CN104359883A - Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6) - Google Patents
Active fluorescence detection method of anthropogenic silent information regulatory factor 6 (Sirtuin6) Download PDFInfo
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- CN104359883A CN104359883A CN201410641045.7A CN201410641045A CN104359883A CN 104359883 A CN104359883 A CN 104359883A CN 201410641045 A CN201410641045 A CN 201410641045A CN 104359883 A CN104359883 A CN 104359883A
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- polypeptide
- sirtuin6
- substrate
- regulatory factor
- lysine
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Abstract
The invention discloses an active fluorescence detection method of an anthropogenic silent information regulatory factor 6 (Sirtuin6). A polypeptide containing fluorophore and quenching group (FRET (fluorescence resonance energy transfer) effect) and long-chain fatty acylation lysine residue is used as a substrate, the fluorescence intensity is associated with the enzymatic activity of Sirtuin6, and the Sirtuin6 recognition site (long-chain fatty acylation lysine) of the substrate is located in the middle of the substrate by the FRET effect, thus, the substrate (polypeptide) is close to a natural substrate of Sirtuin6, and the combination is close; more fluorophores can be selected, and reliable screening result can be obtained. The method can be applied to screening of the Sirtuin6 regulator or is used for active detection of Sirtuin6 in a biological sample, has the characteristics of miniaturization, automation, reliability, rapidness and the like, is suitable for high-reflux application, and also can be used for development of the novel Sirtuin6 active detection kit and screening of the Sirtuin6 regulator.
Description
Technical field
The present invention relates to pharmaceutical field, especially a kind of people source silent message regulatory factor 6(Sirtuin 6 or SIRT6) activity fluorescence detection method.
Background technology
Histon deacetylase (HDAC) is the protease family of the acetyl group hydrolysis of a class catalysis various substrate protein lysine residue side chain.The histon deacetylase (HDAC) in the people source of current discovery comprises 4 subclass totally 18 subspecies.By the difference of catalytic mechanism, they are divided into classical I class, II class and IV class to take zinc as histon deacetylase (HDAC) (HDAC has 11 kinds to be HDAC1 to HDAC11 respectively) [A. J. de Ruijter, the et al. of coenzyme
biochem. J. 2003,
370, 737]; In addition, the histon deacetylase (HDAC) that more special III class is coenzyme with nicotinamide adenine dinucleotide (NAD), is also called silent message regulatory factor (Sirtuin has 7 kinds to be SIRT1 to SIRT7 respectively).According to similarity and the classification of evolving of protein sequence, the Sirtuin of all species is divided into again four large classes: SIRT1, SIRT2, SIRT3 belong to I class; SIRT4 belongs to II class; SIRT5 belongs to III class; SIRT6 and SIRT7 belongs to IV class [A. A. Sauve, et al.
annu. Rev. Biochem. 2006,
75, 435].
Research shows, hdac inhibitor can inhibition tumor cell growth, promote its differentiation and apoptosis, and on normal cell almost no impact.Therefore, they have become star's targeted anticancer medicine of new generation, and current multiple hdac inhibitor is in [G. Giannini, et al. in preclinical phase and clinical testing
future Med. Chem. 2012,
4, 1439; S. Minucci, et al.
nature Reviews Cancer 2006,
6, 38].Vorinostat and FK228(Figure 10) be used for the treatment of intractable CTCL [M. Duvic, et al. by U.S. FDA respectively at 2006 and approval in 2009
blood 2007,
109, 31; R. L. Piekarz, et al.
j. Clin. Oncol. 2009,
27, 5410].These two medicines gone on the market are all the HADC inhibitor of mechanism type, but they are not optionally only suppress some HDAC.These some spinoffs likely causing these two medicines can bring over the course for the treatment of, as tired, nauseating, diarrhoea, pulmonary embolism and decrease of platelet etc., other cardiac toxic even extremely individual [G. Giannini, et al.
future Med. Chem. 2012,
4, 1439; S. Minucci, et al.
nature Reviews Cancer 2006,
6, 38].
Relative to hdac inhibitor, the antitumor research of Sirtuin inhibitor relatively lags behind, few [S. Sanchez-Fidalgo, et al. of report
curr. Med. Chem. 2012,
19, 2414].A nonspecific inhibitor for Sirtuin (EX-527) is only had to enter first phase clinical research [Y. Zhang, et al. in Europe at present
oncogene 2009,
28, 445].
The main cause that the exploitation of Sirtuin inhibitor relatively lags behind is, in the middle of the Sirtuin of this four large class, only has I class Sirtuin(and SIRT1, SIRT2 and SIRT3) there is the work of significant deacetylase; Other II, III, IV class Sirtuin(SIRT4, SIRT5, SIRT6 and SIRT7) deacetylase work be then very faint [E. Michishita, et al.
mol. Biol. Cell 2005,
16, 4623; G. Liszt, et al.
j. Biol. Chem. 2005,
280, 21323; M. C. Haigis, et al.
cell 2006,
126, 941; A. Schuetz, et al.
structure,
2007,
15, 277; E. Michishita et al.
nature,
2008,
452, 292].These are only had to the Sirtuin of faint deacetylation activity, we are difficult to develop the correctives that reliable Sirtuin method for screening active ingredients goes to develop them.
Recent newest research results shows, a member SIRT6 in IV class Sirtuin be NAD rely on remove long-chain fat acylase (NAD-dependent defatty-aclyase)
Go this current research result of long-chain fat acylase activity according to SIRT6, developed the fluorogenic substrate of cumarin coupled peptide and established the method for screening active ingredients [CN103403553A of relevant SIRT6; J. Hu, et al.
org. Biomol. Chem. 2013, 11,5213].But; the cumarin that these methods rely on can only be connected to the C end of polypeptide; make this SIRT6 recognition site of substrate long-chain fat acylated lysine can only be positioned at C end; thus the affinity that result in substrate polypeptide and SIRT6 declines; on the other hand; coumarin group serves misleading [K. T. Howitz, et al. also may to follow-up drug screening band
nature 2003,
425, 191; M. Kaeberlein, et al.
j. Biol. Chem. 2005,
280, 17038; M.T. Borra, et al.
j. Biol. Chem. 2005,
280, 17187; D. Beher, et al.
chem. Biol. Drug Des. 2009,
74, 619; M, Pacholec, et al.
j. Biol. Chem. 2010,
285, 8340].
Therefore, the novel active screening technique of SIRT6 is is urgently researched and developed.
Summary of the invention
The object of the invention is: the active fluoro screening technique providing a kind of people source silent message regulatory factor 6, its screening effect is more accurate, and simple, with low cost, to overcome the deficiencies in the prior art.
The present invention is achieved in that the active fluoro screening technique of people source silent message regulatory factor 6, with the polypeptide containing fluorophor and fluorescent quenching group and containing long-chain fat acylated lysine residue for substrate, 1) described substrate and people source silent message regulatory factor 6 are hatched, the long-chain fat acidylate of sloughing on the lysine residue of substrate is modified, and obtains literalness lysine polypeptide; 2) described literalness lysine polypeptide and proteolytic enzyme cleaves liquid are hatched, from the C end check solution peptide bond of lysine; Fluorescence intensity is associated with above-mentioned literalness lysine polypeptide, and the enzymatic activity that this detection obtains fluorescence intensity and people source silent message regulatory factor 6 has correlativity.
Described with the polypeptide containing fluorophor and fluorescent quenching group and containing long-chain fat acylated lysine residue for substrate has following structural formula:
In formula, R1 and R2 all represents that 3 with upper amino acid; Described polypeptide is the amino acid of more than 3 be connected by peptide bond; In this amino acid sequence, there is no literalness lysine or arginine, and long-chain fat acylated lysine residue can not be positioned at the two ends of polypeptide.
Fluorophor and the fluorescent quenching group of described pairing comprise Edans/Dabcyl, Trp/Dansyl, Trp/DNP, MCA/DNP, Abz/DNP or Abz/Tyr (NO
2).
Described proteolytic enzyme cleaves liquid is its effective constituent of protein cleavage liquid is trypsase, carboxypeptidase or gluc-C.
The volume ratio of described substrate and people source silent message regulatory factor 5 is 10:1, and incubation time is 30 minutes; The literalness lysine polypeptide of gained and the volume ratio of proteolytic enzyme cleaves liquid are 10:1, and incubation time is 1 hour; Above-mentioned incubation temperature is 25-37 DEG C.
The present invention as substrate polypeptide contained by two groups, belong to the relation of fluorogenic donor-fluorescent quenching acceptor.According to Fluorescence Resonance Energy transfer (fluorescence resonance energy transfer, FRET) principle, when fluorogenic donor-fluorescent quenching acceptor these two groups by covalent bond (peptide bond) connect maintain a certain distance in, the fluorescence that fluorophor sends can by fluorescent quenching group absorptions, cause the fluorescence intensity of substrate very weak, when after the process of proteolytic enzyme cleaves liquid, the peptide bond rupture of polypeptide, fluorophor and fluorescent quenching group lose the coverage of FRET, the fluorescence intensity of fluorophor strengthens greatly, fluorescence intensity and described literalness lysine polypeptide associate, and described literalness lysine polypeptide and people source silent message regulatory factor 6(are hereinafter referred to as Sirtuin6 or SIRT6) activity relevant, therefore, FRET signal reduces, fluorescence signal strengthens, with Sirtuin6 activity, there is correlativity.
Owing to have employed technique scheme, compared with prior art, the polypeptide of the present invention's utilization containing fluorophor and fluorescent quenching group (FRET effect) and with long-chain fat acidylate modification group is substrate, fluorescence intensity is associated with the enzymatic activity of Sirtuin6, FRET effect can make this Sirtuin6 recognition site of substrate long-chain fat acylated lysine be positioned at the middle part of substrate, so, make substrate polypeptide closer to the natural substrate of Sirtuin6, in conjunction with tightr; Meanwhile, the fluorophor of more more options can be given, finally, more reliable the selection result can be obtained.Such method can be applicable to the screening of Sirtuin6 correctives, or the Sirtuin6 Activity determination in biological specimen, there is the features such as microminiaturization, robotization, fast and reliable, be applicable to high-throughout application, also can be applicable to the exploitation of New type of S IRT6 activity detection kit and carry out the screening service of Sirtuin6 correctives.And screening effect of the present invention is accurate, simple, with low cost.
Accompanying drawing explanation
Fig. 1: use the polypeptide containing Dabcyl, Edans and long-chain fat acylated lysine as the substrate of Sirtuin6 Activity determination;
The FRET effect of Fig. 2: (Dabcyl) ISGASEKDIVHSE (Edans) G polypeptide;
The long-chain fat acylated lysine of Fig. 3: Fmoc protection and the synthesis of FRET polypeptide;
Fig. 4: (Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G is the substrate of Sirtuin6;
Fig. 5: measure the kinetic curve of (Dabcyl) ISGASEK (Myristoyl) KDIVHSE (Edans) G polypeptide under SIRT6 catalysis; Kinetic parameter is Km be respectively 10.8uM, Kcat is 0.0030S
-1be 277S with Kcat/Km
-1m
-1;
Fig. 6: measure the kinetic curve of (Dabcyl) ISGASEK (Acetyl) KDIVHSE (Edans) G polypeptide under SIRT6 catalysis; Kinetic parameter is Km be respectively 178.5uM, Kcat is 0.0024S
-1be 14S with Kcat/Km
-1m
-1;
Fig. 7: use (Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G to screen the inhibitor of SIRT6;
Accompanying drawing 8 uses (Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G to measure the IC50 of SIRT6 inhibitor.
Embodiment
Embodiments of the invention 1: the activity fluorescence detection method of people source silent message regulatory factor 6:
reagent and instrument
All reagent is all bought from Aldrich or Acros company and is analyzed pure rank, does not do further process; VARIAN INOVA 400M Hz nuclear magnetic resonance analyser; Liquid plasmogamy is set to Shimadzu-LC20A and Thermo LCQ FLEET mass spectrum, analytical column is Sprite TARGA C18 column (40 × 2.1 mm, 5 μm, Higgins Analytical, Inc.), determined wavelength is 215 and 280 nanometers, adopts the binary gradient type of elution of 0.1% formic acid solution, 0.1% formic acid acetonitrile solution; The multi-functional microplate reader of BIO-TEK Synergy H4 (excitation wavelength 336 nm, emission wavelength 490 nm).
the synthesis of polypeptide and purifying
As shown in Figure 3, long-chain fat acyl polypeptide and other polypeptide are all by Fmoc-Wang resin to synthetic route, adopt the Fmoc/tBu Peptide systhesis of standard to lead to (Du et al., Biochemistry 48:2878-2890,2009) of method synthesis.Containing the amino acid that long-chain fat acidylate is modified, and the amino acid that other Fmoc protect, connect successively according to implementation sequence order.Finally, by containing phenol (5%), THIOANISOLE (5%), the resin that dithioglycol (2.5%) has been connected with amino acid with the trifluoroacetic acid solution of water (5%) hatches 2 ~ 4 hours, scales off from resin, obtains unshielded polypeptide crude product.Through reverse HPLC-purified, freeze dryer freeze-drying, obtains the target polypeptides of white solid.(Dabcyl) ISGASEKDIVHSE (Edans) G polypeptide (SEQ ID NO:1), molecular formula C
86h
122n
22o
27s, LC-MS (ESI) calculated value [M+H
+]=1927.9, measured value [M+H
+]=1928.3.(Dabcyl) ISGASEK (Acetyl) DIVHSE (Edans) G polypeptide (SEQ ID NO:2), molecular formula C
88h
124n
22o
28s, LC-MS (ESI) calculated value [M+H
+]=1969.9, measured value [M+H
+]=1970.1.(Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G polypeptide (SEQ ID NO:3), molecular formula C
100h
148n
22o
28s, LC-MS (ESI) calculated value [M+H
+]=2138.1, measured value [M+H
+]=2138.7.
(Dabcyl) structural formula of ISGASEKDIVHSE (Edans) G polypeptide (SEQ ID NO:1) is as follows:
(Dabcyl) structural formula of ISGASEK (Acetyl) DIVHSE (Edans) G polypeptide (SEQ ID NO:2) is as follows:
(Dabcyl) structural formula of ISGASEK (Myristoyl) DIVHSE (Edans) G polypeptide (SEQ ID NO:3) is as follows:
the clone of the deacetylase (Sirtuin) that people source NAD relies on, expression and purification
The SIRT6 of deacetylase (Sirtuin) that people source NAD relies on carries out cloning as described in document, expression and purification (J. Hu, et al.
org. Biomol. Chem. 2013, 11,5213).Use TOPO and GATEWAY clone technology (Invitrogen Corp., Carlsbad, CA) to be cloned in pDEST-F1 expression vector, express in e. coli bl21 (DE3).The protein lysate obtained passes through HisTrap
tMhP chromatographic column (GE Healthacare, US) carries out purifying.The protein solution obtained measures its protein concentration by Bradford reagent.
the kinetic parameter of polypeptide under SIRT6 catalysis
Detect acidylate and the detection of removing acidylate FRET polypeptide by HPLC, calculate the kinetic parameter of SIRT6.The SIRT6(of purifying adopts 0.2 uM for long-chain fat acyl polypeptide; 1uM is adopted for acetylated polypeptides) (2-256uM is adopted for long-chain fat acyl polypeptide with 1mM NAD, 1mM DTT, 20mM Tris-HCl damping fluid (PH 7.4) and FRET polypeptide; 10-500uM is adopted for acetylated polypeptides) reactant liquor that forms, 37
o60 minutes are hatched under C.Described reactant liquor 100mM hydrochloric acid and 160mM acetic acid stopped reaction.By the gradient elution of HPLC, be separated acidylate with remove acidylate FRET polypeptide, reaction yield according to the absorption peak areal calculation under 336nm, assuming that this two peptide species molar absorptivity is the same at that wavelength.Kcat and Km value is by using KaleidaGraph curve Vinitial/ [E] to obtain than [S].Described test all repeats once to carry out.Experimental result as shown in Figure 5 and Figure 6.
the FRET effect of polypeptide
The reactant liquor (simultaneously establishing one group not add trypsase (Trypsin) as a control group) FRET polypeptide literalness described in 10uM (SEQ ID NO:1), 20mM Tris-HCl damping fluid (PH 7.4) and 6.25U trypsase (Trypsin) formed, 37
oafter hatching 1 hour under C, add isopyknic 100mM hydrochloric acid and 160mM acetic acid stopped reaction.Utilize the multi-functional microplate reader of BIO-TEK Synergy H4 (excitation wavelength 336 nm, emission wavelength 490 nm), detect the fluorescence signal intensity of each sample.Experimental result as shown in Figure 2.
the fluorescence experiments of polypeptide under SIRT6 catalysis
By modify containing long-chain fat acidylate described in 10uM FRET polypeptide (SEQ ID NO:3), 20mM Tris-HCl damping fluid (PH 7.4), 1mM NAD, 1mM DTT respectively with variable concentrations SIRT6(0.2,0.5,1,2uM) reactant liquor that forms, 37
oafter hatching 1 hour under C, add 6.25U trypsase (Trypsin) and 2mM niacinamide and continue 37
o1 hour is hatched under C.Add isopyknic 100mM hydrochloric acid and 160mM acetic acid stopped reaction.Utilize the multi-functional microplate reader of BIO-TEK Synergy H4 (excitation wavelength 336 nm, emission wavelength 490 nm), detect the fluorescence signal intensity of each sample.Experimental principle as shown in Figure 1.Experimental result as shown in Figure 4.
Learn according to Fig. 1, use the polypeptide containing Dabcyl, Edans and long-chain fat acylated lysine as the substrate of SIRT6 Activity determination.
Learn according to Fig. 2, aforementioned polypeptides (Dabcyl) ISGASEKDIVHSE (Edans) G can produce FRET effect.Under being with or without trypsase (Trypsin), aforementioned polypeptides hatches 1 hour.Compared with the contrast not having Trypsin, Trypsin(6.25U) hatch after fluorescence intensity enhance 15 times.
Learn according to Fig. 3, the long-chain fat acylated lysine of Fmoc protection, the route of synthesis of FRET polypeptide.
Learn according to Fig. 4, SIRT6 exist or not in the presence of, 1 hour is hatched with (Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G polypeptide, then, the trypsase (Trypsin) adding 6.25U hatches 1 hour, compared with the non-existent negative control of SIRT6,0.2, fluorescence intensity under 0.5,1,2uM SIRT6 exists all increases by more than 10 times.
Learn according to Fig. 5, SIRT6 goes long-chain fat acidylate active, and enzyme kinetics parameter is Km be respectively 10.8uM, Kcat is 0.0030S
-1be 277S with Kcat/Km
-1m
-1.
Learn according to Fig. 6, the deacetylation of SIRT6 is active, and enzyme kinetics parameter is Km be respectively 178.5uM, Kcat is 0.0024S
-1be 14S with Kcat/Km
-1m
-1.
The screening technique of embodiments of the invention 2:Sirtuin 6 correctives:
(Dabcyl) ISGASEK (Myristoyl) DIVHSE (Edans) G screens the inhibitor of SIRT6
Here step is identical with upper described step (fluorescence experiments of (Dabcyl) ISGASEK (Succinyl) DIVHSE (Edans) G polypeptide under SIRT6 catalysis), except adding different testing compound (concentration is 30uM) in reaction, in first step reaction, finally add SIRT6 and start reaction.Experimental result as shown in Figure 7.
measure the IC50 of SIRT6 inhibitor
Here step is identical with upper described step ((Dabcyl) ISGASEK (Succinyl) DIVHSE (Edans) G screens the inhibitor of SIRT6), except the Nicotinamide(concentration adding variable concentrations in reaction is respectively 50,100,200,500,1000,2000uM).Experimental result as shown in Figure 8.
Learn according to Fig. 7, use without SIRT6 and only add SIRT6 as a control group.All micromolecular concentration is 300uM.Nicotinamide shows significant SIRT6 inhibit activities.
Learn according to Fig. 8, use without SIRT6 and only add SIRT6 as a control group.All the other each group adds variable concentrations respectively, and the concentration of Nicotinamide is respectively 50,100,200,500,1000,2000uM.By fluorescence intensity, calculate the IC50 value of Nicotinamide to SIRT6 and be about 235uM.
Conclusion:
Successful design of the present invention and to develop containing fluorophor and fluorescent quenching group and polypeptide with long-chain fat acidylate modification group is substrate; utilize FRET fluorescent effect; the enzymatic activity of fluorescence intensity with Sirtuin 6 is associated; fluorescence intensity is more than ten times of background fluorescence, establishes Sirtuin 6 activity test method.
This SIRT6 recognition site of long-chain fat acylated lysine is positioned at the middle part of substrate, and make substrate polypeptide closer to the natural substrate of SIRT6, kinetic constant Kcat/Km reaches
277s
-1m
-1.
Detection method of the present invention can be applicable to the Sirtuin6 Activity determination in biological specimen, or is applied to the preliminary screening of Sirtuin6 correctives and the mensuration of IC50.
The screening effect of the present invention to the Activity determination of Sirtuin 6 and correctives thereof is accurate, simple, with low cost.
sequence table
<110> Guiyang Medical College
The active fluoro screening technique of <120> people source silent message regulatory factor 6
<130> nm:
<160> 3
<170> PatentIn version
<210> 1
<211> 14
<212> PRT
<213> artificial sequence
<220>
Can <223> Lys 7 without modification, and contains the polypeptide of fluorophor and fluorescent quenching group, produce FRET effect for examining or check this peptide sequence
<400> 1
Ile Ser Gly Ala Ser Glu Lys Asp Ile Val His Ser Glu Gly
1 7 14
<210> 2
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> Lys 7 acetylation modification, and the polypeptide containing fluorophor and fluorescent quenching group, for examining or check Sirtuin6 activity
<400> 2
Ile Ser Gly Ala Ser Glu Lys Asp Ile Val His Ser Glu Gly
1 7 14
<210> 3
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> Lys 7 long-chain fat acidylate is modified, and contains the polypeptide of fluorophor and fluorescent quenching group, for examining or check Sirtuin6 activity
<400> 3
Ile Ser Gly Ala Ser Glu Lys Asp Ile Val His Ser Glu Gly
1 7 14
Claims (5)
1. the active fluoro screening technique of a people source silent message regulatory factor 6, it is characterized in that: with the polypeptide containing fluorophor and fluorescent quenching group and containing long-chain fat acylated lysine residue for substrate, 1) described substrate and people source silent message regulatory factor 6 are hatched, the long-chain fat acidylate of sloughing on the lysine residue of substrate is modified, and obtains literalness lysine polypeptide; 2) described literalness lysine polypeptide and proteolytic enzyme cleaves liquid are hatched, from the C end check solution peptide bond of lysine; Fluorescence intensity is associated with above-mentioned literalness lysine polypeptide, and the enzymatic activity that this detection obtains fluorescence intensity and people source silent message regulatory factor 6 has correlativity.
2. the active fluoro screening technique of people source according to claim 1 silent message regulatory factor 6, is characterized in that: described with the polypeptide containing fluorophor and fluorescent quenching group and containing long-chain fat acylated lysine residue for substrate has following structural formula:
In formula, R1 and R2 all represents that 3 with upper amino acid; Described polypeptide is the amino acid of more than 3 be connected by peptide bond; In this amino acid sequence, there is no literalness lysine or arginine, and long-chain fat acylated lysine residue can not be positioned at the two ends of polypeptide.
3. the active fluoro screening technique of people source according to claim 1 silent message regulatory factor 6, it is characterized in that: fluorophor and the fluorescent quenching group of described pairing comprise Edans/Dabcyl, Trp/Dansyl, Trp/DNP, MCA/DNP, Abz/DNP or Abz/Tyr (NO
2).
4. the active fluoro screening technique of people source according to claim 1 silent message regulatory factor 6, is characterized in that: described proteolytic enzyme cleaves liquid is its effective constituent of protein cleavage liquid is trypsase, carboxypeptidase or gluc-C.
5. the active fluoro screening technique of people source according to claim 1 silent message regulatory factor 6, is characterized in that: the volume ratio of described substrate and people source silent message regulatory factor 5 is 10:1, and incubation time is 30 minutes; The literalness lysine polypeptide of gained and the volume ratio of proteolytic enzyme cleaves liquid are 10:1, and incubation time is 1 hour; Above-mentioned incubation temperature is 25-37 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112179883A (en) * | 2020-09-24 | 2021-01-05 | 贵州医科大学 | Active fluorescence detection method of humanized silent information regulatory factor 4 |
| CN113880922A (en) * | 2021-08-26 | 2022-01-04 | 深圳大学 | Fluorescent polypeptide substrate for detecting SIRT7 enzyme activity |
Citations (4)
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|---|---|---|---|---|
| WO2003066889A2 (en) * | 2002-02-07 | 2003-08-14 | Axys Pharmaceuticals, Inc. | Assay for acytyltransferase or deacetylase activity |
| US20090117572A1 (en) * | 2002-09-27 | 2009-05-07 | Allergan, Inc. | Cell-Based Fluorescence Resonance Energy Transfer (FRET) Assays For Clostridial Toxins |
| CN102958503A (en) * | 2010-06-29 | 2013-03-06 | Isp投资公司 | SIRTUIN-6-activating peptides, and cosmetic or pharmaceutical compositions including same |
| CN103097545A (en) * | 2010-07-07 | 2013-05-08 | 康奈尔大学 | Modulators for Sirt5 and assays for screening same |
-
2014
- 2014-11-13 CN CN201410641045.7A patent/CN104359883A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003066889A2 (en) * | 2002-02-07 | 2003-08-14 | Axys Pharmaceuticals, Inc. | Assay for acytyltransferase or deacetylase activity |
| US20090117572A1 (en) * | 2002-09-27 | 2009-05-07 | Allergan, Inc. | Cell-Based Fluorescence Resonance Energy Transfer (FRET) Assays For Clostridial Toxins |
| CN102958503A (en) * | 2010-06-29 | 2013-03-06 | Isp投资公司 | SIRTUIN-6-activating peptides, and cosmetic or pharmaceutical compositions including same |
| CN103097545A (en) * | 2010-07-07 | 2013-05-08 | 康奈尔大学 | Modulators for Sirt5 and assays for screening same |
Non-Patent Citations (2)
| Title |
|---|
| JING HU.ET AL: "A fluorogenic assay for screening Sirt6 modulators", 《ORGANIC & BIOMOLECULAR CHEMISTRY》 * |
| PATRICK A. MARCOTTE.ET AL: "Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction", 《ANALYTICAL BIOCHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112179883A (en) * | 2020-09-24 | 2021-01-05 | 贵州医科大学 | Active fluorescence detection method of humanized silent information regulatory factor 4 |
| CN113880922A (en) * | 2021-08-26 | 2022-01-04 | 深圳大学 | Fluorescent polypeptide substrate for detecting SIRT7 enzyme activity |
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