CN103994989B - A kind of acetyl transferase and the method for detecting specificity of inhibitor thereof - Google Patents
A kind of acetyl transferase and the method for detecting specificity of inhibitor thereof Download PDFInfo
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- CN103994989B CN103994989B CN201410249779.0A CN201410249779A CN103994989B CN 103994989 B CN103994989 B CN 103994989B CN 201410249779 A CN201410249779 A CN 201410249779A CN 103994989 B CN103994989 B CN 103994989B
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- acetyl
- expeptidase
- acetyl transferase
- quencher
- inhibitor
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Abstract
The invention provides the method for detecting specificity of a kind of acetyl transferase and inhibitor thereof.The substrate polypeptide of the fluorophor labelling and its inhibitor acetylizad through acetyl transferase suppressed hatches catalytic degradation in expeptidase, then adsorbs with carbon nanomaterial quencher, finally uses spectrofluorophotometer detection.Obstruction expeptidase degraded due to acetyl group; cause the quencher of nanoscale respectively to different with the absorption quenching effect effect of the fluorescent labeling substrate polypeptide of inhibited dose of suppression before and after acetylation, by the change of its fluorescence intensity, acetyl transferase and its inhibitor are detected.The method proposes the acetylation sites inhibitory action to expeptidase activity first, and combines the fluorescent quenching character that carbon nanomaterial is excellent, has features such as exempting from antibody recognition, pollution-free, high sensitivity and end point determination；Meanwhile, the detection time is short, sample treatment simple, and application prospect is good.
Description
Technical field
The present invention relates to realize acetyl transferase and the simplicity of inhibitor, quick, high-sensitivity detecting method.
Background technology:
Protein post-translational modification plays vital effect in life entity, and it makes the structure of protein the most multiple
Miscellaneous, regulate the finest, function is more perfect, acts on more single-minded.The wherein protein under acetyl transferase catalytic action
Acetylation is one of important way of post translational modification in organism.Conservative relying on nucleosome core histone N-terminal tail
Propylhomoserin is the site of acetylation of histone, and acetyl transferase can be by the acetyl grouptransfer of S-acetyl-coenzyme-A to its lysine residue
Epsilon-amino on.In eukaryotic cell, acetylation of histone and reversible process deacetylation thereof, chromatin Structure and gene are turned
The regulation and control of record are multi-level, complex processes for multi-step.Therefore, the simplicity of acetyl transferase, detection quick, highly sensitive
Study the mechanism for being further elucidated with gene expression regulation, specify molecular mechanism and the treatment of the relevant disease morbidities such as tumor
The aspects such as method have great meaning.The detection of traditional acetyl transferase is to utilize3H-or14C-marking substrates acetyl is auxiliary
The radioelement autography technology of enzyme A, there is the shortcoming such as radioactive material contamination and operating procedure complexity in the method.Therefore adopt
Detect with the acetylase based on antibody recognition of non radioactive element labelling and carried out tentative exploratory development, but outstanding behaviours
For the defect such as less stable, batch wise differences, intersection identification, price be high, and it is currently based on and exempts from antibody acetylation recognition mechanism
Report less.
Summary of the invention
It is an object of the invention to provide a kind of easy, quickly, exempt from antibody recognition, highly sensitive detection acetyl transferase and
The method of inhibitor.
Realize the object of the invention technical scheme to comprise the following steps:
A. by having the acetyl transferase substrate polypeptide of fluorophor labelling and S-acetyl-coenzyme-A in acetyl transferase or
In Acetylase and acetyltransferase inhibitors, hatch catalysis, make S-acetyl-coenzyme-A under acetyl transferase effect or
Shift under Acetylase and acetyltransferase inhibitors effect or suppress to shift acetyl group to the binding site of substrate polypeptide；
B. a. step reactant liquor that obtains of reaction is hatched in expeptidase digestion hydrolysis, then with carbon nanomaterial cancellation
Agent carries out adsorption；
C. finally by fluorescence spectrum detect, due to acetyl group expeptidase is degraded obstruction, fluorophor mark
The substrate polypeptide being modified with acetyl group of note is effectively combined with carbon nanomaterial quencher, causes fluorescent quenching, and through described
The substrate polypeptide of the acetyl group unmodified of the fluorophor labelling of inhibitor effect is still protected through expeptidase Degradation, fluorescence
Hold；Strong and weak according to fluorescence signal realizes Acetylase or the detection of acetyltransferase inhibitors.Fluorescence in described a. step
Group includes the derivant that a series of fluorescein maybe can fluoresce.
Hatching catalysis time in described a. step is 20-100min, and temperature is 20-40 DEG C.
Hatching digestion time with expeptidase in described b. step is 20-60min, and hydrolysis temperature is 20-30 DEG C.
In described b. step, carbon nanomaterial quencher includes graphene oxide, multi-walled carbon nano-tubes or SWCN.
In described b. step, quencher adsorption time is 2-30min, and temperature is 20-40 DEG C.
The method of the present invention proposes the acetylation sites inhibitory action to expeptidase activity first, and combines carbon and receive
The fluorescent quenching character that rice material is excellent, has features such as exempting from antibody recognition, pollution-free, high sensitivity and end point determination；Meanwhile,
The detection time is short, sample treatment simple, and application prospect is good.
The present invention devises a kind of quencher based on acetylated polypeptides peptide for inhibiting exopeptidase enzymic digestion/many by such scheme
The nano combined fluorescent probe of peptide thus can efficiently realize acetyl transferase and the simplicity of inhibitor, detection quick, highly sensitive.
It is by acetyl transferase substrate polypeptide and S-acetyl-coenzyme-A that acetyl group is modified on the binding site of substrate polypeptide
In acetyl transferase, hatch catalysis, make acetyl grouptransfer realize to the amino of substrate polypeptid specificity lysine sites
's.
Current existing protein post-translational modification sensing analytical method majority is confined to above protein phosphorylation, and this is main
If because protein phosphorylation is prone to labelling and detection, and process is relatively easy.But due to the electricity before and after acetylation modification
Lotus change, bio-identification character and enzyme interaction property etc. are all difficult to be detected, therefore exploitation acetyl transferase and suppression thereof
The method for detecting specificity of agent has very important significance.And the detection of existing acetyl transferase utilizes radioactivity unit more
, there is radioactive material contamination, operating procedure complexity, less stable, intersection in element autography technology and the method for antibody recognition
The defects such as identification.In view of the bottleneck of above-mentioned acetylation recognition detection, we are found that acetylation sites can effectively suppress first
The digestion Degradation of expeptidase, utilizes the fluorescent quenching of the recognition mechanism incorporating carbon nanostructure materials that this is new
Matter, constructs and exempts from antibody recognition sensor, effectively achieves acetyl transferase and inhibitor detection thereof.Detection method
Building sensing interface, strengthen the sensitivity of sensing response and selectivity has the performance of uniqueness, have simplicity, quickly, exempt to resist
The excellent properties such as body identification.
The principle of the present invention is as shown in Figure 1.The acetyl transferase substrate polypeptide of fluorophor labelling can be with carbon nanometer material
There is effective fluorescent quenching in material quencher.Expeptidase can become amino acid fragment by hydrolyzed peptide.The most acetylizad end
Thing polypeptide, the digestion hydrolysis through expeptidase discharges free fluorophor, after carbon nanomaterial quencher effect
Fluorescence signal keeps；And after acetyl transferase and S-acetyl-coenzyme-A acetylation, owing to expeptidase is degraded by acetyl group
Obstruction so that the polypeptide of fluorophor labelling be easier to is combined and fluorescent quenching with carbon nanomaterial quencher.Such as Fig. 2 institute
Showing, fluorescence intensity significant difference before and after acetylation, the acetylation identification for acetyl transferase effect provides feasibility.
The obstruction that expeptidase is degraded by the present invention by acetyl group, causes carbon nanomaterial quencher respectively to acetyl
Before and after change different with the absorption quenching effect effect of fluorophor labeled substrate polypeptide of inhibited dose of suppression, strong by its fluorescence
Acetyl transferase and inhibitor thereof are detected by the change of degree.This fluorescent polypeptide/nanometer quencher body that we build
System, proposes acetylation sites first and effectively suppresses the digestion hydrolysis new principle of expeptidase, preferably achieve acetyl
Change transferring enzyme and the simplicity of its inhibitor, quick, specific detection.Compared with traditional method, the method has no pollution, higher
Sensitivity and exempt from the advantages such as antibody recognition, the method operation simultaneously process simple, the detection time is short, carry for histone acetylation research
Supply powerful measure.Acetyl transferase lowest detectable limit in the catalyst system and catalyzing as a example by graphene oxide is about 1nM (such as figure
3)；Additionally, the method for the present invention may further be used to detect acetylation inhibitor, its semi-inhibit agent concentration IC50(press down when being suppressed half
The concentration of preparation) value is about 41.61 μMs (such as Fig. 4).In sum, illustrate that the inventive method is that one is easy, quick, exempts from antibody
Identification, the new method of highly sensitive detection acetyl transferase.
Accompanying drawing explanation
Fig. 1 is the principle schematic of the present invention；
Fig. 2 is the fluorescence spectrum figure before and after the acetylization reaction as a example by graphene oxide；
Fig. 3 be variable concentrations acetyl transferase (from top to bottom: 0,1,2,4,6,8,10,20,40,60,80,100,
500,1000nM) fluorescence spectrum figure；
Fig. 4 be variable concentrations inhibitor (from bottom to top: 0,0.1,0.5,1.0,2.5,5.0,7.5,10,20,40,60,
80,100,500,1000,5000 μMs) fluorescence spectrum figure and its fluorescence intensity logarithmic relationship figure (illustration) at 517nm.
Detailed description of the invention
The present invention is further illustrated below in conjunction with embodiment, and the unrestricted present invention.
Utilize the inventive method inventor to acetyl transferase and the simplicity of inhibitor, detection quick, highly sensitive.
Embodiment 1
1, the acetylization reaction of acetyl transferase catalysis
The initiator system in S-acetyl-coenzyme-A by acetyl transferase substrate polypeptide and acetyl transferase, at 20-40 DEG C
In the range of hatch 40min.
2, expeptidase catalysis end solution polypeptide-carbon nanomaterial quencher complex fluorescence probe
Above-mentioned reactant liquor and expeptidase are hatched in the range of 20-30 DEG C 30min digestion hydrolysis, afterwards at 20-
5min is adsorbed with quencher (graphene oxide) in the range of 40 DEG C.
3, fluorescence spectrum detection
Above-mentioned reaction uses spectrofluorophotometer detection.According to the feature of fluorophor used, testing conditions is: excite
Wavelength is 485nm, and transmitting wave-length coverage is 500-620nm, and exciting and launching slit is 5nm.Variable concentrations acetyl transferase
The fluorescence spectrum figure that detection obtains can be found in Fig. 3.Acetyl transferase is about 1nM in the lowest detectable limit of this catalyst system and catalyzing.
Embodiment 2
1, the inhibitor inhibitory action to acetyl transferase
The substrate polypeptide modified by fluorophor and acetyl transferase, acetylation inhibitor are in the range of 20-40 DEG C
Effect 10min, afterwards initiator system in S-acetyl-coenzyme-A, hatch 40min in the range of 20-40 DEG C.
2, expeptidase catalyzing hydrolysis polypeptide-carbon nanomaterial quencher complex fluorescence probe
Above-mentioned reactant liquor and expeptidase are hatched in the range of 20-30 DEG C 30min digestion hydrolysis, afterwards at 20-
5min is adsorbed with quencher (graphene oxide) in the range of 40 DEG C.
3, fluorescence spectrum detection
Above-mentioned reaction uses spectrofluorophotometer detection.According to the feature of fluorophor used, testing conditions is: excite
Wavelength is 485nm, and transmitting wave-length coverage is 500-620nm, and exciting and launching slit is 5nm.Variable concentrations inhibitor fluorescence light
Spectrogram and its fluorescence intensity logarithmic relationship figure (illustration) at 517nm can be found in Fig. 4.Acetylation inhibitor is at this catalyst system and catalyzing
Semi-inhibit agent concentration IC50Value is about 41.61 μMs.
Claims (5)
1. an acetyl transferase and the method for detecting specificity of inhibitor thereof, it is characterised in that comprise the following steps:
A. will there be the acetyl transferase substrate polypeptide of fluorophor labelling and S-acetyl-coenzyme-A in acetyl transferase or in second
Acyltransferase and acetyltransferase inhibitors hatch catalysis, makes S-acetyl-coenzyme-A under acetyl transferase effect or at acetyl
Shift under transferring enzyme and acetyltransferase inhibitors effect or suppress to shift acetyl group and modify on the binding site of substrate polypeptide；
B. the reactant liquor that a. step reaction obtains is hatched digestion hydrolysis in expeptidase, then enter with carbon nanomaterial quencher
Row adsorption；
C. finally by fluorescence spectrum detect, due to acetyl group expeptidase is degraded obstruction, fluorophor labelling
The substrate polypeptide being modified with acetyl group is effectively combined with carbon nanomaterial quencher, causes fluorescent quenching, and through described suppression
The substrate polypeptide of the acetyl group unmodified of the fluorophor labelling of agent effect still keeps through expeptidase Degradation, fluorescence；
Strong and weak according to fluorescence signal realizes Acetylase or the detection of acetyltransferase inhibitors；
In described b. step, carbon nanomaterial quencher includes graphene oxide, multi-walled carbon nano-tubes or SWCN.
Method the most according to claim 1, it is characterised in that described a. step in fluorophor include a series of fluorescein or
The derivant that can fluoresce.
Method the most according to claim 1, it is characterised in that hatching catalysis time in described a. step is 20-100 min,
Temperature is 20-40 ° of C.
Method the most according to claim 1, it is characterised in that hatching digestion time with expeptidase in described b. step is
20-60 min, hydrolysis temperature is 20-30 ° of C.
Method the most according to claim 1, it is characterised in that in described b. step, quencher adsorption time is 2-30 min,
Temperature is 20-40 ° of C.
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| CN110763742B (en) * | 2019-10-14 | 2022-07-26 | 宁波大学 | Preparation method and application of electrochemical sensor based on high-order G4 and acetyl antibody |
| CN111549096A (en) * | 2020-05-06 | 2020-08-18 | 浙江大学 | Method for detecting protein kinase A activity based on carbon nano material fluorescence |
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