CN102879390A - Method for screening protease inhibitor - Google Patents
Method for screening protease inhibitor Download PDFInfo
- Publication number
- CN102879390A CN102879390A CN2012104169700A CN201210416970A CN102879390A CN 102879390 A CN102879390 A CN 102879390A CN 2012104169700 A CN2012104169700 A CN 2012104169700A CN 201210416970 A CN201210416970 A CN 201210416970A CN 102879390 A CN102879390 A CN 102879390A
- Authority
- CN
- China
- Prior art keywords
- solution
- reaction
- triketohydrindene hydrate
- tris
- hcl
- 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.)
- Granted
Links
Images
Abstract
The invention provides a method for screening a protease inhibitor. The method comprises the following steps of: (1) adding a substance to be tested in protease solution to be uniformly mixed and meanwhile setting the protease solution in which the substance to be tested is not added as a blank control; (2) adding substrate protein solution into the protease solution to react so as to obtain reaction solution; (3) adding reaction end solution in the reaction solution; (4) adding ninhydrin solution to react; and (5) adding double distilled water, judging a result according to an absorbance value OD570 nm, and ensuring that a sample to be tested is positive if the absorbance of the solution of the matter to be tested is less than that of the blank control. By using the method, the protease inhibitor can be conveniently and quickly screened, a substrate is not required to be manually synthesized, the cost is low, the flexibility is high, and a new approach is provided for finding a medicament for inhibiting tumor invasiveness and metastasis.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to a kind of method of screening protease inhibitors.
Background technology
Cancer is one of major disease that has a strong impact on human life quality and existence, and for cancer cell infiltration, be transferred to its hetero-organization and organ, the mankind there is no feasible method and stoped.Matrix metalloproteinase (matrix metalloproteinases, MMP) is one of Major Enzymes that participates in cancer metastasis, and the tissue around the degradable cancer cell diffuses in a big way cancer cell or enter blood circulation to be transferred to other organs.The activity that suppresses matrix metalloproteinase can be controlled the transfer of cancer cell effectively, so from known or new synthetic compound, filter out the focus that the inhibitor that can suppress matrix metal proteinase activity is current cancer therapy drug research, must set up effective, feasible, economic NMPI evaluation system for this reason.
The high flux screening system mainly contains fluorescence method and spectrophotometric method at present.The former utilizes the FRET(FRET (fluorescence resonance energy transfer) of two fluorophors) change and to obtain the inhibitor capacity of water, its reaction substrate is artificial synthetic fragments of peptides, and on this substrate two ends connect two fluorescent dyes, when used substrate is complete, because the fluorescent energy that sends when first fluorescent dye is excited is absorbed by second neighbour fluorescent dye, thereby make the fluorescent quenching of first fluorophor.When having matrix metalloproteinase to exist, substrate is degraded, so that two fluorophors separate, recovers the fluorescence of first fluorophor.Thereby the reacting condition of fluorescence the size of matrix metalloproteinase vigor: when inhibitor was arranged, change in fluorescence was slow; During the unrestraint agent, change rapidly.Thereby obtain the half-inhibition concentration of inhibitor according to change in fluorescence.Spectrophotometric method also is to utilize the artificial synthetic fragments of peptides that contains sulfydryl (oligopeptides) as substrate, after matrix metalloproteinase is hydrolyzed this substrate, discharges the fragment with sulfydryl.Utilize the Ellman ' s Reagent reaction of sulfydryl to form yellow compound, the yellow depth has been reacted the repressed degree of matrix metalloproteinase.
Although fluorescence method (or spectrophotometric method) has been used for the screening of NMPI, have higher sensitivity and large flux screening, but still have some shortcoming: 1) because excessively sensitive, artificial false appearance is common in the experiment, control group reasonable in design; 2) if inhibitor has fluorescence or energy cancellation fluorescence, can not use; 3) need artificial synthetic substrate, higher cost; 4) need special instrument; 5) fluorescence more easily by other impurity or the institute's cancellation of reaction buffer component or enhancing, creates a false impression.
Triketohydrindene hydrate (ninhydrin) is an organic compound, is widely used in life science, bioengineering, food industry and scene of a crime fingerprint analysis, and it and amino acid can form the purple compound and be used for Contents of Amino Acids.When amino acid and triketohydrindene hydrate hydrate heat under mild acid conditions altogether, the oxidized deamination of amino acid, decarboxylation, and the triketohydrindene hydrate hydrate is reduced, its reduzate can add the ammonia that thermal decomposition produces with amino acid and be combined, become the bluish violet compound with another molecule triketohydrindene hydrate condensation again, be called Luo Manzi (Ruhemann's purple).This compound maximum absorption band is at 570nm wavelength place.Simultaneously, triketohydrindene hydrate also is used for determining the protein quantity, but be the albumen complete hydrolysis is become amino acid (digestion hydrolysis in 24 hours), and again with ninhydrin reaction, early stage, reaction was wasted time and energy, and had increased the cost of reaction, was unfavorable for albumen and the ninhydrin reaction of large flux.
Summary of the invention
For above-mentioned deficiency, the invention provides a kind of method of screening protease inhibitors.
Formed amino acid or fragments of peptides and ninhydrin reaction when the present invention utilizes its substrate of protease hydrolytic-protein, generate the purple compound and carry out colorimetric (seeing Fig. 1 ~ 4), the depth with absorbance (OD) representative color, and then reflection repressed degree during protease hydrolytic, thereby obtain the half-inhibition concentration (IC of inhibitor
50Or LD
50).
A kind of protease inhibitors screening technique provided by the invention comprises step:
(1) determinand is added to mixing in the protein enzyme solution, reaction; Establishing simultaneously the protein enzyme solution that does not add determinand is blank;
(2) contain and add respectively substrate protein solution in the protein enzyme solution of determinand and the blank and react, obtain reactant liquor;
(3) add reaction terminating liquid in the reactant liquor;
(4) adding ninhydrin reaction liquid reacts again;
(5) add distilled water, according to absorbance OD
570nmJudged result is lower than blank if contain the absorbance of determinand solution, and then testing sample is positive.
Described proteinase is hydrolysising protease.
Preferably, described proteinase is matrix metalloproteinase or pancreatin.
Wherein, step (1) is added determinand in protein enzyme solution mixing equilibration time 〉=5min.
Wherein, the protein enzyme solution preparation method is in the step (1): the aqueous solution of protease that with concentration is 1mg/ml is mixed to get protein enzyme solution for 1:16 ~ 30 by volume with reaction buffer solution; The substrate protein solution manufacturing method is in the step (2): the substrate protein aqueous solution of 15-30mg/ml is mixed to get substrate protein solution for 1:5 ~ 10 by volume with reaction buffer solution.
Further, in the methods of the invention, when the reaction buffer of selecting is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, during pH=7.5, reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25 ~ 100mM, contains 12% ~ 20% polyglycol;
When the reaction buffer of selecting is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5, when containing 3% ~ 6% polyglycol, reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25 ~ 100mM.
Described polyglycol is molecular weight polyethylene glycol 400 ~ 8000.
Preferably, described polyglycol is Macrogol 6000.
In the inventive method, the final concentration of determinand adding protein enzyme solution is 0.1 ~ 300 μ M in the step (1).
Wherein, step (2) protein enzyme solution mixes 1:2 ~ 4 by volume with substrate protein solution.
Wherein, in the step (3) reaction terminating liquid by volume 0.25 ~ 1:1 be added in the reactant liquor.
Preferably, reactant liquor is to mix on ice with stop buffer in the step (3).
In the inventive method, the described ninhydrin reaction solution of step (4) is mixed to get by solution A and solution B equal-volume,
Described solution A is stannous chloride-sodium citrate solution, contains 0.1 ~ 0.3M citric acid, and 7 ~ 10mM stannous chloride is 5.0 with the NaOH adjust pH;
Described solution B is triketohydrindene hydrate-dimethyl sulfoxide (DMSO) (DMSO) solution, triketohydrindene hydrate-ethylene glycol solution or triketohydrindene hydrate-N, dinethylformamide (DMF) solution, it is for to be dissolved in triketohydrindene hydrate among DMSO or ethylene glycol or the DMF, and making the triketohydrindene hydrate final concentration is 40 ~ 70g/L.
Preferably, the triketohydrindene hydrate final concentration is 50g/L.
Wherein, in the step (4) ninhydrin reaction liquid by volume 5 ~ 8:1 be added in the mixed solution of step (3).
Wherein, step (4) reaction conditions is 80-100 ℃, 10-15min, pH=5.0.
Preferably, step (4) reaction conditions is 80 ℃, 10min, pH=5.0.
Wherein, step (5) distilled water by volume 1:1.2 join in the mixed solution of step (4).
The invention provides the application of method in the medicine of preparation inhibition tumor invasion, transfer of above-mentioned screening protease inhibitors.
The invention provides a kind of kit of screening, contain ninhydrin reaction solution, described ninhydrin reaction solution is mixed to get by solution A and solution B equal-volume;
Described solution A is stannous chloride-sodium citrate solution, contains 0.1 ~ 0.3M citric acid, and 7 ~ 10mM stannous chloride is 5.0 with the NaOH adjust pH;
Described solution B is triketohydrindene hydrate-dimethyl sulphoxide solution, triketohydrindene hydrate-ethylene glycol solution or triketohydrindene hydrate-N, dinethylformamide solution, it is for to be dissolved in triketohydrindene hydrate in dimethyl sulphoxide solution or ethylene glycol solution or the DMF solution, and making the triketohydrindene hydrate final concentration is 40 ~ 70g/L.
Preferably, among the mentioned solution B, the triketohydrindene hydrate final concentration is 50g/L.
The invention provides a kind of kit that screens protease inhibitors, contain above-mentioned ninhydrin reaction solution, reaction buffer, reaction terminating liquid;
Described reaction buffer is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5 contains 3% ~ 6% polyglycol;
Reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25-100mM.
The invention provides the kit of another kind of screening protease inhibitors, it contains above-mentioned ninhydrin reaction solution, reaction buffer, reaction terminating liquid;
Described reaction buffer is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5;
Described reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25 ~ 100mM, contains 12% ~ 20% polyglycol.
Polyglycol of the present invention can be PEG400 ~ 8000.
The present invention is by optimizing all many-sides such as buffer system, pH, temperature, reaction reagent preparation condition and colour stabilizer, select optimum temperature (80 ℃), pH(5.0) and add colour stabilizer (polyglycol, the polymerizable molecular amount is from 400-8000) etc. condition, make the color reaction of triketohydrindene hydrate and amino acid or fragments of peptides more stable, the method greatly improved the repeatability of experiment, so that can be used for the especially screening of NMPI of protease inhibitors.In addition, in the inventive method, the hydrolysate of proteinase and protein need not with high concentrated acid (HCl, H
2SO
4Deng) or alkali (NaOH, Ba (OH)
2) breaks down into amino acids (traditionally, all needing albumen, peptide complete hydrolysis are become amino acid), measure by ninhydrin reaction again, effectively reduced operation steps, save time.When the screening protease inhibitors, reaction product and unreacted albumen, carry out precipitate and separate (the common need with these sour protein precipitations without trichloroacetic acid or perchloric acid, centrifugal, supernatant neutralizes with alkali again), can be directly and ninhydrin reaction, obtain the amount of relative product, reduce equally operation steps, shortened the running time, improved the effect ratio.
The advantage that the inventive method has is as follows: (1) is colour-stable, and is highly sensitive, do not have the sensitive stutter bands that causes; (2) damping fluid forms color reaction impact little (except the buffer system that contains amino or ammonium salt); The substrate that (3) need not manually synthesize, with the natural substrate of proteinase---protein is substrate, need not chemical modification, more meets the truth of biological respinse; (4) because artificial synthetic substrate that need not be expensive, with low cost, the simple sample expense only is 1/10 of current commercial kit; (5) need not specific installation, only with laboratory spectrophotometer or microplate reader commonly used, simple and feasible; (6) the present invention measures simple sample and only needs 30 minutes, compares about the used 1 hour time with current kit measurement, not only fast but also save time and human cost.
Description of drawings
Fig. 1 is ninhydrin reaction (quality) schematic diagram.
Fig. 2 is gelatin is hydrolyzed formed product and ninhydrin reaction by clostridiopetidase A spectrum change.
The graph of a relation in the reaction time of absorbance and hydrolysis when Fig. 3 is the unrestraint agent; Absorbance and the time of 570 nanometers are linear, and along with the increase in reaction time, the product of hydrolysis increases gradually, and corresponding absorbance also increases, and linear.
Fig. 4 is the relation in reaction time of absorbance and hydrolysis that has or not inhibitor (Phen) when existing.When having inhibitor to exist, the slope of the slope of straight line (speed) during less than the unrestraint agent, thereby can be used for inhibitor screening.
Fig. 5 is that the absorbance of variable concentrations Phen when existing changes spectrum.Along with increasing (0-160 μ M) absorbance, inhibitor concentration progressively descends.
Fig. 6 is that Phen is to the inhibiting effect (half-inhibition concentration is 67.1 ± 4.2 μ M) of clostridiopetidase A.
Fig. 7 is the spectrum change of variable concentrations Z-pro-leu-gly when existing, and progressively descends along with Z-pro-leu-gly increases (0-80 μ M) absorbance.
Fig. 8 is Z-pro-leu-gly, and (half-inhibition concentration mean value is 52.4 ± 2.2 μ M to the inhibiting effect of clostridiopetidase A.
Fig. 9 is that the screening of 96 orifice plates is to the inhibiting effect of clostridiopetidase A.The 570nm absorbance measurement obtains in microplate reader.Right half part is Z-pro-leu-gly, 30-80 μ M, and left-half is Phen, 20-120 μ M.
The spectrum change of the formed product of Figure 10 pancreatin hydrolysis gelatin and ninhydrin reaction, the product amount of hydrolysis increases progressively trend along with time lengthening is.
The relation of Figure 11 pancreatin hydrolysis gelatin and time.The product amount that time increases hydrolysis also increases, and is linear proportional relation.When Phenylmethylsulfonyl chloride (PMSF) being arranged when existing, pancreatin is suppressed, and corresponding linear gradient reduces (speed decline).
Figure 12 is that 0.1% glycerine is on the figure that affects of degraded by collagenase gelatin.
Figure 13 be have, the reaction rate variation diagram during without glycerine, without impact, absorbance does not change glycerine on the degraded by collagenase gelatin.
Figure 14 Bradford method is measured albumen (BSA) typical curve, 100-1500mg/ml between linear zone.
Figure 15 ninhydrin method of the present invention is measured bovine serum albumin(BSA) (BSA) typical curve, 100-3200 μ g/ml between linear zone.
Figure 16 ninhydrin method of the present invention and Bradford method are relatively.The scope of ninhydrin method protein determination (100-3200 μ g/ml) is greater than Bradford (100-1500 μ g/ml).
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the conventional means that used technological means is well known to those skilled in the art among the embodiment.If do not specialize, used reagent is commercially available among the embodiment.
The reagent and the instrument that use in the embodiment of the invention are as follows: gelatin, and available from Sangon Biotech (Shanghai) Co., Ltd., lot number 1786B510 is made into the aqueous gelatin solution of 30mg/ml; (collagenase IV also claims MMP-2 to clostridiopetidase A, and MMP-2), available from invitrogen company, lot number 17104-019 is made into the solution of 1mg/ml; Pancreatin is made into the solution of 1mg/ml; Phen (phenanthrolin) is sent Buddhist nun's chemical reagent factory available from Zhengzhou, and lot number 2005-6-2 is made into the solution of 1mM; Triketohydrindene hydrate is available from Tianjin moral grace chemical reagent company limited, lot number 2011-8-27.Citric acid, Zhengzhou are sent Buddhist nun's chemical reagent factory, lot number: 20100619.Two hydrated stannous chlorides, Chemical Reagent Co., Ltd., Sinopharm Group, lot number: F20090324.Polyglycol, ethylene glycol, DMF (DMF) are all sent Buddhist nun's chemical reagent factory available from Zhengzhou, lot number: 20070615.Spectroanalysis instrument (Japanese Shimadzu, model: UV-2450).
The preparation (1) of embodiment 1 solution
1, ninhydrin reaction solution
Solution A: stannous chloride-sodium citrate solution, contain the 0.2M sodium citrate, the 7mM stannous chloride, pH 5.0.
Solution B: triketohydrindene hydrate-dimethyl sulphoxide solution, it is for to be dissolved in DMSO solution with triketohydrindene hydrate, and making the triketohydrindene hydrate final concentration is 50.0g/L.
Solution A and solution B equal-volume are mixed to get ninhydrin reaction solution.
2, reaction buffer
50mM?Tris-HCl，150mM?NaCl，3mM?CaCl
2，1μM?ZnCl
2，pH=7.5
3, reaction terminating liquid is
The EDTA-Tris-HCl damping fluid of 50mM contains 12% Macrogol 6000.
The preparation (2) of embodiment 2 solution
1, ninhydrin reaction solution
Solution A: stannous chloride-sodium citrate solution, contain the 0.1M sodium citrate, the 8mM stannous chloride, pH 5.0.
Solution B: triketohydrindene hydrate-DMF solution, it is for to be dissolved in DMF (DMF) solution with triketohydrindene hydrate, and making the triketohydrindene hydrate final concentration is 40.0g/L.
Solution A and solution B equal-volume are mixed to get ninhydrin reaction solution.
2, reaction buffer
50mM?Tris-HCl，100mM?NaCl，1mM?CaCl
2，1μM?ZnCl
2，pH=7.5
3, reaction terminating liquid
The EDTA-Tris-HCl damping fluid of 25mM contains 18% Macrogol 4000.
The preparation (3) of embodiment 3 solution
1, ninhydrin reaction solution
Solution A: stannous chloride-sodium citrate solution, contain the 0.2M sodium citrate, the 8mM stannous chloride, pH 5.0.
Solution B: triketohydrindene hydrate-ethylene glycol solution, it is for to be dissolved in ethylene glycol solution with triketohydrindene hydrate, and making the triketohydrindene hydrate final concentration is 60.0g/L.
Solution A and solution B equal-volume are mixed to get ninhydrin reaction solution.
2, reaction buffer
50mM?Tris-HCl，250mM?NaCl，5mM?CaCl
2，1μM?ZnCl
2，pH=7.5，
Contain 3% Macrogol 6000
3, reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 100mM.
The preparation (4) of embodiment 4 solution
1, ninhydrin reaction solution
Solution A: stannous chloride-sodium citrate solution, contain the 0.3M sodium citrate, the 10mM stannous chloride, pH 5.0.
Solution B: triketohydrindene hydrate-dimethyl sulphoxide solution, it is for to be dissolved in DMSO solution with triketohydrindene hydrate, and making the triketohydrindene hydrate final concentration is 70.0g/L.
Solution A and solution B equal-volume are mixed to get ninhydrin reaction solution.
2, reaction buffer
50mM Tris-HCl, 200mM NaCl, 2.5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5 contains 6% Macrogol 2000.
3, reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 75mM.
The screening technique (1) of embodiment 5 collagenase inhibitors
Ninhydrin reaction solution, reaction buffer and reaction terminating liquid are got the solution combination among the embodiment 1.
The blank group: (1) gets the aqueous gelatin solution 22 μ l of 30mg/ml, is positioned in 1.5 milliliters of Ependoff pipes, adds 321 μ l reaction buffers, mixing again.(2) clostridiopetidase A (1mg/ml) solution with 7 μ l joins in the above-mentioned gelatin solution 37 ℃ of reactions.Behind 0min, 2min, 4min, 6min, 8min, 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 50 μ l reaction terminating liquids.
Negative control group: get 22 μ l gelatin (30mg/ml) and be positioned over respectively in the Ependoff pipe, add 200 μ l reaction buffers, mixing; Other gets a series of Ependoff pipes, adds 7 μ l clostridiopetidase As (1mg/ml), 117.5 μ l reaction buffers, and 3.5 μ l glycerine, mixing.Then mix with gelatin solution, final concentration is that the 1%(cumulative volume is 350 μ l), behind 0min, 2min, 4min, 6min, 8min, 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 50 μ l reaction terminating liquids.(seeing Figure 12).
Experimental group: (adding Phen (phenanthrolin)): get respectively 22 μ l aqueous gelatin solutions (30mg/ml) and be positioned in the different Ependoff pipes, and add 120 μ l reaction buffers, mixing; Other gets some Ependoff pipe, adds respectively 7 μ l clostridiopetidase As (1mg/ml) in the pipe, and the Phen (1mM) of press the volume adding reaction buffer shown in the table 1 and different volumes is in collagenase solution, mixing.Then mix with gelatin solution, make its final concentration (cumulative volume is 350 μ l) be respectively 10 μ M, 20 μ M, 40 μ M, 60 μ M, 80 μ M, 100 μ M, 120 μ M, 140 μ M, 160 μ M, the reaction system of variable concentrations Phen is packed in the different sample hoses.The Phen test group of each concentration extracts respectively 50 μ l reactant liquors and mixes cessation reaction on ice with 50 μ l reaction terminating liquids behind 0min, 2min, 4min, 6min, 8min, 10min.
Table 1 preparation variable concentrations Phen damping fluid
Behind the reaction terminating, add 500 μ l ninhydrin reaction liquid in each sample hose, the vortex mixing is put into 80 ℃ of water baths and is reacted 10min; Then each sample hose adds 500 μ l distilled waters, and (400～700nm), acquisition is at the absorbance of 570nm to carry out spectral scan behind the mixing.Interpretation of result is as follows: in the identical time interval, the absorbance changing value (deducting 0 minute absorbance (or 2 minutes) in 10 minutes) that adds Phen absorbance changing value of (blank group) when not adding Phen, the inhibiting rate of acquisition when this concentration, thereby obtain the inhibiting rate of variable concentrations Phen, take concentration as horizontal ordinate, inhibiting rate is the ordinate mapping at last.Half inhibitor concentration (IC
50) obtain through the recurrence of mathematics nucleoid.See Fig. 5 and Fig. 6.Fig. 5 demonstration, gelatin can be suppressed by Phen by the clostridiopetidase A hydrolysis, and along with the concentration increase of Phen, absorbance progressively descends.Fig. 6 is through mathematics manipulation, and the inhibition degree is to the mapping of Phen concentration, and then acquisition half-inhibition concentration (IC
50).
This experimental result shows: the ninhydrin reaction that the amino acid product that clostridiopetidase A decomposition gelatin produces can utilize the present invention to optimize detects, after in screening system of the present invention, adding the Phen inhibited to matrix metalloproteinase, the absorbance of its 570nm is lower than blank group and negative control group, confirmed the effect of the protease inhibitors of Phen, reliable results, repeatability is high; Further by the impact of variable concentrations Phen on absorbance, calculate Phen to the half-inhibition concentration of clostridiopetidase A, its IC through the recurrence of mathematics nucleoid
50=67.1 μ M.
The screening technique (2) of embodiment 6 collagenase inhibitors
Ninhydrin reaction solution, reaction buffer and reaction terminating liquid are got the solution combination among the embodiment 2.
The blank group: (1) gets the aqueous gelatin solution 22 μ l of 30mg/ml, is positioned in 1.5 milliliters of Ependoff pipes, adds 321 μ l reaction buffers, mixing again.(2) clostridiopetidase A (1mg/ml) solution with 7 μ l joins in the above-mentioned gelatin solution 37 ℃ of reactions.Behind 0min, 2min, 4min, 6min, 8min, 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 50 μ l reaction terminating liquids.
Negative control group: get 22 μ l gelatin (30mg/ml) and be positioned over respectively in the Ependoff pipe, add 200 μ l reaction buffers, mixing; Other gets a series of Ependoff pipes, adds 7 μ l clostridiopetidase As (1mg/ml), and 117.5 μ l reaction buffers, and 3.5 μ l ethanol, mixing.Then mix with gelatin solution, cumulative volume is 350 μ l, extracts respectively 50 μ l reactant liquors and mix cessation reaction on ice with 50 μ l reaction terminating liquids behind 0min, 2min, 4min, 6min, 8min, 10min.
Experimental group: add Z-Pro-Leu-Gly (available from U.S. Sigma company): get 22 μ l gelatin (30mg/ml) and be positioned in the different Ependoff pipes, and add 120 μ l reaction buffers, mixing; Other gets some Ependoff pipes, adds respectively 7 μ l clostridiopetidase As (1mg/ml), and presses the Z-Pro-Leu-Gly(1mM that the volume shown in the table 2 adds reaction buffer and different volumes) in collagenase solution, mixing.Then mix with gelatin solution, make its final concentration (cumulative volume is 350 μ l) be respectively 10 μ M, 20 μ M, 30 μ M, 40 μ M, 50 μ M, 60 μ M, 70 μ M, 80 μ M, the reaction system of variable concentrations Z-Pro-Leu-Gly is packed in the different sample hoses.The Z-Pro-Leu-Gly test group of each concentration extracts respectively 50 μ l reactant liquors and mixes cessation reaction on ice with 50 μ l reaction terminating liquids behind 0min, 2min, 4min, 6min, 8min, 10min.
Composing system when table-2Z-Pro-Leu-Gly inhibiting effect is measured
Behind the reaction terminating, add 600 μ l ninhydrin reaction liquid in each sample hose, the vortex mixing is put into 80 ℃ of water baths and is reacted 10min; Then each sample hose adds 584 μ l distilled waters, and (400～700nm), acquisition is at the absorbance of 570nm to carry out spectral scan behind the mixing.Interpretation of result is as follows: in the identical time interval, the absorbance changing value of the absorbance changing value (deducting 0 minute absorbance (or 2 minutes) in 10 minutes) that adds Z-Pro-Leu-Gly when not adding Z-Pro-Leu-Gly, the inhibiting rate of acquisition when this concentration, thereby obtain the inhibiting rate of variable concentrations Z-Pro-Leu-Gly, take concentration as horizontal ordinate, inhibiting rate is the ordinate mapping at last.Half inhibitor concentration (IC
50) obtain through the recurrence of mathematics nucleoid.See Fig. 7 and Fig. 8.Fig. 7 demonstration, Z-Pro-Leu-Gly suppresses clostridiopetidase A to the hydrolytic action of gelatin, and along with the concentration increase of Z-Pro-Leu-Gly, absorbance progressively descends, and absorbance all is lower than the absorbance of blank and negative control.Fig. 8 is through mathematics manipulation, and the inhibition degree is to the mapping of Z-Pro-Leu-Gly concentration, and then acquisition half-inhibition concentration (IC
50=52.4 μ M).See Fig. 7,8.
This experimental result shows: the ninhydrin reaction that the amino acid product that clostridiopetidase A decomposition gelatin produces can utilize the present invention to optimize detects, after in screening system of the present invention, adding the Z-Pro-Leu-Gly inhibited to clostridiopetidase A, the absorbance of its 570nm is lower than blank group and negative control group, has confirmed the effect of the protease inhibitors of Z-Pro-Leu-Gly; Further by the impact of variable concentrations Z-Pro-Leu-Gly on absorbance, calculate Z-Pro-Leu-Gly to the half-inhibition concentration of clostridiopetidase A, its IC through the recurrence of mathematics nucleoid
50=52.4 μ M.
The IC of Z-Pro-Leu-Gly
50With literature value (Curtis A.Spilburg, Chesterfield; William M.Moore, St, Charles, United States Patent, patent number:4720486 (Jan.19,1988)) identical.
The screening technique of embodiment 7 pancreatin inhibitors
Ninhydrin reaction solution, reaction buffer and reaction terminating liquid are got the solution combination among the embodiment 3.
The blank group: (1) gets the aqueous gelatin solution 22 μ l of 30mg/ml, is positioned in 1.5 milliliters of Ependoff pipes, adds 321 μ l reaction buffers, mixing again.(2) pancreatin (1mg/ml) solution with 7 μ l joins in the above-mentioned gelatin solution 37 ℃ of reactions.Behind 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 50 μ l reaction terminating liquids.
Negative control group: get 22 μ l gelatin (30mg/ml) and be positioned over respectively in the Ependoff pipe, add 200 μ l reaction buffers, mixing; Other gets a series of Ependoff pipes, adds 7 μ l clostridiopetidase As (1mg/ml), 117.5 μ l reaction buffers, and 3.5 μ l glycerine, mixing.Then mix with gelatin solution, final concentration is that the 1%(cumulative volume is 350 μ l), behind 0min, 2min, 4min, 6min, 8min, 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 25 μ l reaction terminating liquids.(seeing Figure 12).
Experimental group: get 22 μ l gelatin (30mg/ml) and be positioned over respectively in the Ependoff pipe, add 200 μ l reaction buffers, mixing; Other gets a series of Ependoff pipe, adds 7 μ l pancreatin (1mg/ml), and 120 μ l reaction buffers, and 1 μ l(100mM) the PMSF(phenylmethylsulfonyl fluoride, can suppress trypsase, available from U.S. AMRESCO company) solution, mixing.Then mix with gelatin solution, its final concentration is that 285 μ M(cumulative volumes are 350 μ l), behind 0min, 2min, 4min, 6min, 8min, 10min, extract respectively 50 μ l reactant liquors and mix cessation reaction on ice with 25 μ l reaction terminating liquids.Behind (seeing Figure 10,11) reaction terminating, add 450 μ l ninhydrin reaction liquid in each sample hose, the vortex mixing is put into 80 ℃ of water baths and is reacted 10min; Then each sample hose adds 437.5 μ l distilled waters, and (400～700nm), acquisition is at the absorbance of 570nm to carry out spectral scan behind the mixing.
The result shows: the ninhydrin reaction that the amino acid product that pancreatin decomposition gelatin produces can utilize the present invention to optimize detects, after in screening system of the present invention, adding the PMSF inhibited to pancreatin, the absorbance of its 570nm is lower than blank group and negative control group, has confirmed the effect of the pancreatin inhibitor of PMSF.
The mensuration of embodiment 8 protein contents
Standard curve making: standard bovine serum albumin(BSA) (BSA) (5mg/ml, Sangon Biotech (Shanghai) Co., Ltd., production code member: SK3061) proportional diluted (with the 10mMTrs-HCl that contains Macrogol 6000, pH7.5 damping fluid) is: 100 μ g/ml, 200 μ g/ml, 400 μ g/ml, 800 μ g/ml, 1600 μ g/ml, 2000 μ g/ml, 3200 μ g/ml.
Method:
1) get the BSA 30 μ l of each concentration, add Bradford reactant liquor 1500 μ l, room temperature is placed after 5 minutes and is carried out photometric detection (Super-Bradford Protein Assay Kit, health is century, CW0013, range of linearity 100-1500 μ g/ml).See figure-14
2) get the BSA 30 μ l of each concentration, add ninhydrin reaction liquid 500 μ l, 80 ℃ of reaction 10min in constant water bath box add distilled water 500 μ l after being chilled to room temperature, record the absorption value of 570nm with spectrophotometric method.The result obtains straight line as follows with BSA concentration-absorption value mapping, R2=0.9980, and the range of linearity of typical curve reaches 100～3200 μ g/ml.See figure-15 and figure-16.
Example: 1) with the K562 cell (1 * 10 of exponential phase
6) collect centrifugally, abandon supernatant.Add endochylema lysate (on ice) suspension cell, then transfer in the lysis device pull 20 times.1 * 10
4Left the heart 10 minutes, supernatant is cell cytosol albumen.
The endochylema total protein content is measured: 30 μ l endochylema extracts are mixed with 500 μ l ninhydrin reaction liquid, and 80 ℃ were reacted 10 minutes, added water to 1 milliliter after being chilled to room temperature again.Colorimetric obtains absorbance, obtains its concentration from above-mentioned typical curve.
Same program detects the cell cytosol total protein with Bradford method (Super-Bradford Protein Assay Kit, health is century, CW0013, range of linearity 100-1500 μ g/ml), obtains identical result (error is 1.8%)
Based on the above results, the inventive method can be used for the quantitative detection of albumen, has the advantages such as easy and simple to handle, highly sensitive, that stability is strong, and the range of linearity of detection is at 100～3200 μ g/ml.
Claims (10)
1. a protease inhibitors screening technique is characterized in that, comprises step:
(1) determinand is added to mixing in the protein enzyme solution; Establishing simultaneously the protein enzyme solution that does not add determinand is blank;
(2) add respectively substrate protein solution in the protein enzyme solution that contains determinand of step (1) and the blank and react, obtain reactant liquor;
(3) add reaction terminating liquid in the reactant liquor;
(4) adding ninhydrin reaction liquid reacts again;
(5) add distilled water, according to absorbance OD
570nmJudged result is lower than blank if contain the absorbance of determinand solution, and then testing sample is positive.
2. the method for claim 1 is characterized in that, described proteinase is matrix metalloproteinase or pancreatin.
3. the method for claim 1 is characterized in that, the protein enzyme solution preparation method is in the step (1): the aqueous solution of protease that with concentration is 1mg/ml is mixed to get protein enzyme solution for 1:16 ~ 30 by volume with reaction buffer solution; The preparation method of substrate protein solution is in the step (2): the substrate protein aqueous solution of 15-30mg/ml is mixed to get substrate protein solution for 1:5 ~ 10 by volume with reaction buffer solution.
4. method as claimed in claim 3 is characterized in that, described reaction buffer is for containing 50mMTris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5;
Or 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5 contains 3% ~ 6% polyglycol.
5. method as claimed in claim 4 is characterized in that, when containing polyglycol in protein enzyme solution and the substrate protein solution, the described reaction terminating liquid of step (3) is the EDTA-Tris-HCl damping fluid of 25 ~ 100mM;
When not having polyglycol in proteinase buffer solution and the albumen buffer solution, the described reaction terminating liquid of step (3) is the EDTA-Tris-HCl damping fluid of 25 ~ 100mM, contains 12% ~ 20% polyglycol.
6. the method for claim 1 is characterized in that, the described ninhydrin reaction solution of step (4) is mixed to get by solution A and solution B equal-volume,
Described solution A is stannous chloride-sodium citrate solution, contains 0.1 ~ 0.3M citric acid, and 7 ~ 10mM stannous chloride is 5.0 with the NaOH adjust pH;
Described solution B is triketohydrindene hydrate-dimethyl sulphoxide solution, triketohydrindene hydrate-ethylene glycol solution or triketohydrindene hydrate-N, dinethylformamide solution is about to triketohydrindene hydrate and is dissolved in dimethyl sulfoxide (DMSO) or is dissolved in ethylene glycol, or is dissolved in N, in the dinethylformamide solution, making the triketohydrindene hydrate final concentration is 40 ~ 70g/L.
7. the method for claim 1 is characterized in that, the middle ninhydrin reaction liquid of step (4) by volume 5 ~ 8:1 is added in the mixed solution of step (3).
8. the method for claim 1 is characterized in that, step (4) reaction conditions is 80-100 ℃, 10-15min, pH=5.0.
9. the application of the arbitrary described method of claim 1 ~ 8 in the medicine of preparation inhibition tumor invasion, transfer.
10. a kit that screens protease inhibitors is characterized in that, contains ninhydrin reaction solution, reaction buffer, reaction terminating liquid;
Described ninhydrin reaction solution is mixed to get by solution A and solution B equal-volume;
Described solution A is stannous chloride-sodium citrate solution, contains 0.1 ~ 0.3M citric acid, and 7 ~ 10mM stannous chloride is 5.0 with the NaOH adjust pH;
Described solution B is triketohydrindene hydrate-dimethyl sulphoxide solution, triketohydrindene hydrate-ethylene glycol solution or triketohydrindene hydrate-N, dinethylformamide solution, it is for to be dissolved in triketohydrindene hydrate in dimethyl sulphoxide solution or ethylene glycol solution or the DMF solution, and making the triketohydrindene hydrate final concentration is 40 ~ 70g/L;
When described reaction buffer is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, during pH=7.5,
Described reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25-100mM, contains 12% ~ 20% polyglycol; Or
When described reaction buffer is 50mM Tris-HCl, 100 ~ 250mM NaCl, 1 ~ 5mM CaCl
2, 1 μ M ZnCl
2, pH=7.5 is when containing 3% ~ 6% polyglycol;
Described reaction terminating liquid is the EDTA-Tris-HCl damping fluid of 25-100mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210416970.0A CN102879390B (en) | 2012-10-26 | 2012-10-26 | Method for screening protease inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210416970.0A CN102879390B (en) | 2012-10-26 | 2012-10-26 | Method for screening protease inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102879390A true CN102879390A (en) | 2013-01-16 |
| CN102879390B CN102879390B (en) | 2015-04-22 |
Family
ID=47480796
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210416970.0A Expired - Fee Related CN102879390B (en) | 2012-10-26 | 2012-10-26 | Method for screening protease inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102879390B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458668A (en) * | 2013-09-12 | 2015-03-25 | 中国药科大学 | High flux screening method for screening cathepsin B inhibitor |
| CN111235221A (en) * | 2020-01-22 | 2020-06-05 | 北京大学第一医院 | Method for detecting activity of FAP inhibitor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590497A (en) * | 2012-01-13 | 2012-07-18 | 宁波美康生物科技股份有限公司 | Cysteine protease inhibitor C test kit |
-
2012
- 2012-10-26 CN CN201210416970.0A patent/CN102879390B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590497A (en) * | 2012-01-13 | 2012-07-18 | 宁波美康生物科技股份有限公司 | Cysteine protease inhibitor C test kit |
Non-Patent Citations (2)
| Title |
|---|
| ETSUSHIRO DOI等: "Modified Colorimetric Ninhydrin Methods for Peptidase Assay", 《ANALYTICAL BIOCHEMISTRY》 * |
| STANFORD MOORE等: "A modified ninhydrin reagent for the photometric determination of amino acids and related compounds", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458668A (en) * | 2013-09-12 | 2015-03-25 | 中国药科大学 | High flux screening method for screening cathepsin B inhibitor |
| CN111235221A (en) * | 2020-01-22 | 2020-06-05 | 北京大学第一医院 | Method for detecting activity of FAP inhibitor |
| CN111235221B (en) * | 2020-01-22 | 2022-08-05 | 北京大学第一医院 | Method for detecting activity of FAP inhibitor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102879390B (en) | 2015-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bantan-Polak et al. | A comparison of fluorescamine and naphthalene-2, 3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids | |
| Wang et al. | A highly specific fluorescent probe for hypochlorite based on fluorescein derivative and its endogenous imaging in living cells | |
| Muckenschnabel et al. | Quantitation of hyaluronidases by the Morgan–Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers | |
| Yang et al. | Simple 1, 8-diaminonaphthalene-based fluorescence chemosensor for hypochlorites and its practical application | |
| Du et al. | Dual-site fluorescent probe for multi-response detection of ClO− and H2O2 and bio-imaging | |
| CN108752331A (en) | Synthesis and application a kind of while that distinguish detection Cys, Hcy and GSH Multifunction fluorescent molecular probe | |
| CN104502585A (en) | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor | |
| Jones et al. | Simplified endoproteinase assays using gelatin or azogelatin | |
| CN102879390B (en) | Method for screening protease inhibitor | |
| Jannat et al. | Liquid crystal-enabled protease inhibition assays developed in a millifluidic device | |
| CN106290326B (en) | Detect colorimetric sensor, preparation method and the application of lipopolysaccharides | |
| Chao et al. | Fluorescence detection and imaging in zebrafish and Arabidopsis thaliana based on Cys/Hcy breaking space effect | |
| CN108181274A (en) | Bacterial fluorescence probe containing CdSe quantum dot is to the highly selective detection method of copper ion in water and blood plasma | |
| Li et al. | Fluorimetric determination of albumin and globulin in human serum using tetra-substituted sulphonated aluminum phthalocyanine | |
| He et al. | A water-soluble two-photon fluorescent probe for rapid and reversible monitoring of redox state | |
| CN105758833A (en) | Microballoon-based cup-type time resolution fluorescent troponin I analysis kit as well as preparation method and application thereof | |
| EP2295980A1 (en) | Molecules and methods using same for measuring non-transferrin bound iron | |
| Li et al. | A novel strategy of designing neutrophil elastase fluorescent probe based on self-immolative group and its application in bioimaging | |
| Yang et al. | Rational construction of a new water soluble turn-on colorimetric and NIR fluorescent sensor for high selective Sec detection in Se-enriched foods and biosystems | |
| US20060141551A1 (en) | Analytical method for detecting alkaline sphingomyelinase and kit for use in such method | |
| AU748663B2 (en) | Method of measuring bone resorption rate | |
| EP2859343A1 (en) | Method for measurement of peptidic degradation products of a proteolytic cascade in blood samples | |
| Koshti et al. | A new azo dye for the selective detection of glycine | |
| CN107003303A (en) | For composition of enzyme immunoassay (EIA) using immunofluorescence and application thereof | |
| Mednis et al. | A sensitive fluorometric assay for the determination of cathepsin D |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Youxun Inventor after: Zhang Yanfang Inventor after: Fu Yun Inventor after: Zhou Sufeng Inventor after: Kang Lixia Inventor after: Li Changzheng Inventor after: Sun Fang Inventor before: Li Changzheng Inventor before: Zhang Yanfang Inventor before: Fu Yun Inventor before: Zhou Sufeng Inventor before: Kang Lixia Inventor before: Sun Fang |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: LI CHANGZHENG ZHANG YANFANG FU YUN ZHOU SUFENG KANG LIXIA SUN FANG TO: LIU YOUXUN ZHANG YANFANG FU YUN ZHOU SUFENG KANG LIXIA LI CHANGZHENG SUN FANG |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150422 Termination date: 20151026 |
|
| EXPY | Termination of patent right or utility model |