CN111198185A - Enzyme catalysis detection method of selenium monosaccharide - Google Patents
Enzyme catalysis detection method of selenium monosaccharide Download PDFInfo
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Abstract
The invention relates to cytochrome P450 monooxygenase (CYP236A20), ferredoxin (FoX), ferredoxin reductase (FoR) and Tetramethylbenzidine (TMB)red) The invention also provides a new detection method of the selenium monosaccharide, namely CYP236A20 is used as a catalyst and a receptor of a substrate, FoX and FoR are used as electron transfer media, and reduced TMB is usedredIs a proton donor and a color developing agent; the ligand is removed by enzyme recognition of methyl selenium functional group in the analyte, and the process is accompanied with oxidation of tetramethyl benzidine (TMB)ox) And the color of the solution is changed from colorless transparency to blue-purple, so that the sensitive indication of the selenium monosaccharide is realized.
Description
Technical Field
The invention relates to a method for detecting selenium monosaccharide, in particular to an enzyme catalysis detection method for selenium monosaccharide.
Background
According to the known selenium anabolism, the structure conservation of selenoprotein P serving as a human plasma selenium bank is poor and exists at an expression threshold, and the expression of a series of methylated products and selenium-substituted sulfur-containing amino acids in different tissues and cells is inconsistent and the measurement error is large, so that the selenoprotein P is not suitable to be used as a marker for health monitoring, the latest species formation analysis research finds that one selenium excretion with the absolute dominant content exists in human urine, the chemical name of the selenium excretion is 1-1 β -methylselenium-2-N-acetyl-D-galactosamine (1 β -noomesyl-2-N-acetyl-D-galactosamine, the chemical name of the selenium excretion is Gallacomine 1, and the potential biological terminal point of the selenium metabolism is further clarified as follows, namely the potential biological terminal point of the selenoamine-1 β:
at present, the detection of a specific selenium-doped compound is generally carried out by adopting large-scale equipment such as high performance liquid chromatography (or capillary electrophoresis) tandem mass spectrometry, which necessarily requires a specially-assigned person to operate, has a long test period, and also needs to prepare a proper mobile phase to realize baseline separation of specific components. On the other hand, while lectin-DSA recognizes the protein glycosyl N-acetyl-D-galactosamine (abbreviated as GalNAc) on the surface of a cell membrane, it can also recognize N-acetyl-D-glucosamine (abbreviated as GlcNAc), and thus lacks specificity; the cost for screening monosaccharide polypeptide aptamers is very high, no ready sequence library can be used for reference, and therefore the monosaccharide polypeptide aptamers are not suitable for capturing free GalNAc monosaccharides, and the two molecular aptamers need to be marked by extra dyes. Therefore, it is necessary to develop a simple, mild and effective analysis means for the rapid and sensitive detection of GalNAc1SeMe in future complex biological samples.
Recently, chemists have found that bacterial cytochrome P450 monooxygenase (CYP 450) can be used as demethylase (demethylase) FoR methoxy-protected polysaccharides (e.g. 6-O-methyl-D-galactose, 6-O-methyl-D-galactose) with the help of a combination of ferredoxin (FoX), ferredoxin reductase (FoR fon) and Nicotinamide Adenine Dinucleotide (NADH) coenzymes. Considering that selenium also belongs to the oxygen group element and the covalent bond of selenomethyl (l [ CH ]3-Se]198pm) longer than methyl ether (l [ CH ]3-O]143pm), the former bond is weaker and therefore it can be attempted to dissociate it following a similar approach. However, no detection method with simple process and high detection efficiency has been developed yet for detecting the selenium monosaccharide GalNAc1SeMe by using the above reaction.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an enzyme catalysis detection method of selenium monosaccharide, which utilizes cytochrome P450 monooxygenase to recognize methyl selenium functional groups in the selenium monosaccharide and remove the ligands, and the process is accompanied with the oxidation of tetramethyl benzidine, so that the sensitive indication of the selenium monosaccharide is realized according to the color conversion of a solution.
The present invention discloses cytochrome P450 monooxygenases, ferredoxins (FoX), ferredoxinsReductase (FoR) and reduced Tetramethylbenzidine (TMB)red) The application of the selenium monosaccharide in detecting the selenium monosaccharide or preparing a selenium monosaccharide detection reagent is that the selenium monosaccharide is 1 β -methyl selenium-2-N-acetyl-D-galactosamine (GalNAc1 SeMe).
Further, cytochrome P450 monooxygenase is recombinantly expressed by Escherichia coli (Escherichia coli), is derived from a bacterium Formosa agariphia with the NCBI sequence number WP _038530297.1, and has the cytochrome P450 monooxygenase code CYP236A 20.
Further, the concentration ratio of CYP236A20, ferredoxin reductase and reduced tetramethylbenzidine is 0.01-1.0: 1.0-90.0: 0.2-18.0: 250-3000.
In the present invention, the selenium monosaccharide GalNAc1SeMe is the main metabolic discharge in human urine. Cytochrome P450 monooxygenase acts as a catalyst and a receptor for selenium monosaccharides; ferredoxin and ferredoxin reductase as electron transfer mediators; reduced tetramethylbenzidine was used as proton donor and color developer.
The invention also discloses an enzyme catalysis detection method of selenium monosaccharide, wherein the selenium monosaccharide is 1 β -methylselenium-2-N-acetyl-D-galactosamine, and the method comprises the following steps:
providing a test solution comprising cytochrome P450 monooxygenase, ferredoxin, and ferredoxin reductase;
and adding a reduced tetramethylbenzidine solution into the solution to be detected, and judging whether the solution to be detected contains selenium monosaccharide or not according to the color change of the solution to be detected.
Further, the cytochrome P450 monooxygenase is derived from bacteria (Formosa agariphila), the NCBI of the bacteria (Formosa agariphila) has the sequence number of WP _038530297.1 and the code number of CYP236A 20; the concentration of CYP236A20 in the solution to be tested is 0.01-1.0 mu M.
Further, the concentration of the iron redox protein in the solution to be detected is 1.0-90.0 mu M.
Further, the concentration of the ferredoxin reductase in the solution to be detected is 0.2-18.0 mu M. Ferrodoxin and ferredoxin reductase are proteins essential for a subset of bacteria in the CYP superfamily, and are used as an electron transfer mediator pair for CYP236a 20-dominated enzyme-catalyzed reactions expressed from a polysaccharide utilizing gene site co-localized with CYP236a20 conserved sequences (NCBI sequence numbers are WP _038530300.1 and WP _038530304.1, respectively).
Furthermore, the concentration of the reduced tetramethylbenzidine in the solution to be tested is 0.25-3.0 mM. Reduced tetramethylbenzidine serves as a proton donor and indicator for the CYP236a 20-dominated, enzyme-catalyzed reaction. The present invention selects a proton donor having a color-changing property for visually distinguishing the occurrence of the reaction.
Further, the pH value of the solution to be detected is 7.0-8.0.
Further, the solvent in the solution to be tested comprises sodium phosphate buffer, camphor, mercaptoethanol and sodium chloride.
Further, oxygen is also dissolved in the solution to be measured.
Further, the method for judging whether the solution to be detected contains selenium monosaccharide comprises the following steps: when the solution to be detected contains selenium monosaccharide GalNAc1SeMe, under the action of cytochrome P450 monooxygenase, methyl selenium is removed from the selenium monosaccharide, electrons are transferred to reduced tetramethylbenzidine by ferrodoxin and ferrodoxin reductase, and the reduced tetramethylbenzidine is oxidized (TMB)ox) The color of the solution is changed from colorless transparency to blue-violet. Only when selenium monosaccharide, cytochrome P450 monooxygenase, ferredoxin and ferredoxin reductase coexist, reduced tetramethylbenzidine is oxidized to change color, and any single component or double components cannot start the enzyme catalytic reaction chain. The above reaction principle is as follows:
when the solution to be detected does not contain the selenium monosaccharide GalNAc1SeMe, the change is not generated, and the color of the solution is kept colorless and transparent all the time.
Reduced form TMBredAnd its oxidized form TMBoxThe principle of the morphological transformation and the concomitant proton gain/loss reaction between the following:
further, the color change process of the absorbance test solution at 420nm can be recorded by an ultraviolet-visible spectrophotometry, and when the absorbance exists at 420nm, the solution contains selenium monosaccharide, otherwise, the solution does not contain the selenium monosaccharide.
Based on the above principle, the present invention also provides an indicator of 1 β -methylselenium-2-N-acetyl-D-galactosamine, which comprises cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine.
By the scheme, the invention at least has the following advantages:
the invention provides a novel method for detecting 1 β -methylselenium-2-N-acetyl-D-galactosamine based on a CYP450 catalytic system, which takes cytochrome P450 monooxygenase as a catalyst and an acceptor of a substrate, takes ferrodoxin and ferredoxin reductase as electron transfer media, takes tetramethylbenzidine as a proton donor and a color developing agent, removes a ligand by utilizing the recognition and conversion of enzyme and a methylselenium functional group in a substance to be detected, and converts the color of a solution from colorless transparency to bluish purple along with the oxidation of the tetramethylbenzidine, thereby realizing the sensitive indication of the selenium monosaccharide.
The invention not only simplifies the identification process of the selenium monosaccharide metabolic marker, saves the labor cost and hardware equipment, but also improves the inspection efficiency, and is beneficial to promoting the design and development of related instant inspection products.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
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FIG. 1 is a qualitative photographic comparison of the results of the analysis of selenium monosaccharides from different test groups according to the invention;
FIG. 2 is a graph showing the UV absorption kinetics at a wavelength of 420nm for different test groups according to the present invention.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Examples
In order to verify the capability of detecting selenium monosaccharide GalNAc1SeMe by cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine, hereinafter, a selenium monosaccharide GalNAc1SeMe solution is used as a substrate, cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine are added into the solution, and if the solution is discolored, the detection of selenium monosaccharide is indicated.
The preparation method of the selenium monosaccharide GalNAc1SeMe solution is as follows:
powder sample standards of GalNAc1SeMe were vortexed and dissolved with 50mM sodium phosphate buffer pH7.4 and formulated into 100. mu.M stock solution and allowed to sit overnight at 4 ℃ in a refrigerator to give a substrate solution.
The cytochrome P450 monooxygenase is recombined and expressed by Escherichia coli (E.coli for short), is derived from bacteria Formosa agariphila, and has a corresponding gene retrieval sequence number of WP _038530297.1 and a recombinant protein code number of CYP236A20 in an NCBI database. Before use, CYP236A20 was dispersed by vortex with 100. mu.M camphene in 50mM sodium phosphate buffer pH7.4 to prepare a 0.1. mu.M stock solution, which was stored at 4 ℃ until use.
FoX and FoR are electron transport protein pairs necessary FoR bacterial subclasses in the CYP superfamily to undergo a single oxidation reaction, and are useful in the present invention as electron transport mediators FoR the enzyme-catalyzed reaction dominated by CYP236A 20; the corresponding sequence choose is selected from polysaccharide utilization gene sites (PUL) which are co-located with CYP236A20 conserved sequence, and the corresponding gene retrieval sequence numbers in NCBI database are WP _038530300.1 and WP _038530304.1 respectively, and are also produced by standard E.coli system expression. Before use, FoX was vortexed and dispersed in 50mM sodium phosphate buffer pH7.4 containing 10mM mercaptoethanol to make a 5. mu.M stock solution; FoR was dispersed by vortexing with 50mM sodium phosphate pH7.4 buffer containing 100mM sodium chloride to prepare 1. mu.M stock solutions, all stored at 4 ℃ until use.
Reduced Tetramethylbenzidine (TMB)red) Characterized in that it acts as a proton donor and indicator for CYP236a20 dominated, enzyme catalyzed reactions. Before use, the TMB is mixedredVortex and dissolve in 50mM pH7.4 sodium phosphate buffer solution to make a 1mM solution.
For comparison, several parallel sets of experiments were performed simultaneously:
(a) groups include CYP236A20 (0.01. mu.M), FoX (2.5. mu.M), FoR (0.5. mu.M) and TMBred(1mM)；
(b) Groups included GalNAc1SeMe (3. mu.M), FoX (2.5. mu.M), FoR (0.5. mu.M) and TMBred(1mM)；
(c) Groups included GalNAc1SeMe (3. mu.M), CYP236A20 (0.01. mu.M) and TMBred(1mM)；
(d) Groups included GalNAc1SeMe (3. mu.M), CYP236A20 (0.01. mu.M), FoX (2.5. mu.M) and FoR (0.5. mu.M);
(e) groups included GalNAc1SeMe (3. mu.M), CYP236A20 (0.01. mu.M), FoX (2.5. mu.M), FoR (0.5. mu.M) and TMBox(1mM)。
Solutions were prepared according to the concentrations of the components of the above test groups, and were obtained by mixing the solutions with different volumes of mother liquors using 50mM sodium phosphate pH7.4 as a solvent. (e) The group preparation method comprises the following steps:
pipette 1mM TMMB with micropipettered200. mu.L of the stock solution was used as a mother solution, and the stock solutions of CYP236A20, FoX and FoR prepared above were added thereto in this order, and each transfer was vortexed at room temperature FoR 30 seconds to mix them thoroughly, and finally a substrate solution was added to prepare a mixed solution. After addition of the substrate GalNAc1SeMe, the release was repeated for 5 seconds with pipette suction to mix well.
After the solutions of all the test groups were mixed, the resulting mixture was transferred to a microcuvette with a total volume of 320. mu.L, and the change in absorbance at a wavelength of 420nm was scanned using a kinetic module in UV Probe software carried in a UV-3600 UV-visible-near-infrared spectrophotometer manufactured by Shimadzu corporation, 50mM pH7.4 sodium phosphate buffer as a reference, and continuously recorded for 120 seconds.
FIG. 1 is a qualitative photographic comparison of the results of the analysis of selenium monosaccharides from the different test groups. The beakers which are arranged side by side from left to right correspond to the groups (a), (b), (c), (d) and (e) in turn. As can be seen, only the solution in the group (e) is colored, and the solutions in other test groups are colorless and transparent, which indicates that the enzyme catalysis system of the invention can detect the selenium monosaccharide.
FIG. 2 shows the UV absorption kinetics curves of the above different test groups at a wavelength of 420nm, wherein the curves (a), (b), (c), (d) and (e) correspond to the UV absorption kinetics curves of the above test groups (a), (b), (c), (d) and (e) in sequence. The results show that the ultraviolet absorption signals of the groups (a), (b), (c) and (d) are not changed in the whole test process, the ultraviolet absorption curves of the four groups are overlapped, and only the group (e) generates the ultraviolet absorption signals and changes along with time, which shows that the solution only has the ultraviolet absorption signals when the solution simultaneously contains selenium monosaccharide, cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine. The Michaelis-Menten coefficient K of the demethylation of GalNAc1SeMe catalyzed by CYP236A20 can be calculated by combining the kinetic curve and the ultraviolet-visible absorption spectrumMCatalytic reaction rate constant k ═ 0.63. + -. 0.16 mMcat＝(43.4±0.5)s-1。
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The application of cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine in detecting selenium monosaccharide or preparing a selenium monosaccharide detection reagent is disclosed, wherein the selenium monosaccharide is 1 β -methylselenium-2-N-acetyl-D-galactosamine.
2. Use according to claim 1, characterized in that: the cytochrome P450 monooxygenase is recombinantly expressed by Escherichia coli (Escherichia coli), is derived from bacterium Formosaagaiphila with NCBI serial number WP _038530297.1, and has cytochrome P450 monooxygenase code CYP236A 20.
3. Use according to claim 2, characterized in that: the concentration ratio of CYP236A20, ferredoxin reductase and reduced tetramethylbenzidine is 0.01-1.0: 1.0-90.0: 0.2-18.0: 250-3000.
4. An enzyme catalysis detection method of selenium monosaccharide, wherein the selenium monosaccharide is 1 β -methylselenium-2-N-acetyl-D-galactosamine, and is characterized by comprising the following steps:
providing a test solution comprising cytochrome P450 monooxygenase, ferredoxin, and ferredoxin reductase;
and adding a reduced tetramethylbenzidine solution into the solution to be detected, and judging whether the solution to be detected contains the selenium monosaccharide or not according to the color change of the solution to be detected.
5. The detection method according to claim 4, characterized in that: the cytochrome P450 monooxygenase is derived from bacteria Formosa agariphila, the NCBI of the bacteria Formosa agariphila has the sequence number of WP _038530297.1 and the code number of CYP236A 20; the concentration of CYP236A20 in the solution to be detected is 0.01-1.0 mu M.
6. The detection method according to claim 4, characterized in that: the concentration of the iron redox protein in the solution to be detected is 1.0-90.0 mu M.
7. The detection method according to claim 4, characterized in that: the concentration of the ferredoxin reductase in the solution to be detected is 0.2-18.0 mu M.
8. The detection method according to claim 4, characterized in that: the concentration of the reduced tetramethylbenzidine in the solution to be detected is 0.25-3.0 mM.
9. The detection method according to claim 4, characterized in that: the pH value of the solution to be detected is 7.0-8.0.
10. An indicator of 1 β -methylselenium-2-N-acetyl-D-galactosamine comprising cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine.
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| CN116217638A (en) * | 2023-05-10 | 2023-06-06 | 南京大学 | Seleno-non-natural sugar and preparation method and application thereof |
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