CN108486220A - A kind of active method of catechol-chlB5 in detection blood - Google Patents
A kind of active method of catechol-chlB5 in detection blood Download PDFInfo
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- CN108486220A CN108486220A CN201810269583.6A CN201810269583A CN108486220A CN 108486220 A CN108486220 A CN 108486220A CN 201810269583 A CN201810269583 A CN 201810269583A CN 108486220 A CN108486220 A CN 108486220A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
- G01N2333/91011—Methyltransferases (general) (2.1.1.)
- G01N2333/91017—Methyltransferases (general) (2.1.1.) with definite EC number (2.1.1.-)
Abstract
The present invention relates to a kind of methods of catechol oxygen methyl transferase active in detection blood, belong to biomedicine technical field.The present invention is by high performance liquid chromatography and advantage of the fluoroscopic examination combined instrument in antibiont matrix interference and detection sensitivity, 3 benzothiazole, 7,8 dihydroxycoumarin for selecting chemical stability good（3‑BTD）For the fluorogenic substrate of COMT enzymes, in combination with high performance liquid chromatography detection technique of fluorescence, develop a kind of efficient, highly sensitive and can active assay methods of trace amounts of CO MT in quantitative analysis blood.Fluorescence probe substrate of the present invention can be obtained through chemical synthesis, and synthesis technology is simple and practicable and property is stablized.This method has many advantages, such as that high sensitivity, accuracy is high, antibiont matrix interference ability is strong, reproducible, it cannot be only used for the detection of COMT enzyme activity in blood, it can be additionally used in the quantitative determination of COMT enzyme activity and the screening and evaluation of COMT derivants/activator in the cell or tissue prepared product of various mammal sources.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of High Performance Liquid Chromatography with Fluorescence Detection is for examining
Survey the activity of catechol-chlB5 in blood.
Background technology
Catechol-oxygen-methylated transferase (Catechol-O-methyltransferase, COMT) is weight in human body
The II phase transfer enzyme of one kind wanted, is distributed widely in tissue such as liver, kidney, lung, mammary gland, in erythrocyte and brain.
COMT is responsible for scavenging capacity and the catechol of toxicity in human body, such as neurotransmitter dopamine, adrenaline and goes first
Adrenaline and catechol estrogen class compound such as estradiol etc..In addition, COMT enzymes also participate in exogenous pyrocatechol
Drug（Such as carbidopa, benserazide, apomorphine, dobutamine, fenoldopam, Alpha-Methyl-L-DOPA, isoproterenol
Element and Rimiterol etc.）Metabolite clearance.
COMT enzymes are in gene pleiomorphism in human body, and low activity (COMT is shown as respectively according to its genotype differenceLL), it is medium
Activity (COMTLH) and high activity (COMTHH), there is the difference of 4 times of highest between low activity and high activity.And a large amount of clinical samples
Analysis is it has proven convenient that COMT is horizontal in tissue and schizophrenia, breast cancer etc. have higher correlation.Clinical practice is
It confirms, the quantitative determination of COMT contents for the early diagnosis of clinically relevant disease and having tracked for disease more afterwards in human body
Highly important reference value.In addition, an important target spot of COMT or treatment of Parkinson disease, using probe reaction and by
In vitro metabolism incubation system can be high-throughput screening and evaluation COMT inhibitor, for the discovery meaning of Antiparkison Drugs
It is great.Meanwhile the disturbances in patients with Parkinson disease with high activity COMT, in levodopa treatment, " on-off " fluctuation effect becomes apparent from, because
This COMT activity height Quantitative evaluation has important directive function for the dosage adjustment of levodopa in different patients.
Currently, the probe substrate of common COMT enzymes mainly has levodopa, the catechols such as dopamine and adrenaline
Aminated compounds, but the chemical stability of these substrates is poor, needs that reducing agent is added and is protected from light operation, and needs to use
Mass-spectrometric technique completes COMT Activity determinations micro in biological sample.The present invention is directed to this problem, is being detected by fluorescence analysis
Advantage on sensitive, the 3- benzothiazoles-Daphnelin stablized with property（3-BTD）For the fluorescence probe bottom of COMT
Object develops a kind of efficient, highly sensitive and can be in quantitative analysis blood in combination with high performance liquid chromatography-fluorescence technology
Trace amounts of CO MT activity determination methods.
Invention content
The purpose of the present invention is intended to overcome prior art defect, provides a kind of efficient, highly sensitive and can quantitative analysis blood
In the micro active method of catechol O-methyltransferase.Specifically with 3- benzothiazole -7,8- dihydroxycoumarins（3-
BTD）For the fluorescence probe substrate of COMT, catechol-in blood is detected using High Performance Liquid Chromatography with Fluorescence Detection high sensitivity
The active method of chlB5.
Technical scheme of the present invention：The active side of catechol-chlB5 in a kind of highly sensitive detection blood
Method, it is characterised in that this method is combined using high performance liquid chromatography and fluoroscopic examination and is analyzed, comprise the steps of for：
（1）Sample pretreatment：
Appropriate anticoagulation adds normal saline, mixing, 2000 r/min to centrifuge 5min, remove supernatant;Appropriate red blood cell is taken to add 9
Volumes of deionized water, shakes mixing, and 3000 r/min centrifugations are spare.
（2）The establishment of high performance liquid chromatography-fluorescence analysis condition：
Detecting system includes efficient liquid phase system and fluorescence detector two parts.Liquid phase systems of the present invention are Shimadzu
20A XR systems, configured with system controller CBM-20A, two LC-20AD pumps, CTO-20A column ovens, autosampler SIL-
20ACHT, a DGU-20A3 vacuum degassing machine.Fluorescence detector is RF-20A xs fluorescence detectors.Analysis condition is as follows：
Analytical column is Hedera C18 reverse-phase chromatographic columns（150.0 mm×2.1 mm, 3 μm）, column oven temperature is set as 40 °C.Flowing
Phase A is acetonitrile, and B is water（Containing 0.2% formic acid）, flow velocity is 0.4 ml/min.Condition of gradient elution is：0-10.0 min, 90%
B-5% B; 10.0-13.0 min, 5% B;16.0 min of 13.0-, equilibrate to 90% B.Fluoroscopic examination condition is：Excitation
390 nm of optical wavelength, launch wavelength are 520 nm.
（3）Titer configures and Specification Curve of Increasing：
With 3- benzothiazoles -7-hydroxy-8-methoxycoumarin（3-BTMD）For standard items, series concentration is accurately configured, with it
Fluorescent value maps to standard concentration, calculates regression equation y=a*x+b, obtains standard curve.
（4）COMT determinations of activity in sample
With 3- benzothiazole -7,8- dihydroxycoumarins（3-BTD）For COMT methylation reaction probe substrates, COMT enzyme reactions exist
It is carried out in the 50 mM Tris-HCl buffer solutions of pH 7.4, contains 10 μ L of sample to be tested and 5 mM magnesium chlorides（MgCl2）, 2
MM dithiothreitol (DTT)s（DTT）, 0.2 mM S-adenosylmethionines（SAM）, overall reaction system volume is 0.2 mL.37 °C of items
It after incubating 3 min in advance under part, is originated and is reacted with SAM, after being incubated 10 min, reaction is terminated by the way that 200 μ l acetonitriles are added.Abundant whirlpool
Supination centrifuges 20 minutes under 4 °C under the conditions of 20000 × g, and supernatant carries out high performance liquid chromatography-fluorescence analysis, will
The fluorescent value of sample to be tested substitutes into the activity of standard curve conversion COMT enzymes.
It is provided by the present invention to obtain in technical solution, with 3- benzothiazoles-Daphnelin（3-BTD）For probe bottom
The active principles of analyte detection COMT are as shown in Fig. 1, which can be catalyzed by COMT enzyme specificities and generate 8- methoxy based products（3-
BTMD）And under 390nm excitations fluorescence signal is shown at 520 nm, it can be quantitative determined by the variation of fluorescence intensity
The activity of COMT.
In technical solution provided by the present invention, sample determination condition：The concentration of probe substrate is between 1/10 ~ 10 Km
Between, preferentially select Km；The pH of incubation system is between 6.5 ~ 10.5, and preferably 7.4；Reaction temperature between 20 ~ 60 DEG C,
It is preferred that 37 DEG C；5 ~ 30 min of reaction time, preferably 10 min.
The active method of catechol-chlB5 in a kind of highly sensitive detection blood provided by the present invention,
It is further characterized in that this method can be additionally used in various recombinant C OMT, COMT in the cell or tissue prepared product of various mammal sources
The quantitative determination of enzyme activity.
The active method of catechol-chlB5 in a kind of highly sensitive detection blood provided by the present invention,
It is characterized in that this method can be additionally used in screening and inhibition or the quantitative assessment of inducibility of COMT enzyme inducers or activator.
Beneficial effects of the present invention：Probe substrate 3-BTD in the present invention is off-on type fluorescence probes, can be by COMT high
It is selectively metabolized to a metabolite, and there is good fluorescence emission spectral property, passes through off-on type standard curves
Foundation quantitative determine 8-O- methylates, meanwhile, so that it is not easy in COMT Activity determination processes by high performance liquid chromatography
It is interfered by biosystem matrix and impurity, the Monitoring lower-cut of COMT enzymes is made to reach 10 ng/L, can be good at realizing biology
Micro COMT Activity determinations in sample can especially avoid the interference of ferroheme and hemoglobin etc., realize blood sample
In micro COMT Activity determinations.In addition, 3- benzothiazoles-Daphnelin can be obtained through chemical synthesis, work is synthesized
Skill is simple and practicable and property is stablized.It has a clear superiority compared with the method for detection COMT in the past, this method, which has, preferably may be used
By property and selectivity, and has the characteristics that hypersensitivity.In addition, the method can be additionally used in various recombinant C OMT, the various food in one's mouths
The quantitative determination of COMT enzyme activity in the cell or tissue prepared product of newborn animal origin.
Description of the drawings
Fig. 1 3- benzothiazole -7,8- dihydroxycoumarins detect COMT active principle figures；
Fig. 2 3- benzothiazole -7,8- dihydroxycoumarins1H-NMR spectrum；
Fig. 3 3- benzothiazole -7,8- dihydroxycoumarins13C-NMR spectrograms；
Fig. 4 3- benzothiazole -7,8- dihydroxycoumarins are inhaled with 3- benzothiazoles -7-hydroxy-8-methoxycoumarin fluorescence
Receive spectrogram；
Fig. 5 3- benzothiazole -7,8- dihydroxycoumarins are sent out with 3- benzothiazoles -7-hydroxy-8-methoxycoumarin fluorescence
Penetrate spectrogram；
Fig. 6 quantitation curves figures
COMT activity in 10 blood samples of Fig. 7；
COMT activity and COMT protein content correlation analysis figures in 10 blood samples of Fig. 8.
Specific implementation mode
The following examples will be further described the present invention, but not thereby limiting the invention.
Embodiment 1
The synthesis of 3- benzothiazole -7,8- dihydroxycoumarins：It weighs 1.0 g of 2,3,4- tri hydroxybenzaldehydes and is dissolved in methanol 25
In mL, 2- (2-[4-morpholinodithio) ethyl acetate 1.5 mL is added, 0.1 mL of piperidines is stirred at room temperature after twenty minutes, is heated to 65 DEG C,
4 h are reacted, TLC detection reactions terminate.After reaction solution is cooled down, 10 mL of water is added, filter cake, as crude product are collected in filtering.It will
Acetonitrile 20 mL reflux 1h are added in crude product, and cooled and filtered is dried in vacuo to obtain product 3- benzothiazoles-Daphnelin
1.8 g, yellow-brown solid, yield 88%.1H NMR spectras and13C NMR spectras are as shown in Figures 2 and 3；
1H NMR (400 MHz, DMSO-d 6) δ: 10.59 (s, 1H), 9.68 (s, 1H), 9.11 (s, 1H),
8.16 (d, J = 7.5 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.56 (td, J=7.2 Hz, J=1.2
Hz, 1H), 7.45 (td, J=8.0 Hz, J=0.8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.95
(d, J = 8.5 Hz, 1H).
13C NMR (100 MHz, DMSO-d 6) δ: 112.7, 114.2, 114.7, 122.0, 122.5, 122.6,
125.4, 126.9, 132.6, 136.1, 143.7, 144.1, 152.5, 160.2, 161.0;
ESI-MS:M/z 310.1 is [M-H]。
Embodiment 2.
The measurement of substrate and product fluorescent absorption and emission spectrum:Accurately weigh 3- benzothiazole -7,8- dihydroxycoumarins simultaneously
Be dissolved in chromatographic grade dimethyl sulfoxide solution, be configured to the standard mother liquor of 50 mmol/L, then with the acetonitrile solution of 0.5% formic acid with go
Ionized water（1:1）It is diluted to the titer of final concentration of 1.0 mmol/L, it is glimmering using its is measured using Synergy H1 microplate reader
Light absorption and launching light spectrogram.3- benzothiazoles -7-hydroxy-8-methoxycoumarin is configured to same concentrations using same method
Titer, using using using Synergy H1 microplate reader measuring its fluorescent absorption and launching light spectrogram.Benzothiazole -7 3-,
8- dihydroxycoumarins and 3- benzothiazoles -7-hydroxy-8-methoxycoumarin fluorescent absorption and launching light spectrogram such as attached drawing 4 with
Shown in attached drawing 5.
Embodiment 3.
The establishment of high performance liquid chromatography-fluorescence analysis condition：Liquid phase systems Shimadzu 20A XR systems are controlled configured with system
Device CBM-20A, two LC-20AD pumps, CTO-20A column ovens, autosampler SIL-20ACHT, a DGU-20A3 vacuum are de-
Mechanism of qi, a RF-20A xs fluorescence detector.Analytical column is Hedera C18 reverse-phase chromatographic columns（150.0 mm×2.1 mm,
3 μm）, column oven temperature is set as 40 °C.Mobile phase A is acetonitrile, and B is water（Containing 0.2% formic acid）, flow velocity is 0.4 ml/min.Ladder
Spending elution requirement is：0-10.0 min, 90% B-5% B; 10.0-13.0 min, 5% B;16.0 min of 13.0- are put down
It weighs to 90% B.Fluoroscopic examination condition is：390 nm of excitation wavelength, launch wavelength are 520 nm.
Embodiment 4.
Titer configures and Specification Curve of Increasing：Precision weighs standard reference material 3- benzothiazole -7- hydroxyl -8- methoxyl group tonka-beans
Element（3-BTMD）And it is dissolved in chromatographic grade dimethyl sulfoxide solution, it is configured to the standard mother liquor of 50 mmol/L.Again with 0.5% formic acid
Acetonitrile solution and deionized water（1:1）Be diluted to final concentration of 500,250,100,50,25,10,5,2.5,1.0 nmol/L
Titer sample introduction, each sample are repeated 3 times, and linear regression are carried out to concentration with peak area, as a result in 1.0-500 nmol/L
In the range of linear relationship it is good, regression equation be Y=37227x-101967, coefficient R2=0.998, P<0.0001, it is such as attached
Shown in Fig. 6.
Embodiment 5.
COMT determinations of activity in blood:200 μ L of anticoagulation add normal saline, mixing, 2000 r/min to centrifuge 5min,
Remove supernatant;It takes 10 μ L of red blood cell to add 90 μ L deionized waters, shakes mixing, 3000 r/min centrifugations.Take 10 μ of sample to be tested
L is added in the 50 mM Tris-HCl buffer solutions of pH 7.4 and carries out, and is configured to contain final concentration of 5 mM magnesium chlorides
（MgCl2）, 2 mM dithiothreitol (DTT)s（DTT）, 0.2 mM S-adenosylmethionines（SAM）End reaction liquid, overall reaction system
Volume is 0.2 mL.It after incubating 3 min in advance under the conditions of 37 °C, is originated and is reacted with SAM, after being incubated 10 min, by the way that 200 μ l are added
Acetonitrile terminates reaction.It after being fully vortexed, is centrifuged 20 minutes under the conditions of 20000 × g under 4 °C, supernatant carries out high-efficient liquid phase color
The fluorescent value of sample to be tested is substituted into the activity of standard curve conversion COMT enzymes by spectrum-fluorimetric analysis.In 10 blood
COMT activity is as shown in Fig. 7.
Embodiment 6.
COMT activity and COMT protein content correlation analysis in blood：Using commercially available COMT ELISA kits to blood
COMT contents are measured in sample.Kit operating process of the operation with reference to purchase.First, add diluted measuring samples 0.1
Blank well and negative control hole is arranged in being coated in reacting hole in ml, and 37 DEG C are incubated 1 hour.It is new that 0.1 ml is added in washing
In each reacting hole, 37 DEG C are incubated 0.5~1 hour fresh diluted enzyme labelled antibody.The bottoms TMB that 0.1 ml is newly prepared are added in washing
Object solution, 37 DEG C 10~30 minutes.0.05 ml, 2 M sulfuric acid is added and terminates reaction.In 450 nm of microplate reader, (ABTS colour developings are selected
410 nm) blank control wells zeroing is sentenced, each hole sample OD values are detected, standard curve is done, calculate the content of COMT in sample.With
GraphPad Prism 6 (GraphPad Software, San Diego, CA) map, to COMT activity in blood sample
It is analyzed with COMT protein content correlations, analysis result is as shown in Fig. 8, correlation R2Reach 0.91, illustrates this patent
Method can be good at measuring COMT activity in blood.
Claims (4)
1. a kind of active method of catechol-chlB5 in detection blood, it is characterised in that this method uses efficient liquid
Phase chromatography analyzes target enzyme with fluoroscopic examination combination method, and detecting step is as follows：
（1）Sample pre-treatments：
Appropriate anticoagulation adds normal saline, mixing, 2000 r/min to centrifuge 5min, remove supernatant;Appropriate red blood cell is taken to add 9
Volumes of deionized water, shakes mixing, and 3000 r/min centrifugations are spare；
（2）The establishment of high performance liquid chromatography-fluorescence analysis condition：
Detecting system includes efficient liquid phase system and fluorescence detector two parts；Analysis condition is as follows：Analytical column is Hedera
C18 reverse-phase chromatographic columns（150.0 mm×2.1 mm, 3 μm）, column oven temperature is set as 40 °C；Mobile phase A is acetonitrile, and B is water
（Containing 0.2% formic acid）, flow velocity is 0.4 ml/min, and condition of gradient elution is：0-10.0 min, 90% B-5% B; 10.0-
13.0 min, 5% B;16.0 min of 13.0-, equilibrate to 90% B, and fluoroscopic examination condition is：390 nm of excitation wavelength,
Launch wavelength is 520 nm；
（3）Titer configures and Specification Curve of Increasing：
With 3- benzothiazoles -7-hydroxy-8-methoxycoumarin（3-BTMD）For standard items, series concentration is accurately configured, with it
Fluorescent value maps to standard concentration, calculates regression equation y=a*x+b, obtains standard curve；
（4）COMT determinations of activity in sample
With 3- benzothiazole -7,8- dihydroxycoumarins（3-BTD）For COMT methylation reaction probe substrates, COMT enzyme reactions exist
It is carried out in the 50 mM Tris-HCl buffer solutions of pH 7.4, contains 10 μ L of sample to be tested and 5 mM magnesium chlorides（MgCl2）, 2
MM dithiothreitol (DTT)s（DTT）, 0.2 mM S-adenosylmethionines（SAM）, overall reaction system volume is 0.2 mL, 37 °C of conditions
It after incubating 3 min in advance down, is originated and is reacted with SAM, after being incubated 10 min, terminated reaction by the way that 200 μ l acetonitriles are added, be fully vortexed
Afterwards, it is centrifuged 20 minutes under the conditions of 20000 × g under 4 °C, supernatant carries out high performance liquid chromatography-fluorescence analysis, will wait for
The fluorescent value of test sample sheet substitutes into the activity of standard curve conversion COMT enzymes.
2. the active method of catechol-chlB5 in a kind of highly sensitive detection blood as described in claim 1,
It is further characterized in that in its sample determination condition：The concentration of probe substrate is between 1/10 ~ 10K mBetween, it is preferential to selectK m；It is incubated
The pH of system is between 6.5 ~ 10.5, and preferably 7.4；Reaction temperature is between 20 ~ 60 DEG C, preferably 37 DEG C；Reaction time 5 ~ 30
Min, preferably 10 min.
3. the active method of catechol-chlB5 in a kind of highly sensitive detection blood as described in claim 1,
It is further characterized in that this method can be additionally used in various recombinant C OMT, in the cell or tissue prepared product of various mammal sources
The quantitative determination of COMT enzyme activity.
4. the active method of catechol-chlB5 in a kind of highly sensitive detection blood as described in claim 1,
It is characterized in that this method can be additionally used in screening and inhibition or the quantitative assessment of inducibility of COMT enzyme inducers or activator.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110438101A (en) * | 2019-08-28 | 2019-11-12 | 四川农业大学 | A kind of isoflavones O- transmethylase GmIOMT3 and encoding gene and application |
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| CN105624268A (en) * | 2014-11-05 | 2016-06-01 | 中国科学院大连化学物理研究所 | Method for quantitative determination of catechol-o-methyl transferase activity and use thereof |
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| CN110438101A (en) * | 2019-08-28 | 2019-11-12 | 四川农业大学 | A kind of isoflavones O- transmethylase GmIOMT3 and encoding gene and application |
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