Monoamine oxidase activity fluorescent detecting method

A technology of monoamine oxidase activity and monoamine oxidase, which is applied in the field of fluorescence detection of monoamine oxidase activity, can solve problems such as complex preparation, and achieve the effects of simple equipment, simple operation process and stable properties

Inactive Publication Date: 2007-08-22
ZHEJIANG UNIV OF TECH
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Although there are some methods for detecting and analyzing monoamine oxidase through fluorescent probes, their

Method used

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  • Monoamine oxidase activity fluorescent detecting method
  • Monoamine oxidase activity fluorescent detecting method
  • Monoamine oxidase activity fluorescent detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Taking monoamine oxidase extracted from rat liver as an example, the prepared 7-(3-amino-propoxy)-coumarin was used as a fluorescent probe to detect monoamine oxidase activity:

[0027] Dissolve the test sample of monoamine oxidase extracted from rat liver in a 1ml EP tube, take 100uL and put it into 900uL 50mM pH8.4 boric acid buffer, place it in a constant temperature water bath shaker, preheat at 35°C for 5min, add 5μl 7-(3-amino-propoxy)-coumarin at a concentration of 10 mM was reacted with 2 μl of 1 g / mL BSA reaction solution. After the reaction, it was detected in a 96-well sieve plate with a fully functional fluorescence spectrophotometer.

[0028] Experiments have proved that as long as there is a small amount of monoamine oxidase (5-10mmol / L), the fluorescent probe can be efficiently hydrolyzed. The fluorescence intensity measured by the full-featured microplate detection system increases from 1000 to 20000. The change range is large and intuitive. The change ...

Embodiment 2

[0030] Using the same method as in Example 1, the following enzyme preparations are used to act on the substrate: lipase (EC3.1.1.3 Type VII from Candida rugosa, and hog pancreas Sinopharm Chemical Reagent Co., Ltd.), phosphatase (Phosphatase, Alkalinebovine ), multiple proteases (Protease from Aspergillus oryzae, Protease from Aspergillus sojae, Protease from bovine pancreas) and a compound enzyme (BS-031 Jiangmen Lianjiang District Biochemical Factory), the results showed that only monoamine oxidase showed strong fluorescence , the results are shown in Figure 1, only monoamine oxidase can convert the substrate, which also shows that the monoamine oxidase we extracted is responsible for the hydrolysis, while other enzymes have no effect on the substrate.

Embodiment 3

[0032] Take 100 μl of boric acid buffer solution with pH 8.4, add 15 μl of monoamine oxidase extract solution, 2 μl of 0.1 mM monoamine oxidase inhibitor—hydroxylamine hydrochloride solution, place in a constant temperature water bath shaker at 35°C, and after 30 minutes, add according to the amount in Example 1 7-(3-Amino-propoxy)-coumarin and BSA solution react, after the reaction, use a full-featured fluorescence spectrophotometer to detect in a 96-well sieve plate in a full-featured fluorescence spectrophotometer, and detect fluorescence in real time strength.

[0033] Experiments have proved that after the activity of monoamine oxidase is inhibited by hydroxylamine hydrochloride, no activity can be detected with our prepared probe, and the results are shown in Figure 2.

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Abstract

The invention relates to a fluorescent testing method of monoamine oxidase activity. It forms fluorescent material, detects fluorescent intensity and achieves monoamine oxidase activity data by using monoamine oxidase to hydrolyze fluorescent probe. The probe is coumarin compound and its structure as shown in (I), in which R1 and R2 are alkyl of H or C1-C6 independently. Preparation of probe in this invention just needs simple device, convenient operation and easily gained materials. The prepared probe has stable property and high purity (above 99%). Compared to the current method for testing monoamine oxidase activity, its accuracy, sensitivity and testing velocity are all improved.

Description

(1) Technical field [0001] The invention relates to a fluorescence detection method of monoamine oxidase activity, in particular to a fluorescence detection method of monoamine oxidase activity using coumarin compounds as fluorescent probes. (2) Background technology [0002] Monoamine oxidase is a type of mitochondrial membrane-bound enzyme, which catalyzes the oxidative deamination of amine compounds in organisms to generate corresponding aldehydes in the brain and peripheral nervous tissue. Studies have shown that during the process of metabolizing dopamine, monoamine oxidase produces endogenous substances such as free radicals and hydrogen peroxide, and its abnormal expression is closely related to Parkinson's disease and depression. In addition, the determination and analysis of monoamine oxidase activity in the blood of patients is of great value for the early diagnosis of acute and chronic hepatitis and liver cirrhosis. Therefore, finding a highly sensitive detection...

Claims

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Application Information

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IPC IPC(8): G01N21/00C12Q1/26
Inventor 朱勍陆优优戴斌戴一琦
Owner ZHEJIANG UNIV OF TECH
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