12 concentration identical, takes 50 μ l to add 96 orifice plates.A certain amount of apma is added to make its final concentration in serum
For 1mm, 37 DEG C incubate serum 2 hours.The fluorescent peptide substrates solution being 36 μm with reaction buffer compound concentration is placed in 37
DEG C incubate 15min.Add isopyknic fluorescent peptide substrates solution in serum after incubation, set up the reaction system of 100 μ l.
Enzyme-to-substrate is immediately placed in fluorescence microplate reader after mixing, and uses the optical filter of ex/em=485nm/538nm to read fluorescence at 37 DEG C
Intensity, is spaced 5min, reads 12 times.Every part of serum sample detects that result of calculation averages (mean ± sd) twice twice.System
Make 5-fam standard curve (concrete grammar is shown in aforementioned) that fluorescent peptide substrates concentration is 18 μm.Glimmering according to albumen enzyme-substrate reactions
Light rate of change and standard curve calculate the activity of protease, crack the rate representation of substrate, knot with unit mass mmp-12
Fruit is shown in Table 2.
Table 2 test serum sample mmp-12 Activity determination
.
Reacted from fluorescent peptide substrates of the present invention by visible 4 serum samples of Fig. 5 result and obtain different reactions
Curve.The visible fluorescent peptide substrates of the present invention of table 2 result can detect the activity of mmp-12 in serum, and in mmp-
In the identical 4 groups of serum samples of 12 concentration, there are different activities, thus for clinical experimental study mmp-12 activity with coronary heart disease send out
Raw and development relation provides method and thinking.
The correlation properties being only intended to help understand the method for the present invention and fluorescent peptide substrates described above.It should be understood that
Be for those skilled in the art, on the premise of without departing from inventive substrate sequence and experimental technique, can also be right
The present invention improves, modifies or extends the more examples of acquisition, and these improve, modification and example fall within right of the present invention
In the protection domain requiring.
List of references:
⑴bode w, maskos k. structural basis of the matrix metalloproteinases
and their physiological inhibitors, the tissue inhibitors of
metalloproteinases. biol chem.2003 jun;384(6):863-72.
⑵pilcher bk, wang m, qin xj, parks wc, senior rm, welgus hg. role of
matrix metalloproteinases and their inhibition in cutaneous wound healing and
allergic contact hypersensitivity. ann n y acad sci. 1999 jun 30;878:12-24.
⑶kaczmarek l, lapinska-dzwonek j, szymczak s. matrix
metalloproteinases in the adult brain physiology: a link between c-fos, ap-1
and remodeling of neuronalconnections embo j. 2002 dec 16;21(24):6643-8.
⑷liu m, sun h, wang x, koike t, mishima h, ikeda k, watanabe t,
ochiai n, fanj. association of increased expression of macrophage elastase
(matrixmetalloproteinase 12) with rheumatoid arthritis. arthritis rheum. 2004
oct;50(10):3112-7.
⑸kerkelä e, saarialho-kere u. matrix metalloproteinases in tumor
progression:focus on basal and squamous cell skin cancer. exp dermatol. 2003
apr;12(2):109-25.
⑹demedts ik, morel-montero a, lebecque s, pacheco y, cataldo d, joos
gf,pauwels ra, brusselle gg. elevated mmp-12 protein levels in induced sputum
from patients with copd. thorax. 2006 mar;61(3):196-201.
⑺liu p, sun m, sader s. matrix metalloproteinases in cardiovascular
disease. can j cardiol. 2006 feb;22 suppl b:25b-30b.
⑻werb z, gordon s. elastase secretion by stimulated macrophages.
characterization and regulation. j exp med. 1975 aug 1;142(2):361-77.
⑼shapiro sd, kobayashi dk, ley tj. cloning and characterization of a
unique elastolytic metalloproteinase produced by human alveolar macrophages.
j biol chem. 1993 nov 15;268(32):23824-9.
⑽nar h, werle k, bauer mm, dollinger h, jung b. crystal structure of
human macrophage elastase (mmp-12) in complex with a hydroxamic acid
inhibitor. j mol biol. 2001 sep 28;312(4):743-51.
⑾visse r, nagase h. matrix metalloproteinases and tissue inhibitors
of metalloproteinases: structure, function, and biochemistry. circ res. 2003
may 2;92(8):827-39.
⑿edelstein c, shapiro sd, klezovitch o, scanu am. macrophage
metalloelastase, mmp-12, cleaves human apolipoprotein(a) in the linker region
between kringles iv-4 and iv-5. potential relevance to lipoprotein(a)
biology. j biol chem. 1999 apr 9;274(15):10019-23.
⒀johnson jl, george sj, newby ac, jackson cl. divergent effects of
matrix metalloproteinases 3, 7, 9, and 12 on atherosclerotic plaque stability
in mouse brachiocephalic arteries. proc natl acad sci u s a. 2005 oct 25;102
(43):15575-80.
⒁song y, xie y, liu f, zhao c, yu r, ban s, ye q, wen j, wan h, li
x, ma r, meng z. expression of matrix metalloproteinase-12 in aortic
dissection. bmc cardiovasc disord. 2013 may 3;13:34.
⒂annabi b, shédid d, ghosn p, kenigsberg rl, desrosiers rr,
bojanowski mw, beaulieu e, nassif e, moumdjian r, béliveau r. differential
regulation of matrix metalloproteinase activities in abdominal aortic
aneurysms. j vasc surg. 2002 mar;35(3):539-46.
⒃zhang yb, li w, yao lq, zhao xl, wang b, li hk, ning y, song e,
zhang xx. expression changes and roles of matrix metalloproteinases in a rat
model of traumatic deep vein thrombosis. chin j traumatol. 2010 jun 1;13(3):
188-92.
⒄matayoshi ed, wang gt, krafft ga, erickson j. novel fluorogenic
substrates for assaying retroviral proteases by resonance energy transfer.
science. 1990 feb 23;247(4945):954-8.
⒅hickman d, vasavanonda s, nequist g, colletti l, kati wm, bertz r,
hsu a, kempf dj. estimation of serum-free 50-percent inhibitory
concentrations for human immunodeficiency virus protease inhibitors lopinavir
and ritonavir. antimicrob agents chemother. 2004 aug;48(8):2911-7.
sequence listing
<110>the attached First Hospital of Kunming Medical University
<120>a kind of fluorescent peptide substrates of detection human luteinized granulosa cells 2 activity
<140> 201410450501.x
<141> 2014-09-09
<160> 1
<170> patentin version 3.5
<210> 1
<211> 12
<212> prt
<213>artificial sequence
<400> 1
ile lys ser asp leu val asn glu glu ala thr lys
1 5 10
Claims (13)
1. a kind of detection human luteinized granulosa cells 2 activity fluorescent peptide substrates it is characterised in that: include as peptide
Shown complete aminoacid sequence, and the bad ammonia of the amino of isoleucine at the 1st, its aminoacid sequence n end and the 12nd
The amino of acid is respectively in connection with having fluorophor and fluorescent quenching group.
2. the fluorescent peptide substrates of detection human luteinized granulosa cells 2 activity according to claim 1, its feature exists
In: the fluorophor of described combination and fluorescent quenching group be, the fluorophor 5 being combined with the amino of the 1st isoleucine
CF 5(6)-Carboxyfluorescein, the fluorescent quenching group 5 carboxyl tetramethylrhodamin being combined with the amino of the lysine of the 12nd.
3. the fluorescent peptide substrates of detection human luteinized granulosa cells 2 activity according to claim 2, its feature exists
In: the 50mm tris being 7.4 in ph, 20 mm cacl2, 200mm nacl, 0.005%brij-35, temperature is 37 DEG C anti-
Answer under system, the enzyme kineticss constant k of mmp-12 catalytic fluorometry peptide substrate reactionmFor 35 μm, kcatFor 1.24s-1,
kcat/kmFor 35428m-1.s-1.
4. the fluorescent peptide substrates of detection human luteinized granulosa cells 2 activity according to claim 3, its feature exists
In: fluorescent peptide substrates k described in mmp-9 enzyme actioncat/km6.4 times, mmp-1,3 react with described fluorescent peptide substrates
It is closely zero.
5. answering in a kind of fluorescent peptide substrates as claimed in claim 1 mmp-12 active ingredient in preparation detection human serum
With.
6. answering in fluorescent peptide substrates according to claim 5 mmp-12 active ingredient in preparation detection human serum
With it is characterised in that detection method is: take test plasma sample and excessive described fluorescent peptide substrates in reaction buffer
Mixing, and the reaction system of 100 μ l is set up on black 96 orifice plate, then in described fluorescent peptide substrates institute combined with fluorescent group
Under corresponding excitation wavelength and wavelength of transmitted light, the fluorescence intensity of detection reaction system, according to the rate of change of fluorescence intensity,
Calculate proteinase activity from standard curve, represented with the speed μm ol/l.s.mg of unit mass protease cracking substrate.
7. answering in fluorescent peptide substrates according to claim 6 mmp-12 active ingredient in preparation detection human serum
With it is characterised in that: fluorescent peptide substrates concentration used be 18 μm.
8. answering in fluorescent peptide substrates according to claim 6 mmp-12 active ingredient in preparation detection human serum
With it is characterised in that: a length of 485nm of excitation light wave of corresponding fluorescence radiation group 5 CF 5(6)-Carboxyfluorescein, wavelength of transmitted light is
538nm.
9. answering in fluorescent peptide substrates according to claim 6 mmp-12 active ingredient in preparation detection human serum
With it is characterised in that: described reaction buffer includes the 50mm tris that ph is 7.5,20 mm cacl2, 200mm nacl,
0.005%brij-35.
10. answering in fluorescent peptide substrates according to claim 6 mmp-12 active ingredient in preparation detection human serum
With it is characterised in that: test plasma sample with excessive described fluorescent peptide substrates hybrid reaction in reaction buffer be
37 DEG C are carried out.
11. a kind of detection human luteinized granulosa cells 2 activity fluorescent peptide substrates test kits it is characterised in that: include power
Profit requires the fluorescent peptide substrates described in 1.
The fluorescent peptide substrates test kit of 12. detection human luteinized granulosa cells 2 activity according to claim 11, its
It is characterised by: the reaction buffer corresponding to test kit includes the 50mm tris that ph is 7.5,20 mm cacl2, 200mm
Nacl, 0.005%brij-35.
The fluorescent peptide substrates test kit of 13. detection human luteinized granulosa cells 2 activity according to claim 11, its
It is characterised by: the reaction corresponding to test kit is carried out at 37 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410450501.XA CN104792979B (en) | 2014-09-05 | 2014-09-05 | Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410450501.XA CN104792979B (en) | 2014-09-05 | 2014-09-05 | Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104792979A CN104792979A (en) | 2015-07-22 |
| CN104792979B true CN104792979B (en) | 2017-02-01 |
Family
ID=53557957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410450501.XA Active CN104792979B (en) | 2014-09-05 | 2014-09-05 | Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104792979B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750082B (en) * | 2019-03-11 | 2022-02-18 | 昆明医科大学第一附属医院 | Fluorescent polypeptide substrate for detecting human gelatinase MMP-2 and application thereof |
| CN110302400B (en) * | 2019-05-20 | 2022-05-27 | 哈尔滨医科大学 | PET/MRI (positron emission tomography/magnetic resonance imaging) multi-mode molecular imaging nano probe for early diagnosis of atherosclerotic vulnerable plaque and application thereof |
| WO2024090571A1 (en) * | 2022-10-28 | 2024-05-02 | 慶應義塾 | Diagnosis of immune-mediated inflammatory diseases using mmp12 as indicator, and medicine for treating immune-mediated inflammatory diseases via mmp12 inhibition |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NO20053354D0 (en) * | 2005-07-11 | 2005-07-11 | Amersham Health As | Optical imaging contrast agent. |
| AU2010273321A1 (en) * | 2009-07-15 | 2012-03-08 | The Regents Of The University Of California | Peptides whose uptake in cells is controllable |
| CN102813944B (en) * | 2012-08-07 | 2014-08-06 | 上海交通大学 | Protease-sensitive fluorescent light and magnetism dual-response micro-capsule and preparation method thereof |
-
2014
- 2014-09-05 CN CN201410450501.XA patent/CN104792979B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104792979A (en) | 2015-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Frank et al. | Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by α-chymase | |
| Stawowy et al. | Furin-like proprotein convertases are central regulators of the membrane type matrix metalloproteinase–pro-matrix metalloproteinase-2 proteolytic cascade in atherosclerosis | |
| Ohler et al. | Analyzing the protease web in skin: meprin metalloproteases are activated specifically by KLK4, 5 and 8 vice versa leading to processing of proKLK7 thereby triggering its activation | |
| CN104792979B (en) | Fluorescent polypeptide substrate for detecting activity of human matrix metalloproteinase-12 | |
| Lai et al. | Angiotensin-converting enzyme 2 ectodomain shedding cleavage-site identification: determinants and constraints | |
| US20070212344A1 (en) | Pharmaceutical preparation with RNA as hemostasis cofactor | |
| Fitzpatrick et al. | Regulation of plasmin activity by annexin II tetramer | |
| EP2529221B1 (en) | Thrombin generation determination method | |
| Zaragoza et al. | Nitric oxide regulates matrix metalloprotease-13 expression and activity in endothelium | |
| Lauer-Fields et al. | Triple-helical peptide analysis of collagenolytic protease activity | |
| Park et al. | Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker | |
| Ottaiano et al. | Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP | |
| JP2014502492A (en) | Chemoluminescence-based hemostasis assay | |
| Grierson et al. | Utility of combining MMP-9 enzyme-linked immunosorbent assay and MMP-9 activity assay data to monitor plasma enzyme specific activity | |
| Cauchard et al. | Activation of latent transforming growth factor beta 1 and inhibition of matrix metalloprotease activity by a thrombospondin-like tripeptide linked to elaidic acid | |
| Takada et al. | Influence of coagulation on the activation of plasminogen by streptokinase and urokinase | |
| Andrade et al. | Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans | |
| US10131932B2 (en) | Method for assaying a protease | |
| AU2019362596B2 (en) | Novel chemiluminescent substrates for factor Xa | |
| Bodiga et al. | In vitro biological evaluation of glyburide as potential inhibitor of collagenases | |
| Petrovan et al. | Purification and characterization of multisquamase, the prothrombin activator present in Echis multisquamatus venom | |
| Valeri et al. | Reaction of antithrombin with proteases. Evidence for a specific reaction with papain | |
| RU2820234C2 (en) | NOVEL CHEMILUMINESCENT SUBSTRATES FOR Xa FACTOR | |
| Johnson et al. | Progress Curve Analysis of the one stage chromogenic assay for ecarin | |
| JP2008543273A (en) | Use of thiopeptolide for measuring the activity of ADAM-TS protease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |