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Peroxide nanoenzyme and preparation method thereof

A technology of peroxide and nano-enzyme, applied in the fields of biochemical equipment and methods, oxidoreductase, enzyme, etc., can solve the problems of slow pharmaceutical development process, poor stability, limited sources, etc., and achieve good application prospects and stable properties. , easy to operate effect

Active Publication Date: 2016-10-12
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the limited source and poor stability of natural GPx, its medicinal development process is very slow

Method used

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  • Peroxide nanoenzyme and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0017] Example 1: Synthesis of gold nanoparticles

[0018] Sodium citrate-coated gold nanoparticles were obtained by reducing tetrachloroauric acid with sodium citrate (reference J. Phys. Chem. C 2007, 111, 9172-9176). The specific steps are as follows. In a 100mL double-necked round bottom flask (the interface in the middle is used to connect the reflux condenser, and the interface on the side is used to add reagents during the reaction), add 48.5mL of high-purity water and 1.3mL of tetrachloroauric acid solution (0.01M ), heated to boiling and reflux under magnetic stirring (rotating speed 800rpm). Quickly add 0.2mL sodium citrate solution (0.2M) through the side port, continue heating for 10min, stop heating and continue stirring until natural cooling, the maximum plasmon resonance absorption peak is at 520nm. Then the gold nanoparticle solution was centrifuged at 8,000rpm for 30min, and the precipitate was dissolved in high-purity water and diluted until the absorbance va...

Embodiment 2

[0019] Embodiment 2: the preparation of nanozyme

[0020] Take 2mL gold nanoparticle stock solution, add 510μL phosphate buffer solution (10mM, pH 8), shake well and quickly add 40μL URGDC solution (0.15mM) (purchased from Gill Biochemical (Shanghai) Co., Ltd., purity >98%), immediately Shake well and stand at 4°C for 24 hours to obtain the peroxidase nanozyme with glutathione peroxidase activity, which is stored at 4°C.

Embodiment 3

[0021] Example 3: GPx activity of nanozymes

[0022] According to the Wilson method (J.Am.Chem.Soc.1989,111,5936-5939) to measure the GPx activity of biologically active pentapeptides and nanozymes (reaction equation shown in the following formula), the enzyme activity is defined as 37 ℃, per minute The amount of enzyme required to oxidize 1 μmoL NADPH is an activity unit (U), and the specific activity of the enzyme is defined as the number of enzyme activity units possessed by 1 μmoL of the enzyme, expressed as U / μmoL. The GPx activity of the nanozyme was 5.05U / μmoL and the GPx activity of the biologically active pentapeptide URGDC was 0.36U / μmoL at 37°C and pH 7.0. The GPx activity of the classic commercial mimic Ebselen is 0.99U / μmoL (reference Adv.Pharmacol.1997,38,2229-2246). Therefore, the activity of the prepared nanozyme is higher than that of bioactive pentapeptide and Ebselen enzyme mimics, and has high GPx enzyme activity.

[0023]

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Abstract

The invention provides a peroxide nanoenzyme and a preparation method thereof, belonging to the technical field of nano-biotechnology. According to the method, a gold nanoparticle is used as a carrier, biologically active pentapeptide (URGDC) is employed for self-assembly, and a mercapto group in a pentapeptide and the gold nanoparticle undergo coordination and covalent coupling so as to obtain the peroxide nanoenzyme with high GPx activity. The method is simple to operate and has good stability. The amino acid sequence of the biologically active pentapeptide is Sec-Arg-Gly-Asp-Cys (referred as URGDC for short), wherein Sec(U) contains the active center of the nanoenzyme, Arg-Gly-Asp (RGD) can promote bonding between the nanoenzyme and a substrate, and the mercapto group in a Cys (C) side chain can realize covalent connection with the gold nanoparticle. The gold nanoparticle is used as a carrier for immobilizing the biologically active pentapeptide, promotes full contact between the nanoenzyme and the substrate, and improves activity of glutathion peroxidase.

Description

technical field [0001] The invention belongs to the field of nano-biotechnology, and in particular relates to the intersection and combination of nano-technology and the field of artificial simulated enzymes, and prepares a peroxide with high GPx activity by simulating the properties of glutathione peroxidase (GPx) through nano-technology nanozyme. Background technique [0002] Glutathione peroxidase (GPx) has unique redox properties and is one of the important members of antioxidant enzymes in organisms. GPx uses glutathione (GSH) as a reducing agent to decompose various hydroperoxides in the body. Its main physiological function is to protect red blood cells and mitochondria from being damaged by active oxygen, prevent lipid peroxidation in the body, and play an extremely important role in the anti-oxidation of organisms. [0003] GPx can remove active oxygen in the body and prevent lipid peroxidation, so it has the potential to treat various diseases caused by the imbal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N9/08
CPCC12N9/0065C12N11/14C12Y111/01009
Inventor 郭轶徐力沈娜李慧郝翠婷
Owner JILIN UNIV
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