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Enzyme activity detection kit for alpha-oxoglutarate dependent enzyme and application thereof

A technology for detection of ketoglutaric acid and enzyme activity, which is applied in the field of biotechnology detection, and can solve problems such as cumbersome operation, inapplicability, and inapplicability of accurate quantitative determination

Pending Publication Date: 2019-06-25
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] However, although the method involves 1 step of enzymatic cascade reaction, there are at least 4 independent steps, and the operation is cumbersome.
More importantly, this method involves the color reaction of dyes to phosphoric acid, which will lead to low precision and sensitivity of the method, and poor reproducibility
Therefore, this reaction is not suitable for accurate quantitative determination of enzymatic activity and kinetic parameters of this type of enzyme, nor is it suitable for high-throughput drug screening

Method used

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  • Enzyme activity detection kit for alpha-oxoglutarate dependent enzyme and application thereof
  • Enzyme activity detection kit for alpha-oxoglutarate dependent enzyme and application thereof
  • Enzyme activity detection kit for alpha-oxoglutarate dependent enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. Detection of α-ketoglutarate content by isocitrate dehydrogenase (IDH) mutants

[0068] 1. α-ketoglutarate-dependent enzyme enzyme activity detection kit

[0069] The kit includes one of reduced coenzyme II (NADPH) or reduced coenzyme I (NADH), isocitrate dehydrogenase IDH mutant protein and buffer salt, and the IDH mutant protein refers to the specific amino acid mutation of IDH The obtained IDH mutant protein with the novel function of catalyzing α-ketoglutarate.

[0070] As a preferred solution, the kit also includes metal ions that compete for ferrous ions, and the metal ions that compete for ferrous ions include manganese ions, magnesium ions, calcium ions, cobalt ions, nickel ions, copper ions, zinc ions and One or several metal ions of its congener metals. In the preferred scheme, metal ions that compete with ferrous ions are added to ensure that the autocatalytic reaction of the α-ketoglutarate-dependent enzyme has been terminated when the enzyme ac...

Embodiment 2

[0093] Example 2. Taking ferrous iron and the representative enzyme TET (ten-eleventranslocation) protein of the α-ketoglutarate-dependent dioxygenase family as an example, using the isocitrate dehydrogenase mutant enzyme-linked method to detect human Enzyme activity of TET2 catalytic domain (hTET2-CD) and determination of its enzyme kinetic parameters.

[0094] The specific content includes:

[0095] 1. hTET2-CD / IDH-1(R132H) enzyme-linked method to detect the enzymatic activity of hTET2-CD catalytic domain

[0096] ①React TET and substrate DNA in enzyme activity reaction buffer at 37 degrees for one hour.

[0097] ② Add the reaction reagent to the system after the reaction in step ①, and detect the absorption change of NADPH at 340nm with a microplate reader at room temperature.

[0098] Such as image 3 As shown, we use this method to detect the α-ketoglutarate consumed by different concentrations of TET proteins.

[0099] 2. hTET2-CD / IDH-1(R132H) enzyme-linked method to d...

Embodiment 3

[0117] Example 3. Specific details of the optimization of the α-ketoglutarate catalyzed reaction

[0118] 1. Determination of the acquired catalytic activity of human IDH-1(R132H) and IDH-1(R132Q) on α-ketoglutarate according to the literature conditions:

[0119] The reaction system includes: HEPES, NaCl, DTT, MgCl 2 , NADPH, α-ketoglutarate, IDH-1(R132H) or IDH-1(R132Q) mutant protein. The change of the absorbance value at 340nm at 25°C was detected in a microplate reader. Figure 7 It is the 340nm absorption change reflected by the system under different pH conditions. As shown in the figure, IDH-1 mutants can exhibit higher activity under different pH conditions, and the activity of IDH-1(R132Q) is better than that of IDH-1(R132H).

[0120] 2. Metal ion optimization of the reaction system

[0121] The reaction of IDH itself relies on magnesium ions to achieve normal catalysis. In order to determine the enzyme kinetic parameters of α-ketoglutarate-dependent oxygenase, ...

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Abstract

The invention discloses an enzyme activity detection kit for alpha-oxoglutarate dependent enzyme and an application thereof. The kit includes one of reduced coenzyme II (NADPH) or reduced coenzyme I (NADH), an isocitrate dehydrogenase IDH mutant protein and buffer salt, and the IDH mutant protein refers to an IDH mutant protein having a novel function of catalyzing alpha-oxoglutarate (2-OG) obtained after mutation of a specific amino acid of IDH. As a preferred scheme, the kit further includes a metal ion that competes for ferrous ions. The invention provides a method for detecting the contentof the alpha-oxoglutarate with high efficiency and high precision, which can be used for detecting the enzymatic activity of alpha-oxoglutarate dependent enzyme, determining the enzyme kinetic parameters, and performing high-throughput drug screening by targeting the type of enzyme. The method solves the problems of complex operation, low accuracy, reproducibility, low flux, and difficulty in screening high-throughput drugs for the enzyme activity detection of the alpha-oxoglutarate dependent enzyme.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and more specifically relates to an enzyme activity detection kit for α-ketoglutarate-dependent enzymes and applications thereof. Background technique [0002] α-Ketoglutarate is an important metabolic intermediate in the tricarboxylic acid cycle and a key node connecting intracellular carbon-nitrogen metabolism. In this cycle, isocitrate dehydrogenase (IDH) catalyzes isocitrate to generate α-ketoglutarate (2-OG), and α-ketoglutarate is in turn α-ketoglutarate dehydrogenase ( OGDC) is catalyzed to generate succinyl-CoA to participate in downstream metabolism. In addition to being an important metabolite involved in the tricarboxylic acid cycle, α-ketoglutarate, as a key cofactor, also participates in a large number of important enzyme-catalyzed reactions. Among them, the most representative ones are ferrous and α-ketoglutarate-dependent dioxygenase family. This family belongs to non-heme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C12Q1/26
Inventor 张良罗静张会冰孟周文理
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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