CN108277259A - The active detection kits of CE2 in a kind of measurement complex biological system - Google Patents
The active detection kits of CE2 in a kind of measurement complex biological system Download PDFInfo
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Abstract
The invention discloses the active detection kits of CE2 in a kind of measurement complex biological system.The kit includes the reagent A liquid, reagent B liquid and reagent solution C of independent packaging；The reagent A liquid is 2 Specific probe N normal-butyls 4 of carboxy-lesterase（2 chlorine N methylacetamides）1,8 naphthalimides（NCEN）Solution；The reagent B liquid is 100 mM phosphate buffered saline solutions, pH value 7.4；The reagent C is terminate liquid acetonitrile.2 Enzyme activity assay method of carboxy-lesterase based on the kit has many advantages, such as that few sample dosage, easy to operate, high sensitivity, accuracy is good, antibiont matrix interference ability is strong, reproducible, the screening that can be used for the active quantitative detection and carboxy-lesterase carboxy-lesterase CE2 adjusting control agents of carboxy-lesterase 2 in the cell or tissue prepared product of mammal source, there is wide application field in zymetology, life science and course of drug development.
Description
Technical field
The present invention relates to Measurements for Biotechnique.Carboxylic in the biological samples such as especially a kind of detection mammalian cell/tissue
2 active kit of acid esters enzyme carboxy-lesterase.
Background technology
Carboxy-lesterase takes part in metabolite clearance and the activation of a variety of endogenous Esters, esters medicine and environmental poisonous substance,
It is a kind of important drug metabolic enzyme.Carboxy-lesterase is mainly carboxy-lesterase 1 and carboxy-lesterase 2 in human body.In antitumor drug
Leading role, active height have been played in the metabolism activation of Irinotecan, capecitabine and gemcitabine pro-drug
The incidence of performance and the adverse reaction of said medicine antitumor activity or severity etc. can be had an important influence on.Cause
This, the inhibition or inducing action of research clinical medicine or medicinal herb components to carboxy-lesterase to clinical rational drug use and avoid diving
Drug interaction have important directive significance.The expression of 2 enzyme of studies have shown that human carboxylatase and function itself can be by
To human body heredity, age, disease, gender, environment and the influence of many factors such as medication object altogether.Wherein, inherent cause is to cause
One of an important factor for drug effect individual difference, such as carries the patient of 2 heterozygous mutant of carboxy-lesterase, after taking Irinotecan,
Its pharmacokinetic data available will be substantially distinguished from normal person's level.Since CE2 is in pro-drug, such as cocaine, clopidogrel, department difficult to understand
His Wei, procaine and Irinotecan, waits and played an important role in the metabolism of drugs, therefore carboxy-lesterase 2 can be used as this
The main determining factor of a little pro-drug pharmacological activity and toxic reaction.And 2 gene pleiomorphism of carboxy-lesterase can lead to its correspondence
Metabolic enzyme expression quantity or active change and then the behavior of change pro-drug in vivo, so that its curative effect or bad anti-can be influenced
It answers, therefore, 2 active quantitative assessment of carboxy-lesterase is particularly important.The method of 2 Activity determination of carboxy-lesterase is main at present
It is to be detected to the hydrolysate of its fluorogenic substrate with full-automatic fluorescence microplate reader.But due to currently used carboxy-lesterase 2
The Detection wavelength of fluorogenic substrate and its product it is partially short and small in 600 nm, many bio-matrixes or Chinese herbal medicine/Food Chemistry at can pair
Fluorescence signal generates interference.Therefore, the signal for product directly being collected using full-automatic fluorescence microplate reader often causes false positive
Or false carboxy-lesterase negative findings.Therefore, it is necessary to develop more accurate 2 Activity determination of carboxy-lesterase.
The purpose of the present invention is insufficient present in view of the above technology, carboxylic acid in a kind of measurement complex biological system is provided
2 active detection kit of esterase.The present invention is by High Performance Liquid Chromatography-Fluorescence Detection carboxy-lesterase 2, this method tool
Have the advantages that few sample dosage, easy to operate, high sensitivity, accuracy is good, antibiont matrix interference ability is strong, reproducible,
It is one of drug concentration and the most common method of Enzyme activity assay using high performance liquid chromatography, not only there is easy to operate, stability
Well, the features such as sensitive is detected, can be also realized between object and bio-matrix and complex sample ingredient to be measured by chromatographic isolation
Separation, and then realize object accurate detection.It can be used for carboxylate in the cell or tissue prepared product of mammal source
The screening of enzyme 2 active quantitative detection and 2 adjusting control agent of carboxy-lesterase, has extensively in zymetology, life science and course of drug development
Wealthy application field.
The object of the present invention is achieved like this, including A liquid, B liquid and C liquid.It is characterized in that
The reagent A liquid is the Specific probe N- normal-butyls -4- of carboxy-lesterase 2（2- chloro-n-methyl acetamides）-1,8-
Naphthalimide（NCEN）Solution；
The reagent B liquid is 100 mM phosphate buffered saline solutions, pH value 7.4；
The reagent solution C is terminate liquid acetonitrile；
1. the operating process application method of 2 activity detection kit of carboxy-lesterase is as follows:
（1）Sample preprocessing：Biological sample is diluted with reagent B liquid or is handled to suitable concentration, to operate liquid.
（2）Sample incubation：Will operation liquid 37 DEG C it is pre- incubate 5 ~ after ten minutes, it is working solution to add the reagent A liquid, 37
DEG C be incubated 30 minutes；Working solution volumes reagent solution C terminates reaction after reaction etc.；System vortex mixing 3 after the reaction is divided
Clock, 20000 g are centrifuged 10 minutes, and supernatant is taken after centrifugation.
（3）Liquid phase separation：By step（2）The supernatant obtained carries out chromatographic isolation, and chromatographic separation condition is：Chromatography
Column：Analytic type SHIMADZU Shim-pack XR-ODS columns；Mobile phase：Acetonitrile：Contain 0.2% aqueous formic acid;Flow velocity：
0.1-1.0 mL/min, preferably 0.4 mL/min.
（4）Fluoroscopic examination：Substrate and product after liquid chromatogram is detached be respectively adopted fluorescence detection detection, substrate and
The maximum excitation wavelength of product is respectively 354 nm and 430 nm, and best launch wavelength is respectively 432 nm and 542 nm.
（5）Data statistics：The peak face of probe metabolite N- normal-butyl -4- amino -1,8- naphthalimides is obtained from liquid phase
Product, by the production quantity and then characterization biological sample of N- normal-butyls -4- amino -1,8- naphthalimides in the unit of account time
The activity of carboxy-lesterase 2.
（6）The application for measuring 2 activity detection kit of carboxy-lesterase, for detecting mammalian cell/tissue biological's sample
Activity of carboxy-lesterase 2, characterization step are in this：
（1）Draw probe substrate N- normal-butyl -4- amino -1,8- naphthalimide standard curves.
（2）The S9 or microsome of surveyed biological sample mammalian cell/tissue are prepared, and measures protein content.
（3）Utilize step（2）Middle obtained biological sample, according to the operating process of 2 activity detection kit of carboxy-lesterase
It is detected.
Enzyme concentration and time carry out appropriate adjusting according to enzyme source or experiment condition in above step 3, as long as ensureing product life
It is in a linear relationship at amount and enzyme source concentration.
（4）It calculates：Probe peak areas (the Area of acquisitionSample), bring formula scales into, you can obtain mixed biologic sample
The active alpha of carboxy-lesterase 2 in product S9/ microsomes.
Formula 1：
Wherein, α indicates the activity of carboxy-lesterase 2 in Mixed biological sample；;
AreaSampleIndicate the probe peak areas that mixed biologic sample incubation liquid phase detects；
A indicates step（1）N- normal-butyl -4- amino -1,8- naphthalimide slope of standard curve;
Z indicates that the total protein concentration of mixed biologic sample in reaction system, specific calculate are shown in formula 2
Formula 2：
Wherein, q indicates surveyed biological sample（Mammalian cell/tissue）S9 or microsome, and measure protein content；;
VWorking solutionIndicate mixed biologic sample in claim 2 step（2）In total incubation system；
VBiological sampleExpression requires 2 steps（2）In total incubation system in mixed biologic sample mammalian cell/tissue S9 or
The volume of microsome;
It is the screening and detection for 2 inhibitor of carboxy-lesterase 1. measuring the application of 2 activity detection kit of carboxy-lesterase：
（1）It is screened the preparation of micromolecular compound series concentration solvent：0 ~ 20mM (solvent dimethyl sulfoxide (DMSO));
（2）Sample preprocessing：Various concentration micromolecular compound solvent, people's hepatomicrosome and reagent B liquid mixings to be screened are prepared
Operate liquid.
（3）It after obtaining operation liquid, is detected, surveys according to according to the operating process of 2 activity detection kit of carboxy-lesterase
The peak area of metabolite in random sample sheet.
（4）Screen the inhibitor of carboxy-lesterase 2：It brings the detected probe peak areas of liquid phase into formula 3, calculates
Sample residual activity.
Formula 3：Residual activity=
Wherein, AreaCompoundFor the peak area after being screened the reaction of compound sample is added
AreaBlankPeak face after compound replaces addition sample reaction with same volume dimethyl sulfoxide (DMSO) is screened by no addition
Product.
Enzyme concentration and time can carry out appropriate adjusting according to enzyme source or experiment condition in above step 3, as long as ensureing product
Production quantity and enzyme source concentration are in a linear relationship.
（1）Inhibitor detects：Repeat step（3）, residual activity of the compound under various concentration is measured, is brought into
Graphpad softwares are mapped, and IC is obtained50Value, detection compound rejection ability.
（4）Detection is quick：6.5min completes the primary measurement of sample, and the time is short, therefore is suitble to large batch of biological sample
Detection.
（5）Amount of samples is small：Only need 200 μ L samples, you can determine accurate metabolite concentration.
（6）The method of the present invention quickly, accurate, high sensitivity, easy to operate, the inhibition for evaluation drug to carboxy-lesterase enzyme
Foundation is provided with inducing action.This method in a few days, day to day precision relative standard deviation be respectively less than 15%.
（7）Flexibility and changeability of the present invention：Experimental study suitable for a variety of biological samples and detection.
Description of the drawings
The LC- fluorescein ethanedioic acid ester chromatograms of Fig. 1 NCEN and NAH
Fig. 2 CE2 quantitative measurement standard curve graphs
The determination of activity figure of CE2 in Fig. 3 different genera hepatic tissues
Fig. 4 CE2 inhibitor screening figures
Fig. 5 CE2 ligand bindings site process decision chart.
Specific implementation mode
Further invention is done to the present invention with reference to embodiment
Embodiment 1：The preparation of each working solution
（1）The preparation of reagent A liquid：Balance weighs NCEN, and dimethyl sulfoxide (DMSO) mixing is added, fully dissolves.It dispenses to 50 mL browns
Sealing after in reagent bottle, -4 DEG C of storages.
（2）The preparation of reagent B liquid：Prepare 100mM dipotassium hydrogen phosphates and 100mM potassium dihydrogen phosphates respectively with pure water；
With 1：The two is mixed into the 100mM phosphate buffer solutions of pH=7.4 by 4 ratio.In packing to 500 mL reagent bottles, -4 DEG C
Storage.
（3）The preparation of reagent solution C：Trifluoroacetic acid aqueous solution, in packing to 100 mL reagent bottles, -4 DEG C of storages.
Embodiment 2：2 detection kit of carboxy-lesterase is used for the foundation of 2 quantitative detecting method of carboxy-lesterase
（1）The drafting of working curve：Various concentration carboxy-lesterase 2 is prepared with reagent B liquid, shakes under the conditions of 37 DEG C and incubates in advance
3min；The A liquid of 2 μ L is added into reaction system, is reacted with 5 μM of startings of concentration are postponed；After 37 DEG C are incubated 30 min, it is added
The C liquid of 200 μ L terminates laggard liquid phase detection, and the peak area drawing curve based on product is shown in Fig. 2.
（2）Quantitation methodology is investigated：It is careful to be carried out to specificity, linear relationship and the detection error etc. of probe substrate detection
It investigates；It was found that probe substrate and product all have good chemical stability, can be preserved 48 hours under the conditions of 4 DEG C；The detection
Bio-matrix and measured object can be kept completely separate by method, therefore have specificity well to object.In addition, this method is being examined
It is good to survey linear relationship in range（R2>0.999）, detection limit can be down to 0.003 μM；In the daytime poor RSD is less than 2.3%, has good
Repeatability.It is indicated above herein use the active method high sensitivities of kit measurement carboxy-lesterase CE2, it is easy to operate,
Quickly, and there is good separating degree.
Embodiment 3：2 detection kit of carboxy-lesterase is used for the determination of activity of carboxy-lesterase 2 in different genera hepatic tissue
（1）Choose different genera（People, rat, mouse, monkey, dog and pig）Hepatomicrosome prepares 2 metabolic response body of carboxy-lesterase
System, includes the B liquid of 2 μ L hepatomicrosomes and 196 μ L, is shaken under the conditions of 37 DEG C and incubates 3 points in advance.
（2）The A liquid of 2 μ L is added into reaction system, is reacted with 5 μM of startings of concentration are postponed.
（3）After 37 DEG C are incubated 30 min, the C liquid of 200 μ L, liquid phase detection is added.
N- normal-butyl -4- amino -1,8- naphthalimide the peak areas detected obtain different genera after substituting into standard curve
Metabolic rate (Fig. 3) of the carboxy-lesterase 2 to probe NCEN in hepatomicrosome.
Embodiment 4：2 detection kit of carboxy-lesterase is used for the screening of 2 inhibitor of carboxy-lesterase
（1）Preparation is screened compound solution, prepares 2 metabolic response system of carboxy-lesterase, and 2 μ L hepatomicrosomes add 196 μ L reagents B
With 1 μ L by sieve compound/dimethyl sulphoxide solution, is shaken under the conditions of 37 DEG C and incubate 3 points in advance.
（2）The reagent A liquid of 1 μ L is added into reaction system, is reacted with 5 μM of startings of concentration are postponed.
（3）After 37 DEG C are incubated 30min, the C liquid of 200 μ L, liquid phase detection is added.
（4）The residual activity for calculating each sample, is compared with blank sample, sees Fig. 4.
Embodiment 5：2 ligand binding site of carboxy-lesterase judges
The structure-activity relationship of fluorescein ethanedioic acid ester and high molecular weight protein carboxy-lesterase 2
（1）It is 2 that carboxy-lesterase, which is prepared, with reagent B liquid volume ratios:196) operation liquid takes 198 μ L to be respectively added different dense respectively
1 μ L compounds fluorescein ethanedioic acid esters or dimethyl sulfoxide (DMSO) are spent, 37 DEG C incubate 3-10min in advance.
（2）The starting reaction of 1 μ L various concentration reagent A liquid.
（3）30min is incubated at 37 DEG C, 200 μ L acetonitriles terminate, 20000G centrifugations 30min, supernatant taken to be detected into liquid phase.
（4）Data processing：Data are handled with data processing software Graphpad, linear mapping shows fluorescein ethanedioic acid ester
Noncompetition inhibition is presented to NCEN hydrolysis in people's hepatomicrosome, sees Fig. 5.Show that fluorescein ethanedioic acid ester is distinguished with NCEN
In different binding sites, and corresponding key amino acid is also different, then high molecular weight protein carboxy-lesterase 2 is centainly at least
There are two smaller ligand binding sites.
Claims (6)
1. the active detection kits of CE2 in a kind of measurement complex biological system, including A liquid, B liquid and C liquid, are independent packet
Dress, the reagent A liquid are the Specific probe N- normal-butyls -4- of carboxy-lesterase 2（2- chloro-n-methyl acetamides）-1,8-
Naphthalimide（NCEN）Solution；The reagent B liquid is 100 mM phosphate buffered saline solutions, pH value 7.4；The reagent solution C is
Terminate liquid trifluoroacetic acid aqueous solution.
2. a kind of application method measuring the active detection kits of CE2 in complex biological system according to claim 1,
It is characterized in：
Sample preprocessing：Biological sample is diluted with reagent B liquid or is handled to suitable concentration, to operate liquid；
Sample incubation：Will operation liquid 37 DEG C it is pre- incubate 5 ~ after ten minutes, it is working solution, 37 DEG C of incubations to add the reagent A liquid
30 minutes；Working solution volumes reagent solution C terminates reaction after reaction etc.；By the system vortex mixing 3 minutes after the reaction,
20000 g are centrifuged 10 minutes, and supernatant is taken after centrifugation;
Liquid phase separation：By step（2）The supernatant of acquisition carries out chromatographic isolation, and chromatographic separation condition is：Chromatographic column：Analytic type
C18 columns；Mobile phase：Acetonitrile：Contain 0.2% aqueous formic acid;Flow velocity：0.1-1.0 mL/min, preferably 0.4 mL/min;
Fluoroscopic examination：Fluorescence detection detection is respectively adopted in substrate and product after liquid chromatogram is detached, substrate and product
Maximum excitation wavelength is respectively 354 nm and 430 nm, and best launch wavelength is respectively 432 nm and 542 nm;
Data statistics：The peak area that probe metabolite N- normal-butyl -4- amino -1,8- naphthalimides are obtained from liquid phase, passes through
The production quantity of N- normal-butyls -4- amino -1,8- naphthalimides characterizes carboxy-lesterase in biological sample in turn in the unit of account time
2 activity.
3. a kind of use measuring 2 active detection kit of carboxy-lesterase in complex biological system according to claim 1
Method, the activity suitable for detecting carboxy-lesterase 2 biological samples such as mammalian cell/tissue.
4. a kind of use measuring 2 active detection kit of carboxy-lesterase in complex biological system according to claim 1
Method is suitable for the screening and detection of 2 inhibitor of carboxy-lesterase.
5. a kind of use measuring 2 active detection kit of carboxy-lesterase in complex biological system according to claim 1
Method is suitable for the discovery and detection of 2 derivant of carboxy-lesterase.
6. a kind of use measuring 2 active detection kit of carboxy-lesterase in complex biological system according to claim 1
Method is suitable for carboxy-lesterase 2- ligand interaction mechanisms and studies, including the binding site of ligand and carboxy-lesterase 2 judges.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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Application publication date: 20180713 |