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Vitamin K epoxide reductase in-vitro activity determination method and application thereof

A technology for the determination of epoxide and activity, applied in the field of biochemistry, can solve the problems of cumbersome operation, inability to detect activity in real time, inconvenient high-throughput measurement, etc., and achieve the effect of simple method steps, strong operability and small reaction system

Active Publication Date: 2021-07-20
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the traditional method is to measure the activity of VKOR protein by extracting microsomes. This method is cumbersome and inconvenient for high-throughput measurement. It can only detect the activity of catalytic KO, but cannot detect the activity of catalytic K, and the activity cannot be detected in real time (Fasco , M.J., Principe, L.M., Walsh, W.a & Friedman, P.a. Warfarin inhibition of vitamin K 2,3-epoxide reductase in rat liver microsomes. Biochemistry. 22, 5655–60 (1983); Rost, S., Fregin, A., Ivaskevicius, V., Conzelmann, E. & Hortnagel, K. Mutations in VKORC1 cause warfare resistance and multiple coagulationfactor deficiency type 2. Nature. 427, 537–541 (2004).)

Method used

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  • Vitamin K epoxide reductase in-vitro activity determination method and application thereof
  • Vitamin K epoxide reductase in-vitro activity determination method and application thereof
  • Vitamin K epoxide reductase in-vitro activity determination method and application thereof

Examples

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preparation example Construction

[0066] The preparation method of the purified human VKOR protein used in the examples and test examples is as follows: the gene encoding human VKOR protein (Gene ID: 79001) is cloned into the expression vector pPICZ-B (invitrogen) of Pichia pastoris, and its C-terminus is sequentially connected PreScission protease cleavage site and GFP-HIS10 tag; protein expression and purification were performed according to the kit (Product No. K171001, Thermofisher Company) and literature (Ren F, Logeman BL, Zhang X, Liu Y, Thiele DJ, YuanP. X-ray structures of The high-affinity copper transporter Ctr1.NatCommun.2019,27;10(1):1386.doi:10.1038 / s41467-019-09376-7.) was carried out by the method of expressing membrane protein in Pichia pastoris. The purification steps are as follows:

[0067] 1) Yeast cells expressing human VKOR protein were crushed and dissolved in lysate (2% DDM, 150mM NaCl, 50mM Tris-HCl pH 8.0, 10μg / mL DNase I, 2mM PMSF protease inhibitor, solvent is water), Stir and ext...

Embodiment 1

[0072] In this embodiment, the vitamin K epoxide reductase in vitro activity assay method comprises the following steps:

[0073] 1) Preparation of materials:

[0074] Prepare buffer 1, buffer 2, buffer 3 and buffer 4 respectively, and store them for later use;

[0075] The composition of the buffer solution 1 is: Tris-HCl 20mmol / L with a pH value of 7.5, NaCl 100mmol / L, LMNG1g / L, and the solvent is water;

[0076] The composition of the buffer solution 2 is: GSH 0.5mol / L, appropriate amount of NaOH, solvent is water; pH value is 7.5;

[0077] The composition of described buffer solution 3 is: vitamin K epoxide 1mmol / L, and solvent is isopropanol;

[0078] The composition of described buffer solution 4 is: vitamin K1mmol / L, and solvent is isopropanol;

[0079] 2) Preparation of working solution:

[0080] Prepare vitamin K epoxide working solution, vitamin K working solution and VKOR protein working solution respectively for use;

[0081] The preparation method of the vitami...

Embodiment 2

[0094] In this embodiment, the vitamin K epoxide reductase in vitro activity assay method comprises the following steps:

[0095] 1) Preparation of materials:

[0096] Prepare buffer 1, buffer 2, buffer 3 and buffer 4 respectively, and store them for later use;

[0097] The composition of the buffer solution 1 is: Tris-HCl 30mmol / L with a pH value of 7.0, NaCl 120mmol / L, LMNG0.5g / L, and the solvent is water;

[0098] The composition of the buffer solution 2 is: GSH 0.4mol / L, appropriate amount of NaOH, solvent is water; pH value is 7.0;

[0099] The composition of the buffer solution 3 includes: vitamin K epoxide 20mmol / L, and the solvent is isopropanol;

[0100] The composition of described buffer solution 4 comprises: vitamin K 50mmol / L, and solvent is isopropanol;

[0101] 2) Preparation of working solution:

[0102] Take buffer 1, buffer 2 and buffer 3 respectively, and prepare a vitamin K epoxide working solution with a GSH concentration of 60 mmol / L and a vitamin K e...

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Abstract

The invention discloses a vitamin K epoxide reductase in-vitro activity determination method and application thereof, and belongs to the technical field of biochemistry. In a vitamin K cycle, gamma-glutamate carboxylase (GGCX) catalyzes glutamic acid residue gamma carboxylation of a blood coagulation factor Gla structural domain by utilizing dihydrovitamin K (KH2) as a cofactor, and meanwhile, KH2 is converted into vitamin K epoxide (KO); and vitamin K epoxide reductase (VKOR) catalyzes KO to K and K to KH2, so that vitamin K can be recycled. According to the detection principle, the activity of the VKOR protein is reflected by detecting the generation amount of the product KH2 according to the characteristic that the product KH2 can generate fluorescence under 250nm exciting light. The method is simple in step and high in operability, and compared with a traditional microsome method, the method can rapidly detect the activity of VKOR protein in catalyzing KO or K in real time in a high-throughput mode; and by adopting the method, the small-molecule inhibitor targeting the VKOR can be effectively screened out.

Description

technical field [0001] The invention relates to a method for measuring the in vitro activity of vitamin K epoxide reductase and its application, belonging to the technical field of biochemistry. Background technique [0002] Vitamin K epoxide reductase (VKOR) is the specific target of clinical anticoagulant drug warfarin. Warfarin prevents KO from being transformed into K and KH by inhibiting the activity of VKOR 2 , thereby blocking the circulation of vitamin K, and then inhibiting the gamma carboxylation modification of the glutamic acid residue in the Gla domain of the blood coagulation factor, so as to achieve the purpose of anticoagulation. The method of measuring the activity of VKOR protein in vitro is an important means to study the properties and inhibitors of VKOR protein, and can also be used to screen new small molecule inhibitors targeting VKOR. At present, the traditional method is to measure the activity of VKOR protein by extracting microsomes. This method ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26
CPCC12Q1/26
Inventor 沈国民高蒙刘红丽沈滟曹青李三强
Owner HENAN UNIV OF SCI & TECH
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