CN100454002C - Method for measuring content of cozymase I and derivative thereof - Google Patents
Method for measuring content of cozymase I and derivative thereof Download PDFInfo
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- CN100454002C CN100454002C CNB200610050790XA CN200610050790A CN100454002C CN 100454002 C CN100454002 C CN 100454002C CN B200610050790X A CNB200610050790X A CN B200610050790XA CN 200610050790 A CN200610050790 A CN 200610050790A CN 100454002 C CN100454002 C CN 100454002C
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Abstract
The present invention discloses a method for measuring the content of coenzyme I and derivatives thereof. In the method, a sodium pyrophosphate buffer solution with the pH value of 8.0 to 11.0 is prepared, which contains 0.01 to 0.3 vol% of hydrazine hydrate; a substrate solution and a measuring solution of coenzyme I to be measured and derivatives thereof are added in the buffer solution; after the zero adjustment at the maximum absorption peak of reduced coenzyme I and derivatives thereof on a spectrophotometer, 3 alpha-steroid dehydrogenase is added for reaction till the absorbance value does not increase, and according to the change of absorbance values, the content of coenzyme I is calculated, wherein the 3 alpha-steroid dehydrogenase is a raw material commonly used by diagnostic reagents, and the source is wide; hydrazine is added in the reaction buffer solution to cause the reaction to be carried out in the positive direction, the coenzyme I and the derivatives thereof are transformed into reduced coenzyme I and derivatives thereof, and according to the molar absorbance value of the reduced coenzyme I and the derivatives thereof at a specific wavelength, the content of coenzyme I and the derivatives thereof can be quantitatively calculated.
Description
Technical field
The present invention relates to the assay method of a kind of coenzyme I and derivative content thereof.
Background technology
The change of characteristic absorption can appear during the two-way transformation of NAD and reduced diphosphopyridine nucleotide.Under certain condition, can be linear with the concentration of measured object and change, many chemical analysis methods are based on that this principle realizes, but there are many defectives in the chemical property of oxidized form/reduced diphosphopyridine nucleotide.The chemical property of oxidized form/reduced diphosphopyridine nucleotide derivant has then had bigger change, and (mainly showing as maximum absorption band change, the increase of molar absorptivity value, the change of reaction Km value, stability of solution and stable pH range increases greatly.) maximum absorption wavelength of reduced diphosphopyridine nucleotide derivant is between 340-405nm, the oxidation-reduction reaction that oxidized form/reduced diphosphopyridine nucleotide derivant participates in can pass through the variation detection by quantitative biochemical reaction substrate of light absorption value, thereby is the core material of many biochemistry detection reagent.
The purity of raw material can have influence on the quality of diagnostic reagent, and in the process of reagent preparation, what be necessary monitors the raw material quality.In addition, in the production run of coenzyme I and derivant thereof, because processing step is more, it is also very important that each step is carried out purity testing.Coenzyme I and derivant kind thereof are more, lack unified detection method at present.If adopt HPLC (high performance liquid chromatogram) method, process is comparatively loaded down with trivial details, needs standard items, actually applies inconvenience.
In the presence of dehydrogenasa, coenzyme I and derivant thereof participate in reaction as coenzyme, and NAD and derivant will be converted into reduced diphosphopyridine nucleotide and derivant thereof, but reaction generally is reversible reaction, because the product feedback inhibition, coenzyme I and derivant can not transform fully.
Adding semicarbazides in the reaction that alcohol dehydrogenase participates in can fix product, makes reaction transfer unidirectional response to.Utilize this principle to measure the existing report of method of coenzyme I content, but alcohol dehydrogenase derives from yeast, needs oneself to extract, practical operation comparatively bothers.
Summary of the invention
The assay method that the purpose of this invention is to provide a kind of coenzyme I and derivative content thereof.
Measure the method for coenzyme I and derivant thereof: configuration 0.03-0.2mol/L, pH8.0-11.0 sodium pyrophosphate buffer solution, wherein containing percent by volume is the 0.01%-0.3% hydrazine hydrate, add substrate and coenzyme I and derivant liquid to be determined thereof, concentration of substrate is 0.005-0.08mg/ml, and the concentration of coenzyme I and derivant liquid to be determined thereof is 0.01-0.3mg/ml; On spectrophotometer, after the zeroing of the maximum absorption band place of reduced diphosphopyridine nucleotide and derivant thereof, press 3 α-steroid dehydrogenase of reaction volume adding 0.05-1U/ml, react no longer increase to light absorption value till, according to the content of light absorption value change calculations coenzyme I.Calculate coenzyme I and derivative content thereof according to following computing formula, computing formula is: coenzyme I and derivant thereof liquid sample size to be determined (w/v)=(A-A
0) * V
1* Mw/ (ε * V
2) * 100%, wherein A is for adding 3 α-final light absorption value of steroid dehydrogenase enzyme reaction, A
0For not adding the final light absorption value of 3 α-steroid dehydrogenase, V
1Be reaction volume, V
2Long-pending for coenzyme I and derivant liquid to be determined thereof, Mw is a coenzyme I or derivatives thereof molecular weight, and ε is the molar absorptivity value of reduced diphosphopyridine nucleotide or derivatives thereof.
Described substrate is androsterone, sodium taurocholate or NaTDC.Coenzyme I and derivant thereof are coenzyme I, sulfo-coenzyme I or 3-acetylpyridine coenzyme I.
3 α of the present invention-steroid dehydrogenase is the raw material commonly used of diagnostic reagent, wide material sources.This method utilizes coenzyme I and derivant thereof to participate in redox reaction in the presence of 3 α-steroid dehydrogenase, and with hydrazine product is fixed, and reaction is carried out to positive dirction.Coenzyme I and derivant will all be converted into reduced diphosphopyridine nucleotide and derivant, according to reduced diphosphopyridine nucleotide and the derivant molar absorptivity value at specific wavelength, can quantitative calculation go out the content of coenzyme I and derivant.
HPLC (high performance liquid chromatogram) and this method detect such as table
Embodiment
Measuring principle of the present invention
3 α-steroid dehydrogenase is a coenzyme with NAD and derivant, reacts under alkali condition and higher concentration of substrate.Hydrazine hydrate is fixed reaction product, makes coenzyme I and derivant almost all turn to reduced diphosphopyridine nucleotide and derivant, according to reduced diphosphopyridine nucleotide and the derivant molar absorptivity value at specific wavelength, can quantitative calculation goes out the content of coenzyme I and derivant.
Sample determination liquid
The accurate sample about weighing 0.1g, distilled water constant volume to final concentration between 0.01-0.3mg/ml.
Enzyme reaction buffer solution
Add 50% hydrazine hydrate in the sodium pyrophosphate buffer solution, the hydrazine hydrate percent by volume is 0.01%-0.3%.
Substrate solution
Androsterone is dissolved in the methyl alcohol in advance, 2mg/ml;
Cholate or dexycholate can directly add in the enzyme reaction buffer solution.
3 α-steroid dehydrogenase glycerin liquid
3 α-steroid dehydrogenase is dissolved in pH=7.5, and 0.05mol/L phosphate wherein contains 50% (v/v) glycerine in liquid, 100mmol/L NaCl, and EDTA 1mmol/L, the concentration of 3 α-steroid dehydrogenase is 250U/ml.
Embodiment 1: measure coenzyme I
Configuration 0.06mol/L, pH8.9 sodium pyrophosphate buffer solution, wherein containing percent by volume is 0.03% hydrazine hydrate.Add substrate and liquid to be determined, substrate is an androsterone, and concentration is 0.02mg/ml, and the concentration of measuring liquid is 0.01-0.3mg/ml; On spectrophotometer, after 340nm zeroing, press 3 α-steroid dehydrogenase of reaction volume adding 0.3U/ml, react no longer increase to light absorption value till, according to the content of light absorption value change calculations coenzyme I.Calculate coenzyme I and derivative content thereof according to following computing formula, computing formula is: liquid sample size to be determined (w/v)=(A-A
0) * V
1* 663/ (6220 * V
2) * 100%, wherein A is for adding 3 α-final light absorption value of steroid dehydrogenase enzyme reaction, A
0For not adding the final light absorption value of 3 α-steroid dehydrogenase, V
1Be reaction volume, 663 is the coenzyme I molecular weight, and 6220 is the molar absorptivity value of reduced diphosphopyridine nucleotide.
Measurement result
Embodiment 2: measure the sulfo-coenzyme I
Be determined under the lucifuge and carry out, configuration 0.03mol/L, pH8.0 sodium pyrophosphate buffer solution, wherein containing percent by volume is 0.01% hydrazine hydrate.Add substrate and liquid to be determined, substrate is a sodium taurocholate, and concentration is 0.005mg/ml, and the concentration of measuring liquid is 0.01-0.3mg/ml; On spectrophotometer, after 398nm zeroing, press 3 α-steroid dehydrogenase of reaction volume adding 0.05U/ml, react no longer increase to light absorption value till, according to the content of light absorption value change calculations coenzyme I.Calculate coenzyme I and derivative content thereof according to following computing formula, computing formula is: liquid sample size to be determined (w/v)=(A-A
0) * V
1* 680/ (11900 * V
2) * 100%, wherein A is for adding 3 α-final light absorption value of steroid dehydrogenase enzyme reaction, A
0For not adding the final light absorption value of 3 α-steroid dehydrogenase, V
1Be reaction volume, 680 is sulfo-coenzyme I molecular weight, and 11900 is the molar absorptivity value of reduced form sulfo-coenzyme I.
Embodiment 3: measure 3-acetylpyridine coenzyme I
Configuration 0.2mol/L, pH11.0 sodium pyrophosphate buffer solution, wherein containing percent by volume is 0.3% hydrazine hydrate.Add substrate and liquid to be determined, substrate is a NaTDC, and concentration is 0.08mg/ml, and the concentration of measuring liquid is 0.01-0.3mg/ml; On spectrophotometer, after 363nm zeroing, press 3 α-steroid dehydrogenase of reaction volume adding 1U/ml, react no longer increase to light absorption value till, according to the content of light absorption value change calculations coenzyme I.Calculate 3-acetylpyridine coenzyme I content according to following computing formula, computing formula is: liquid sample size to be determined (w/v)=(A-A
0) * V
1* 662/ (7800 * V
2) * 100%, wherein A is for adding 3 α-final light absorption value of steroid dehydrogenase enzyme reaction, A
0For not adding the final light absorption value of 3 α-steroid dehydrogenase, V
1Be reaction volume, 662 is 3-acetylpyridine coenzyme I molecular weight, and 7800 is 3-acetylpyridine coenzyme I molar absorptivity value.
Claims (2)
1. the assay method of coenzyme I and derivative content thereof, it is characterized in that: configuration 0.03-0.2mol/L, pH8.0-11.0 sodium pyrophosphate buffer solution, wherein containing percent by volume is the 0.01%-0.3% hydrazine hydrate, add substrate and coenzyme I and derivant liquid to be determined thereof, concentration of substrate is 0.005-0.08mg/ml, and the concentration of coenzyme I and derivant liquid to be determined thereof is 0.01-0.3mg/ml; On spectrophotometer, after the zeroing of the maximum absorption band place of reduced diphosphopyridine nucleotide and derivant thereof, press 3 α-steroid dehydrogenase that reaction volume adds 0.05-1U/ml, react no longer increase to light absorption value till, content according to light absorption value change calculations coenzyme I, calculate coenzyme I and derivative content thereof according to following computing formula, computing formula is: coenzyme I and derivant thereof liquid sample size to be determined (w/v)=(A-A
0) * V
1* Mw/ (ε * V
2) * 100%, wherein A is for adding 3 α-final light absorption value of steroid dehydrogenase enzyme reaction, A
0For not adding the final light absorption value of 3 α-steroid dehydrogenase, V
1Be reaction volume, V
2Long-pending for coenzyme I and derivant liquid to be determined thereof, Mw is a coenzyme I or derivatives thereof molecular weight, and ε is the molar absorptivity value of reduced diphosphopyridine nucleotide or derivatives thereof, and described substrate is androsterone, sodium taurocholate or NaTDC.
2. the assay method of a kind of coenzyme I according to claim 1 and derivative content thereof is characterized in that: described coenzyme I and derivant thereof are coenzyme I, sulfo-coenzyme I or 3-acetylpyridine coenzyme I.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200610050790XA CN100454002C (en) | 2006-05-17 | 2006-05-17 | Method for measuring content of cozymase I and derivative thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200610050790XA CN100454002C (en) | 2006-05-17 | 2006-05-17 | Method for measuring content of cozymase I and derivative thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1865928A CN1865928A (en) | 2006-11-22 |
| CN100454002C true CN100454002C (en) | 2009-01-21 |
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|---|---|---|---|
| CNB200610050790XA Active CN100454002C (en) | 2006-05-17 | 2006-05-17 | Method for measuring content of cozymase I and derivative thereof |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507463B (en) * | 2011-09-30 | 2013-07-03 | 浙江夸克生物科技有限公司 | Cyclophorase detection method for quantitative detection of thio-nicotinamide-adenine dinucleotide (Thio-NAD) and kit |
| CN103743689B (en) * | 2014-01-03 | 2015-09-09 | 苏州科铭生物技术有限公司 | A kind of cozymase NAD or NADH reagent box for detecting content |
| CN114236012A (en) * | 2021-12-21 | 2022-03-25 | 上海蔚之星生物科技有限公司 | High performance liquid chromatography analysis method for coenzyme I content detection and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5444597A (en) * | 1977-09-14 | 1979-04-09 | Shimadzu Corp | Measuring method of bile acids |
| JPS60137300A (en) * | 1983-12-26 | 1985-07-20 | Dai Ichi Pure Chem Co Ltd | Determination of 3alpha-hydroxysteroid |
| CN1311335A (en) * | 2000-03-02 | 2001-09-05 | 上海复旦张江生物医药有限公司 | Reagent for stabilizing reducing coenzyme I and reducing coenzyme II |
-
2006
- 2006-05-17 CN CNB200610050790XA patent/CN100454002C/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5444597A (en) * | 1977-09-14 | 1979-04-09 | Shimadzu Corp | Measuring method of bile acids |
| JPS60137300A (en) * | 1983-12-26 | 1985-07-20 | Dai Ichi Pure Chem Co Ltd | Determination of 3alpha-hydroxysteroid |
| CN1311335A (en) * | 2000-03-02 | 2001-09-05 | 上海复旦张江生物医药有限公司 | Reagent for stabilizing reducing coenzyme I and reducing coenzyme II |
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|---|---|
| CN1865928A (en) | 2006-11-22 |
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Effective date of registration: 20170106 Address after: 225312 Jiangsu city of Taizhou province Taizhou City Road on the west side, east medicine China Lu Jia Lu G59 building 62 No. three on the eastern side Patentee after: Ali biotechnology Taizhou Co., Ltd. Address before: 427 Hangzhou Wulin Road, Zhejiang, room 100, No. 310006 Patentee before: Hangzhou Li'an Biological Technology Co., Ltd. |