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A method for quantitatively measuring catechol-o-methyltransferase activity and its application

A technology for quantitative determination of methyltransferase, applied in the field of quantitative determination of catechol-O-methyltransferase activity, can solve the problems of inconvenient quantitative detection, difficult separation by liquid chromatography, high cost, etc., and achieve good fluorescence emission The effect of spectral characteristics, simple synthesis process, and low detection cost

Inactive Publication Date: 2019-03-15
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

High-throughput detection of samples is difficult due to expensive chromatography or mass spectrometry methods and long sample analysis times
In addition, the probe substrates used in the above detection methods, including protocatechuic acid, generate two monomethylated products under the catalysis of mammalian COMT enzyme, and the two products are similar in structure and physical and chemical properties, and can be detected by liquid chromatography. It is usually difficult to separate, which brings inconvenience to its quantitative detection

Method used

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  • A method for quantitatively measuring catechol-o-methyltransferase activity and its application
  • A method for quantitatively measuring catechol-o-methyltransferase activity and its application
  • A method for quantitatively measuring catechol-o-methyltransferase activity and its application

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Experimental program
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Effect test

Embodiment 1

[0038] Synthesis of 4-Acetate-7-Hydroxy-8-Methoxycoumarin

[0039] The synthetic route of 4-acetic acid-7-hydroxyl-8-methoxycoumarin is as follows Figure 8 As shown, weigh 0.5g of 2-methoxyresorcinol and place it in a three-necked flask, add 0.6g of acetone-1,3-malonic acid, 1.5ml of perchloric acid, stir at room temperature for 20 minutes, and heat to 55 °C, react for 3 hours, TLC detects that the reaction is complete, the reaction solution is directly filtered, and the filter cake is collected, which is the crude product. The crude product was subjected to silica gel column chromatography to obtain 150 mg of 4-acetic acid-7-hydroxy-8-methoxycoumarin as a white solid. Structural formula such as figure 1 show its 1 H-NMR spectrum and 13 C-NMR spectrum as figure 2 and image 3 shown;

[0040] 1 H NMR (500MHz, d-DMSO) δ: 3.31(s, 2H, CH2), 3.82(s, 3H, OCH3), 6.22(s, 1H, COCH=C), 6.89(d, J=3Hz, 1H, ArH), 7.34 (d, J=8Hz, 1H, ArH);

[0041] 13 C NMR (500MHz, d-DMSO) δ:...

Embodiment 2

[0043] Quantitative assessment of COMT activity in liver microsomes from different individual sources

[0044] (1) Select 12 cases of human liver microsomes (HLM) and dilute them to 10 mg / ml to prepare the COMT metabolic reaction system, including Tris-HCl buffer (50 mM) at pH 7.4, human liver microsomes (0.5 mg / ml), Dithiothreitol 40mM, MgCl 2 50mM, the final concentration of 4-acetic acid-7,8-dihydroxycoumarin is 5μM, pre-incubated with shaking for 3 minutes at 37°C;

[0045] (2) Add 10 μl of SAM with a concentration of 4 mM to the reaction system to initiate the reaction;

[0046] (3) After 15 minutes, add 200 μl of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0047] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant, perform fluorescence detection (Ex=320nm, Em=520nm), and substitute the obtained fluorescence intensity into the standard curve Afterwards, the metabol...

Embodiment 3

[0049] Determination of the lower limit of detection of COMT in vitro

[0050] The experiment was carried out on a microplate reader using a 96-well plate, 4-acetate-7,8-dihydroxycoumarin 5μM, S-adenosylmethionine 200μM, dithiothreitol 2mM, MgCl 2 5mM, COMT single enzyme 5ng / ml~100ng / ml, pH 7.4 Tris-HCl buffer 50mM, the total volume is 100μL, incubated at 37℃ for 1h and analyzed by microplate reader, the average value of each group is the same as that without COMT Comparison of the control group showed that 30 and 50ng of COMT were statistically significant (P<0.05), and the lower limit of detection of COMT was determined to be 30ng.

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Abstract

A method for quantitatively measuring catechol-O-methyltransferase activity and application thereof belong to the technical field of biomedicine. The method uses 4-acetic acid-7,8-dihydroxycoumarin as the specific probe substrate of COMT enzyme, which can be specifically catalyzed by COMT enzyme to generate 8-methoxy product and display at 520nm Fluorescence signal, the activity of COMT can be quantitatively determined by detecting the change of fluorescence intensity. The invention can not only be used for quantitative evaluation of COMT enzyme activity in biological samples from different sources, but also can be used for rapid screening of COMT inhibitors in vitro and evaluation of their inhibitory ability. The method can be used to quantitatively evaluate the real activity of COMT enzymes in various in vitro biological samples, realize rapid screening of inhibitors and quantitative evaluation of inhibitory ability; it can be used to evaluate the catalytic activity of different species of COMT and COMT mutants with different amino acid sequences , and then evaluate its ability to metabolize catechols. The method of the invention has the advantages of (1) high selectivity, (2) cheap and easy to obtain; (3) high sensitivity and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for quantitatively measuring the activity of catechol-O-methyltransferase and its application. Background technique [0002] Catechol-O-methyltransferase (COMT) is a methyltransferase widely distributed in mammals, and its main physiological function is to be responsible for the metabolism of endogenous neurotransmitters (such as epinephrine, norepinephrine hormones and dopamine, etc.) and exogenous catechol drugs (such as carbidopa, benserazide, apomorphine, dobutamine, fenoldopam, α-methyl-L-DOPA, isoproterenol and rimeterol, etc.). In the human body, COMT enzyme exists in two forms, one is soluble COMT (S-COMT) and the other is membrane-bound COMT (MB-COMT). COMT is distributed in various tissues of the human body, such as kidney, liver, lung, etc. In the human brain, it mainly exists in postsynaptic neurons. In most tissues of the human body, the a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/48
Inventor 杨凌夏杨柳葛广波王平钱星凯窦同意
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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