CN111198185B - Enzyme catalysis detection method of selenium monosaccharide - Google Patents
Enzyme catalysis detection method of selenium monosaccharide Download PDFInfo
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Abstract
The present invention relates to a cytochrome P450 monooxygenase (CYP 236A 20), ferredoxin (FoX), ferredoxin reductase (FoR) and Tetramethylbenzidine (TMB) red ) The application in detecting selenium monosaccharide or preparing a selenium monosaccharide detection reagent is characterized in that the detected selenium monosaccharide is 1 beta-methyl selenium-2-N-acetyl-D-galactosamine (GalNAc 1 SeMe), which is metabolic discharge in human urine. The invention also provides a new detection method of the selenium monosaccharide, namely CYP236A20 is taken as a catalyst and a receptor of a substrate, foX and FoR are taken as electron transfer media, and reduced TMB is taken red Is 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 ) The color of the solution is changed from colorless transparency to blue-purple, thereby realizing the sensitive indication of the selenium monosaccharide.
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
Selenium is a trace mineral substance necessary for human bodies, and keshan disease caused by selenium deficiency commonly occurs in northern China and remote areas in the middle and western China; modern clinical medicine further proves that the selenium content of the human body is closely related to the metabolic expression of various disease-related molecules (such as a marker of cardiovascular diseases, homocysteine). Therefore, it is essential to establish biochemical indicators for evaluating selenium nutrition levels. However, according to the known selenium anabolism, selenoprotein P as a human plasma selenium bank has poor structural conservation and an expression threshold, and the expression of a series of methylation products and selenium substituted sulfur-containing amino acids in different tissues and cells is inconsistent and measurement errors are large, so that the selenoprotein P is not suitable to be used as a marker for health monitoring. Recent speciation analysis researches show that selenium excretion with the absolute dominant content exists in human urine, and the selenium excretion is known as 1 beta-methyl selenium-2-N-acetyl-D-galactosamine (1 beta-selenomethyl-2-N-acetyl-D-galactosamine, galNAc1 SeMe), which is expected to become a potential evaluation endpoint and can provide an effective analysis means for further elucidating biological selenium associated metabolism. The hemiacetal structure corresponding to 1 beta-methylselenium-2-N-acetyl-D-galactosamine is as follows:
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 (GalNAc for short) on the surface of cell membrane, but also can be recognized by N-acetyl-D-glucosamine (GlcNAc for short), and has no 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, it has been discovered by chemists 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) and Nicotinamide Adenine Dinucleotide (NADH) coenzymes. Considering that selenium also belongs to the chalcogen and the covalent bond of selenomethyl (l [ CH) 3 -Se]=198 pm) longer than methyl ether (l [ CH ] 3 -O]=143 pm), the former of which the bond is weaker and therefore it may be attempted to dissociate it using 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 a methyl selenium functional group in the selenium monosaccharide and remove the ligand, and the process is accompanied with oxidation of tetramethyl benzidine to realize sensitive indication of the selenium monosaccharide according to color conversion of a solution.
The present invention discloses cytochrome P450 monooxygenase, ferredoxin (FoX), ferredoxin reductase (FoR) and reduced Tetramethylbenzidine (TMB) red ) The application in detecting selenium monosaccharide or preparing a selenium monosaccharide detection reagent, wherein the selenium monosaccharide is 1 beta-methyl selenium-2-N-acetyl-D-galactosamine (GalNAc 1 SeMe).
Further, cytochrome P450 monooxygenase is recombinantly expressed by Escherichia coli (Escherichia coli), is derived from a bacterium Formosa agariphia with the NCBI serial number WP _038530297.1, and has the cytochrome P450 monooxygenase code CYP236A20.
Further, the concentration ratio of CYP236A20, ferredoxin reductase and reduced tetramethylbenzidine is 0.01-1.0.
In the present invention, the selenium monosaccharide GalNAc1SeMe is the major metabolic drain in human urine. Cytochrome P450 monooxygenase is used as a catalyst and a receptor for selenium monosaccharide; 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 beta-methyl selenium-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) is the sequence number WP _038530297.1, and the code CYP236A20; 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 μ M.
Further, the concentration of ferredoxin reductase in the solution to be tested is 0.2-18.0. Mu.M. Ferredoxin and ferredoxin reductase are proteins essential to a subset of bacteria in the CYP superfamily, and are used as a pair of electron transfer mediators for the enzyme-catalyzed reaction dominated by CYP236a20, expressed from a polysaccharide utilizing gene site co-localized with a conserved sequence of CYP236a20 (NCBI sequence numbers 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 CYP236a 20-dominated, enzyme-catalyzed reactions. 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 measured 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, ferrodoxin and ferredoxin reductase transfer electrons to reduced tetramethylbenzidine, 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 TMB red And its oxidized form TMB ox The 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 principle, the invention also provides an indicator of 1 beta-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 new method for detecting 1 beta-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 ferredoxin and ferredoxin reductase as electron transfer mediators, and takes tetramethylbenzidine as a proton donor and a color developing agent; the petunidin is removed by utilizing the recognition and the transformation of the enzyme and the methyl selenium functional group in the substance to be detected. The process is accompanied with the oxidation of tetramethyl benzidine, so that the color of the solution is changed from colorless transparency to bluish purple, 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.
Drawings
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 kinetics of UV absorption at a wavelength of 420nm for different test groups according to the present invention.
Detailed Description
The following examples are given to further illustrate 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 with 50mM sodium phosphate buffer pH 7.4 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, has a corresponding gene retrieval serial number WP _038530297.1 in NCBI database, and has a recombinant protein code CYP236A20. Before use, CYP236A20 was dispersed by vortex with 50mM sodium phosphate buffer pH 7.4 containing 100. Mu.M camphor to prepare a 0.1. Mu.M stock solution, and stored at 4 ℃ until use.
FoX and FoR are electron transport protein pairs necessary FoR the bacterial subclass in the CYP superfamily to undergo a single oxidation reaction, and are used in the present invention as electron conduction mediators FoR the enzyme-catalyzed reaction dominated by CYP236A20; the corresponding sequence 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. Prior to use, foX was vortexed and dispersed in 50mM sodium phosphate buffer pH 7.4 containing 10mM mercaptoethanol to make a 5. Mu.M stock solution; foR was dispersed by vortexing with 50mM sodium phosphate buffer pH 7.4 containing 100mM sodium chloride to make 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 mixed red Vortex and dissolve in50mM pH 7.4 sodium phosphate buffer solution, and a 1mM concentration solution was prepared.
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 TMB red (1mM)；
(b) Groups included GalNAc1SeMe (3. Mu.M), foX (2.5. Mu.M), foR (0.5. Mu.M) and TMB red (1mM)；
(c) Groups included GalNAc1SeMe (3. Mu.M), CYP236A20 (0.01. Mu.M) and TMB red (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 TMB ox (1mM)。
Solutions were prepared according to the concentrations of the components of the above test groups, and were obtained by mixing with different volumes of mother liquors using 50mM sodium phosphate pH 7.4 as solvent. The group (e) preparation method comprises the following steps:
pipette 1mM TMMB with micropipette red 200. 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 mixture was mixed well by repeated pipetting for 5 seconds.
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, based on 50mM pH 7.4 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 sequence. 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 is the UV absorption kinetics curves of the above different test groups at a wavelength of 420nm, and the graphs (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 with time, which indicates that the solution has the ultraviolet absorption signals only when the solution contains selenium monosaccharide, cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine simultaneously. 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 spectrum M = (0.63. + -. 0.16) mM, catalytic reaction rate constant k cat ＝(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 beta-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 Formosa agariphila with NCBI sequence number WP _038530297.1, and has the code number CYP236A20.
3. Use according to claim 2, characterized in that: the concentration ratio of CYP236A20, ferredoxin reductase and reduced tetramethylbenzidine is 0.01-1.0.
4. The enzyme catalysis detection method of selenium monosaccharide is characterized by comprising the following steps of:
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 CYP236A20; 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 tetramethyl benzidine 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 for 1 beta-methylselenium-2-N-acetyl-D-galactosamine comprising cytochrome P450 monooxygenase, ferredoxin reductase and reduced tetramethylbenzidine.
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| EP2639308A1 (en) * | 2012-03-12 | 2013-09-18 | Evonik Industries AG | Enzymatic omega-oxidation and -amination of fatty acids |
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