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Universal sulfo-group transferase activity analysis method

An activity analysis and transferase technology, applied in the field of fluorescence analysis and sulfotransferase activity analysis, can solve the problems of unsuitable sulfonation product separation and detection, time-consuming, complicated operation, etc., so as to overcome radioactive hazards and avoid inhibition. , the effect of good general applicability

Active Publication Date: 2017-11-17
SHAANXI NORMAL UNIV
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  • Description
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Problems solved by technology

However, this method has many disadvantages: cumbersome operation, time-consuming, radioactive contamination, etc., and most importantly, poor versatility. For example, gel electrophoresis is suitable for sulfotransferase activity analysis such as TPST, which uses proteins or polypeptides as exclusive substrates. But it is not suitable for the separation and detection of sulfonated products of substrates such as polysaccharides and small molecules
In addition to radioactive labeling, there are currently some reports using colorimetric and fluorescent methods to measure the activity of sulfotransferases, but these methods are limited to the significant changes in the absorption spectrum or fluorescence properties of the substrate before and after sulfonation
And in these methods, the gradual accumulation of by-product 3'-phosphate adenosine-5'-phosphate in the sulfonation reaction process can produce obvious inhibitory effect on the activity of sulfotransferase, and the accuracy of sulfotransferase activity detection have the inevitable impact

Method used

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Examples

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Embodiment 1

[0024] Taking the detection of sulfotransferase SULT1A3 activity as an example, the specific detection method is as follows:

[0025] 1. 2 μL 100 μmol / L 3'-phosphoadenosine-5'-phosphorylsulfuric acid aqueous solution, 2 μL 1 mmol / L dopamine aqueous solution and sulfotransferase SULT1A3 solution (the enzyme activity of 150 pmol / min / μg sulfotransferase SULT1A3 Diluted with 10mmol / L pH value of Tris-HCl buffer solution of 7.5) dispersed in 10mmol / L pH value of Tris-HCl buffer solution of 7.5, the total volume is 10 μ L, so that the concentration of sulfotransferase SULT1A3 in the mixed system is respectively 0, 0.05, 0.075, 0.1, 0.25, 0.5, 0.65, 0.8 ng / μL, incubate on a constant temperature shaker at 37°C for 20 minutes to carry out sulfonation reaction.

[0026] 2. Add 2 μL 20ng / μL IMPAD1 enzyme and 10mmol / L Tris-HCL buffer solution (containing 2mmol / L MgCl 2 ), with a final volume of 20 μL, incubated at 37°C for 20 minutes to carry out the hydrolysis reaction of 3’-phosphate a...

Embodiment 2

[0030]Taking the detection of sulfotransferase SULT1A1 activity as an example, the specific detection method is as follows:

[0031] 1. 2 μL 100 μmol / L 3'-phosphoadenosine-5'-phosphorylsulfuric acid aqueous solution, 2 μL 1mmol / L SULT1A1 specific substrate 2-naphthol and sulfotransferase SULT1A1 solution (enzyme activity is 30pmol / min / μg of sulfotransferase SULT1A1 diluted with 10mmol / L Tris-HCl buffer solution with a pH value of 7.5) was dispersed in a 10mmol / L Tris-HCl buffer solution with a pH value of 7.5, and the total volume was 10μL, so that the sulfo group in the mixed system The concentrations of the transferase SULT1A3 were 0, 0.2, 0.4, 1, 2 ng / μL, and incubated on a constant temperature shaker at 37° C. for 20 minutes to carry out sulfonation reaction.

[0032] 2. Add 2 μL 20ng / μL IMPAD1 enzyme and 10mmol / L Tris-HCL buffer solution (containing 2mmol / L MgCl 2 ), with a final volume of 20 μL, incubated at 37°C for 20 minutes to carry out the hydrolysis reaction of 3...

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Abstract

The invention discloses a universal sulfo-group transferase activity analysis method. According to the method, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), sulfo-group transferase and a sulfo-group transferase specific substrate are mixed to be subjected to a sulfonation reaction to generate 3'-phosphoadenosine-phosphate, an IMPAD1 enzyme can specifically hydrolyze 3'-phosphoadenosine-5'-phosphosulfate to release a phosphate group (Pi), and Pi is subjected to fluorogenic quantitative detection through a mixed system composed of metal ions (Mn+) which has a high combining capacity with Pi and a specific fluorescent probe (FP), and quantitative analysis of an sulfo-group transferase activity can be achieved. According to the system, the combination of Mn+ and FP causes that a fluorescence system is effectively quenched, Pi generated in the reaction process effectively shields Mn+, therefore the fluorescence signal of FP is restored. Therefore, by monitoring the change situation of the fluorescence signal in the system, fluorescence reinforced sensing of the sulfo-group transferase activity can be achieved. The universal sulfo-group transferase activity analysis method is generally applicable to detection of the sulfo-group transferase activity with PASA being a sulfonic group donor.

Description

technical field [0001] The invention belongs to the technical field of biologically active molecules / protease detection, and specifically relates to a method for quantitatively detecting phosphate radicals produced by the hydrolysis of 3'-phosphate adenosine-5'-phosphate, a by-product of the sulfonation reaction process, so as to realize sulfotransfer Fluorometric assay method for enzyme activity analysis. Background technique [0002] The sulfonation modification process of intracellular proteins and other biomolecules is an extremely important modification mechanism that affects the biological activities of proteins and various small molecules. The sulfonation process in organisms occurs under the catalysis of sulfotransferase, and the sulfonic acid group on the sulfonic acid donor 3'-phosphoadenosine-5'-phosphorylsulfate is transferred to the substrate molecule to make the substrate Sulfonation, which is accompanied by the production of 3'-phosphoadenosine-5'-phosphate b...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 刘成辉李娅张语
Owner SHAANXI NORMAL UNIV
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