CN102703576A - Determination method for S-adenosylmethionine methyltransferase and kit thereof - Google Patents
Determination method for S-adenosylmethionine methyltransferase and kit thereof Download PDFInfo
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- CN102703576A CN102703576A CN2012101696675A CN201210169667A CN102703576A CN 102703576 A CN102703576 A CN 102703576A CN 2012101696675 A CN2012101696675 A CN 2012101696675A CN 201210169667 A CN201210169667 A CN 201210169667A CN 102703576 A CN102703576 A CN 102703576A
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- adenosylmethionine
- methyltransgerase
- homocysteine
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Abstract
The invention discloses a determination method for S-adenosylmethionine methyltransferase and a kit thereof. The determination method for S-adenosylmethionine methyltransferase comprises the step of detecting SAM methyltransferase through the enzyme coupling reaction between S-adenosylhomocysteine hydrolase and adenosine deaminase. The detection technique relieves feedback inhibition of products, has stable results and accurate quantitative reaction, and effectively overcomes the problem of radioactive pollution of the radioactive marking method. With double reagents in the kit, detection can be carried out manually or through a full automatic biochemical analyzer within the visible range, and therefore the use and the fast detection of a plurality of samples are convenient. The determination method is also suitable for detection of the enzyme coupling reaction between S-adenosylhomocysteine hydrolase and adenosine deaminase.
Description
Technical field:
The present invention relates to biological chemistry, testing reagent field; What be specifically related to is the enzyme coupling analysis and detection technology of a kind of S-adenosylmethionine (SAM) methyltransgerase (methyltransgerase that S-adenosylmethionine relies on); And based on the detection kit of this technological development; This test kit also can be prepared into S-SAHH or adenosine deaminase detection kit respectively through improving.
Background technology:
Important effect is being brought into play in the aspects such as being modified at signal conduction, biosynthesizing, albumen reparation, gene silencing and chromosomal expression regulation and control that methylates of biomacromolecule; S-adenosylmethionine (S-adenosyl-L-methionine; SAM) be the methyl donor that is only second to ATP in the body; The S-adenosylmethionine transferring enzyme of its dependence utilizes SAM as methyl donor, thereby with the modification that methylates of DNA and protein, thereby participate in the important physiological activity.Research at present shows that the abnormal level of this enzyme is relevant with multiple diseases such as senile dementia, dysthymia disorders, parkinsonism, cancers, aspect medical diagnosis, has important use and is worth and clinical meaning.
Measure SAM Methyl transporters enzyme activity at present radioactive substrates method, Paper Chromatography are arranged.The radioactive substrates method adopts usually
14C or
3H mark AdoMet after the Methyl transporters reaction is accomplished, observes radiolabeled product amount through the radioisotope scan imager, thereby detects the activity of this enzyme.This method needs reaction product isolated and substrate; Waste time and energy, poor repeatability is difficult to accomplish accurately quantitatively; And reaction product S-adenosine-L-homocysteine (Hcy) has the intensive feedback inhibition to this enzymatic methylation reaction, causes reaction not exclusively.In addition, this method has radioactivity is arranged, and environment and operator are polluted greatly.And Paper Chromatography adopt usually ply of paper analyse the technology reaction substrate and product are separated; Detect the growing amount of product under given conditions, thereby detect the activity of this enzyme, the growing amount that this method can only the half-quantitative detection product; And have the product feedback inhibition equally, be difficult to accurately quantitatively.
The enzyme coupling method measuring method that patent and bibliographical information SAM methyltransgerase are also arranged at present.Patent " detection method of the methyltransgerase that a kind of S-adenosylmethionine (AdoMet) relies on " application number is 201010197808.5; This patent report utilize the S-adenosyl-L-homocysteine nucleosidase of reorganization and the S-Ribosylhomocysteinase catalysis S-adenosyl-L-homocysteine reaction generation homocysteine of reorganization; The latter is with Ellman reagent 5; 5 '-Lian sulphur-2,2 '-two nitrobenzoic acids are quantitative, thus the enzyme of finally measuring the SAM methyltransgerase is lived.But this method also exists the homocysteine quantitative reaction more loaded down with trivial details, and workload is big, and the deficiency that can't on semi-automatic or full automatic biochemical apparatus, operate.
Summary of the invention:
The present invention is directed to the above-mentioned deficiency of prior art; A kind of feedback inhibition of removing product has been proposed; The result is reliable and stable, and quantitative reaction is accurate, has overcome the complex operation that the radio-labeling method exists simultaneously; Waste time and energy and the detection method of the insufficient SAM methyltransgerase that radioactivity is polluted environment and operator.
In order to solve the problems of the technologies described above; The technical scheme that the present invention adopts is: a kind of detection method of SAM methyltransgerase; Process is: through with S-SAHH and adenosine deaminase coupling, the generation product S-adenosyl homocysteine of SAM methyltransgerase is changed into the quinone thing rapidly, and in visible-range through detecting the variation of absorbancy; Confirm the growing amount of quinone thing, thereby confirm the activity of SAM methyltransgerase.
The present invention is directed to the problems referred to above, in conjunction with the deficiency of existing invention, utilize enzyme linked reaction method, a kind of SAM methyltransgerase enzyme activity determination test kit that can be quantitative, easy to detect is provided, reaction principle is following:
The concise and to the point process that the SAM methyltransgerase detects is:
(1) removes methyl group from SAM, generate the S-adenosyl homocysteine.In this reactions step, related to the L-homocysteine.If in reagent, do not add the L-homocysteine, add the SAM methyltransgerase, continue ensuing enzyme linked reaction then, just can process the test kit that is used to detect the L-homocysteine
(2) the S-adenosyl homocysteine is decomposed into L-homocysteine and adenosine by S-adenosine-L-homocysteine lytic enzyme (S-SAHH) fast, has avoided the former (S-adenosine-L-homocysteine lytic enzyme) accumulation to form the product feedback inhibition catalytic reaction process that methylates.The process that methylates itself is a reversible process, and the S-adenosyl homocysteine constantly is decomposed, and could guarantee that the process of methylating constantly carries out to the positive reaction direction.
(3) adenosine is converted into inosine by desaminase (the 3rd enzyme that relates in the detection) with adenosine; Inosine generates xanthoglobulin under the effect of purine nucleoside phosphorylase (PNP), can detect absorbancy at the 265nm place this moment and change the hypoxanthic formation speed of detection.Perhaps continue the vigor of other reaction monitoring of coupling SAM methyltransgerase.Because the existence of this step, our linked reaction also can be made into the test kit that detects adenosine nucleoside desaminase vigor.
(4) because 265nm belongs to ultraviolet wavelength; Be not suitable for full-automatic or semi-automatic biochemical analyzer, it is quick to cause detecting inconvenience, in order to overcome this problem; We proceed the coupling design, and xanthoglobulin generates hydrogen peroxide and uric acid fast under the effect of XOD (XOD).
(5) next be exactly the stronger Trinder reaction of specificity.The Trinder reaction is claimed again " coupling terminal colorimetric analysis ", and its principle is that measured matter passes through the hydrogen peroxide (H that the enzyme effect produces
2O
2) in the presence of 4-amino replaces than woods (4-AAP), px (POD), can generate red quinonimine compound.This reaction is at first proposed in 1969 by Trinder, so name Trinder reaction.The initial phenol that uses is made chromogenic agent, have afterwards many authors propose ten surplus kind of compound substitute phenol (like 2.4-two chlorophenols etc.), make the sensitivity of reaction be able to significantly improve.The Trinder reaction has constituted the basis of enzymatic assays such as glucose, SUV, triglyceride and uric acid, and the use value of outbalance is arranged.We are the generation quinone thing under the effect of px of chromogen, hydrogen peroxide and 4-aminoantipyrene with TOOS, and under the 546nm wavelength, absorption are arranged.The generation concentration of quinone thing absorbancy and hydrogen peroxide is proportional; Promptly proportional with the vigor of SAM methyltransgerase; Thereby the variation through absorbancy detects the enzyme of SAM methyltransgerase and lives, and perhaps is used for other enzyme activity determinations such as L-homocysteine, s-SAHH and adenosine deaminase.
A kind of above-mentioned SAM methyltransgerase enzyme activity determination test kit is specially: this test kit is made up of reagent 1 and reagent 2, wherein:
Reagent 1 is:
20～200mmol/L damping fluid,
10～200mmol/L?MgCl
2
1～100mmol/L S-adenosylmethionine
1～100mmol/L Triphosaden
1～100mmol/L L-homocysteine
0.1～5mmol/L chromogen
1～100g/L stablizer
0.1～100g/L sanitas
1～100ml/L tensio-active agent;
Reagent 2 is:
20～200mmol/L damping fluid
0.1～100mmol/L 4-aminoantipyrene
1～2000U/L S-SAHH
1～2000U/L adenosine deaminase
1～2000U/L purine nucleoside phosphorylase
1～2000U/L XOD
1～2000U/L px
1～100g/L stablizer
0.1～100g/L sanitas
1～100ml/L tensio-active agent.
Above-mentioned damping fluid is one or more in phosphate buffered saline buffer, citric acid-sodium citrate damping fluid, Tris damping fluid, glycine buffer, GOOD ' the S damping fluid.
Reagent 1 and damping fluid in the reagent 2 can be identical also can be different.
Above-mentioned chromogen is N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-monomethylaniline sodium salt (TOOS), phenol, methyl catechol, 3, one or more of 5-two chloro-2-hydroxybenzene phenolic acid sodium.
Above-mentioned stablizer is one or more in polyvalent alcohol, sodium ethylene diamine tetracetate, bovine serum albumin (BSA), N.F,USP MANNITOL, disaccharides, the polysaccharide;
Above-mentioned sanitas is one or more in triazo-compound (like sodium azide etc.), the PC series sanitas;
Above-mentioned tensio-active agent is one or more in tween, the triton x-100.
Stablizer in reagent 1 and the reagent 2, sanitas, tensio-active agent etc. can be identical also can be different.
Mentioned reagent box of the present invention adopts the ordinary method configuration, and promptly reagent 1 and 2 each concentration of component are to add back concentration such as entry such as zero(ppm) water, and promptly each component and water mix and gets final product.
Advantage of the present invention and beneficial effect:
1. the final linked reaction of using of the present invention is reacted as Trinder, and reaction significantly, and is quantitatively simple, and has overcome the reaction of coupling ammonia, has got rid of the interference of ammonia effectively.Biological products are in producing and preparing at present; Tend to bring into a large amount of ammonium ions; The ammonia pollution phenomenon that causes reagent to detect; And this invention has been owing to got rid of the interference of ammonia effectively, so this detection method use range is wider, can be used in the detection of SAM methyltransgerase and related substances or enzyme of most samples.
2. the present invention utilizes the S-adenosyl homocysteine that conjugate enzyme quick catalysis SAM methyltransgerase produces, and has eliminated the product accumulation phenomenon, has removed the feedback inhibition of product, and the result is reliable and stable, and quantitative reaction is accurate.Overcome the complex operation that the radio-labeling method exists simultaneously, wasted time and energy and deficiency that radioactivity is polluted environment and operator, for detecting SAM methyltransgerase simple and fast, and eliminated radiocontamination, safe and reliable.
3. test kit of the present invention can be processed the double reagent box that is applicable to the full automatic biochemical apparatus analyzing and testing, can adopt on craft or the full automatic biochemical apparatus and in visible-range, detect, and is easy to use, is convenient to the rapid detection of a large amount of samples.Main application is the SAM methyltransgerase, but available same coupling method can be prepared into S-SAHH or adenosine deaminase detection kit respectively, applied range.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, but be not limitation of the present invention.Any modification of in spirit of the present invention and right protection scope, being made all falls into protection scope of the present invention.
Embodiment 1:
Damping fluid is used phosphoric acid buffer, and stablizer is used BSA
Reagent 1 is: 100mmol/L phosphoric acid buffer, PH7.4
20mmol/L?MgCl
2
The 50mmol/L S-adenosylmethionine
The 50mmol/L Triphosaden
10mmol/L L-homocysteine
1mmol/L?TOOS
1g/L?BSA
The 1g/L sodium azide
The 10ml/L triton x-100
Reagent 2 is: the 100mmol/L phosphoric acid buffer
10mmol/L 4-aminoantipyrene
100U/L S-SAHH
The 100U/L adenosine deaminase
The 50U/L purine nucleoside phosphorylase
The 50U/L XOD
The 100U/L px
1g/L BSA
The 1g/L sodium azide
The 10ml/L triton x-100;
The mentioned reagent box is used for the enzyme coupling analysis and detection technology of SAM methyltransgerase; Detailed process is: through with S-SAHH and adenosine deaminase coupling; Generation product S-the adenosyl homocysteine of SAM methyltransgerase is changed into the quinone thing rapidly; And in visible-range through detecting the variation of absorbancy, confirm the growing amount of quinone thing, thus the activity of definite SAM methyltransgerase.
When measuring sample, adopt rate method, temperature is 37 ℃, reagent 1: sample: reagent 2=200: 10: 50 (volume ratio); The mensuration wavelength is 546nm, after reagent 1 adds standard substance or sample, adds reagent 2 after hatching 300s; Mixing was hatched one minute again, read first point, continuous detecting 3 minutes; Read second point,, measure the enzyme of SAM methyltransgerase and live then through the relatively variation of absorbancy between 2.
Embodiment 2:
Damping fluid is used the Tris damping fluid, and stablizer is used EDTA-Na
2
Reagent 1 is: 100mmol/LTris damping fluid, PH7.4
20mmol/L?MgCl
2
The 50mmol/L S-adenosylmethionine
The 50mmol/L Triphosaden
10mmol/L L-homocysteine
1mmol/L?TOOS
The 1g/L sodium ethylene diamine tetracetate
The 1g/L sodium azide
The 10ml/L triton x-100
Reagent 2 is: the 100mmol/LTris damping fluid
10mmol/L 4-aminoantipyrene
100U/L S-SAHH
The 100U/L adenosine deaminase
The 50U/L purine nucleoside phosphorylase
The 50U/L XOD
The 100U/L px
The 1g/L sodium ethylene diamine tetracetate
The 1g/L sodium azide
The 10ml/L triton x-100
The measuring method of embodiment 2 is identical with embodiment 1.
Embodiment 3:
Damping fluid is used glycine buffer, and stablizer is used N.F,USP MANNITOL.
Reagent 1 is: 100mmol/L glycine buffer, pH7.4
20mmol/L?MgCl
2
The 50mmol/L S-adenosylmethionine
The 50mmol/L Triphosaden
10mmol/L L-homocysteine
1mmol/L?TOOS
1g/L N.F,USP MANNITOL
The 1g/L sodium azide
The 10ml/L triton x-100
Reagent 2 is: the 100mmol/L glycine buffer
10mmol/L 4-aminoantipyrene
100U/L S-SAHH
The 100U/L adenosine deaminase
The 50U/L purine nucleoside phosphorylase
The 50U/L XOD
The 100U/L px
1g/L N.F,USP MANNITOL
The 1g/L sodium azide
The 10ml/L triton x-100
The measuring method of embodiment 3 is identical with embodiment 1.
We test the performance that reagent of the present invention detects the SAM methyltransgerase, have measured linearity range, precision that SAM methyltransgerase of the present invention detects.
Linearity range is measured: we are diluted to a series of multiple with the SAM methyltransgerase with sterilized water, have measured under the different extension rates, and the enzyme of SAM methyltransgerase is lived, and data are seen table 1:
Table 1: under the different extension rates, SAM methyltransgerase enzyme activity determination
| Extension rate | 1 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 | 0 |
| Enzyme (U/ml) alive | 10.26 | 9.18 | 8.52 | 7.36 | 6.23 | 5.13 | 4.02 | 3.15 | 2.15 | 0.99 | 0.12 |
According to table 1, the equation of measuring between enzyme work and the extension rate is y=10.289x+0.0469, R
2=0.998, obtaining detection linearity range of the present invention is 0～10U/ml, and linearity range is wide.
Precision is measured: we utilize continuous detecting SAM methyltransgerase of the present invention, obtain a series of mensuration results, like table 2:
Table 2: continuous detecting SAM methyltransgerase enzyme activity determination result
| Measure number of times | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Enzyme (U/ml) alive | 8.59 | 8.36 | 8.47 | 8.75 | 8.39 | 8.46 | 8.9 | 8.34 | 8.77 | 8.42 |
According to table 2, continuous detecting SAM methyltransgerase CV (relative variability coefficient)=2.31%, the variation coefficient is little, and it is high to detect precision.
Detection method of the present invention is wide for the detection linearity range of SAM methyltransgerase, and linear good, the variation coefficient is low, and precision is high, demonstrates the premium properties in this enzyme context of detection, has guaranteed accuracy and stability that this enzyme detects.
Claims (8)
1. the measuring method of a S-adenosylmethionine methyltransgerase; It is characterized in that: its process is: through with S-SAHH and adenosine deaminase coupling; Generation product S-the adenosyl homocysteine of S-adenosylmethionine methyltransgerase is changed into the quinone thing rapidly; And in visible-range through detecting the variation of absorbancy, confirm the growing amount of quinone thing, thus the activity of definite S-adenosylmethionine methyltransgerase.
2. the measuring method of S-adenosylmethionine methyltransgerase according to claim 1 is characterized in that: the testing process of S-adenosylmethionine methyltransgerase is specially:
(1) removes methyl group from S-adenosylmethionine, generate the S-adenosyl homocysteine;
(2) the S-adenosyl homocysteine is decomposed into L-homocysteine and adenosine by S-adenosine-L-homocysteine lytic enzyme fast;
(3) adenosine is converted into inosine by adenosine deaminase, and inosine generates xanthoglobulin under the effect of purine nucleoside phosphorylase;
(4) xanthoglobulin generates hydrogen peroxide and uric acid fast under the effect of XOD;
(5) next be exactly the stronger Trinder reaction of specificity: the generation quinone thing under the effect of px that with TOOS chromogen, hydrogen peroxide and 4-aminoantipyrene; And under the 546nm wavelength, absorption is arranged; The generation concentration of quinone thing absorbancy and hydrogen peroxide is proportional; Promptly proportional with the vigor of S-adenosylmethionine methyltransgerase, thereby detecting the enzyme of S-adenosylmethionine methyltransgerase, the variation through absorbancy lives.
3. the used mensuration test kit of measuring method of claim 1 or 2 described S-adenosylmethionine methyltransgerases, it is characterized in that: this test kit specifically is made up of reagent 1 and reagent 2, wherein:
Reagent 1 is:
20～200mmol/L damping fluid
10～200mmol/L?MgCl
2
1～100mmol/L S-adenosylmethionine
1～100mmol/L Triphosaden
1～100mmol/L L-homocysteine
0.1～5mmol/L chromogen
1～100g/L stablizer
0.1～100g/L sanitas
1～100ml/L tensio-active agent;
Reagent 2 is:
20～200mmol/L damping fluid
0.1～100mmol/L 4-aminoantipyrene
1～2000U/L S-SAHH
1～2000U/L adenosine deaminase
1～2000U/L purine nucleoside phosphorylase
1～2000U/L XOD
1～2000U/L px
1～100g/L stablizer
0.1～100g/L sanitas
1～100ml/L tensio-active agent.
4. S-adenosylmethionine Methyl transporters enzymatic determination test kit according to claim 3 is characterized in that: described damping fluid is one or more in phosphate buffered saline buffer, citric acid-sodium citrate damping fluid, Tris damping fluid, glycine buffer, GOOD ' the S damping fluid.
5. S-adenosylmethionine Methyl transporters enzymatic determination test kit according to claim 3; It is characterized in that: described chromogen is N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-monomethylaniline sodium salt, phenol, methyl catechol, 3, one or more of 5-two chloro-2-hydroxybenzene phenolic acid sodium.
6. S-adenosylmethionine Methyl transporters enzymatic determination test kit according to claim 3, it is characterized in that: described stablizer is one or more in polyvalent alcohol, sodium ethylene diamine tetracetate, bovine serum albumin, N.F,USP MANNITOL, disaccharides, the polysaccharide.
7. S-adenosylmethionine Methyl transporters enzymatic determination test kit according to claim 3 is characterized in that: described sanitas is one or more in triazo-compound, the PC series sanitas.
8. S-adenosylmethionine Methyl transporters enzymatic determination test kit according to claim 3, it is characterized in that: described tensio-active agent is one or more in tween, the triton x-100.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104263810A (en) * | 2014-05-09 | 2015-01-07 | 山东博科生物产业有限公司 | A detection agent for homocysteine |
| CN106086160A (en) * | 2016-04-28 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring 5'Nueleotidme and preparation method thereof |
| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
| CN106282309A (en) * | 2016-08-31 | 2017-01-04 | 广东省中医院 | A kind of ADA Adenosine deaminase detection method |
| CN107653298A (en) * | 2017-11-15 | 2018-02-02 | 浙江夸克生物科技有限公司 | Adenosine deaminase determines kit |
| CN105567786B (en) * | 2016-03-10 | 2019-01-25 | 北京中科唯新生物医学研究所有限公司 | A kind of thiopurine methyltransferase enzyme activity detection kit |
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| CN114088692A (en) * | 2021-11-13 | 2022-02-25 | 普十生物科技(北京)有限公司 | Reduction reagent for HCY detection, kit and use method thereof |
| CN116840197A (en) * | 2022-03-23 | 2023-10-03 | 中国科学院苏州生物医学工程技术研究所 | 2' -O-methyltransferase activity detection method, kit and application |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263810A (en) * | 2014-05-09 | 2015-01-07 | 山东博科生物产业有限公司 | A detection agent for homocysteine |
| CN105567786B (en) * | 2016-03-10 | 2019-01-25 | 北京中科唯新生物医学研究所有限公司 | A kind of thiopurine methyltransferase enzyme activity detection kit |
| CN106086160A (en) * | 2016-04-28 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring 5'Nueleotidme and preparation method thereof |
| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
| CN106282309A (en) * | 2016-08-31 | 2017-01-04 | 广东省中医院 | A kind of ADA Adenosine deaminase detection method |
| CN107653298A (en) * | 2017-11-15 | 2018-02-02 | 浙江夸克生物科技有限公司 | Adenosine deaminase determines kit |
| CN112129719A (en) * | 2020-09-10 | 2020-12-25 | 浙江五味和食品有限公司 | Method and kit for measuring histamine in soy sauce and vinegar |
| CN114088692A (en) * | 2021-11-13 | 2022-02-25 | 普十生物科技(北京)有限公司 | Reduction reagent for HCY detection, kit and use method thereof |
| CN116840197A (en) * | 2022-03-23 | 2023-10-03 | 中国科学院苏州生物医学工程技术研究所 | 2' -O-methyltransferase activity detection method, kit and application |
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