CN106970132A - Determine the electrophoresis titration method of peroxidase activity - Google Patents
Determine the electrophoresis titration method of peroxidase activity Download PDFInfo
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- CN106970132A CN106970132A CN201610021350.5A CN201610021350A CN106970132A CN 106970132 A CN106970132 A CN 106970132A CN 201610021350 A CN201610021350 A CN 201610021350A CN 106970132 A CN106970132 A CN 106970132A
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Abstract
The invention discloses a kind of electrophoresis titration method for determining peroxidase activity, methods described includes：The channel part of chip is filled to the mixed solution b of injection chemiluminescence bottom liquid and background electrolyte in the mixed solution a that hydrochloric acid solution and background electrolyte are injected in gel, anode pool, cathode pool；Peroxidase Solution is injected in cathode pool, cataluminescence bottom liquid reaction generation excitation state luminol anion L* is allowed to；After enzymic catalytic reaction is stable, apply electric field, make the excitation state luminol anion L* and hydrogen ion H in anode pool generated in cathode pool+MRB is formed, the MRB of generation shifts to anode；The functional relation existed according to MRB rate travels and peroxidase activity, measures peroxidase activity index.The method of the present invention effectively can be determined quickly to every Enzyme target of peroxidase, and without complicated optical detection apparatus.
Description
Technical field
The present invention relates to bioassay technique and field of medical examination, and in particular to a kind of electrophoresis titration method of measure peroxidase activity.
Background technology
Enzyme is analyzed in clinical medicine (Abraham, E.C.；Huff,T.A.；Cope,N.D.；Wilson,J.B.；Bramsome ELISA,,E.D.Jr.；Huisman, T.H.J.Diabetes 1978,27,931-937.) biology (Tannu, N.S.；Hemby, S.E.Nature Protocols 2006,1,1732-1942.) and drug screening (Li, Juan；Wu,Ling-Jie；Guo,Shan-Shan；Fu,Hua-E；Chen,Guo-Nan；Yang, Huang-Hao, NANOSCALE, 2013,5,619-623.) play the role of in field it is important.Traditional detection method is generally basede on substrate in detection enzyme reaction system and reduces and product generate to carry out catalytic rate, activity, the measure of Michaelis constant.Existing many methods can be used for enzyme detection, such as mass spectrum (Northen, TR at present；Lee,JC；Hoang,L；Raymond,J；Hwang,DR；Yannone,SM；Wong,CH；Siuzdak, G, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2008,105,3678-3683.), electrophoresis electrophoresis (Y F Chen, L L Xu, W W Zhao, L P Guo, L Yang, Anal.Chem., 2012,84,2961-2967.), liquid chromatogram (Lahoz, A；Donato,MT；Castell,JV；Gomez-Lechon, MJ, CURRENT DRUG METABOLISM, 2008,9,12-19.) and electrochemical method (Bernhardt, Paul V.CHEM.COM., 2011,47,1663-1673.) and enzyme linked immunosorbent detection ELISA (Ciaccafava, A；Infossi,P；Ilbert,M；Guiral,M；Lecomte,S；Giudici-Orticoni,MT；Lojou,E,ANGEWANDTE CHEMIE-INTERNATIONAL EDITION,2012,51,953-956.).
Peroxidase is a class oxidoreducing enzyme, can be catalyzed many reactions.Peroxidase is the enzyme that substrate oxidation is catalyzed by electron acceptor of hydrogen peroxide.Be primarily present in the peroxisome of cell, using ferriporphyrin as prothetic group, can catalyzing hydrogen peroxide oxidation phenols and aminated compounds, with eliminating hydrogen peroxide and phenols, the double action of amine toxicity.And for the measure of peroxidase, conventional method is activity (the Volker Herzoga for passing through colorimetric method for determining horseradish peroxidase as hydrogen ion donor with trimethylbiphenyl amine；H.Dariush Fahimia, Analytical Biochemistry, October 1973,554-562.), also have by substrate of para-aminophenol and determine electrochemical method (the Wei Suna of peroxidase by determining the front and rear conductivity variations of reaction；Kui Jiao,Analytica Chimica Acta,434,2001,43–50.).The above method has many good qualities, but faces some shortcomings simultaneously, and such as flux is low, and cost is high and sample consumption is big etc..
The content of the invention
For defect of the prior art, the invention provides a kind of electrophoresis titration method (moving reaction boundary electrophoresis titration, MRBET), specially a kind of electrophoresis titration method for determining peroxidase activity.The present invention has carried out the theory and technology development of novelty, is successfully realized the measure of peroxidase activity.
The purpose of the present invention is achieved through the following technical solutions：
The present invention relates to a kind of electrophoresis titration method for determining peroxidase activity, it the described method comprises the following steps：
A, the mixed solution b that the channel part of chip is filled to injection chemiluminescence bottom liquid and background electrolyte in the mixed solution a that hydrochloric acid solution and background electrolyte are injected in gel, anode pool, cathode pool；
B, Peroxidase Solution injected in cathode pool, be allowed to cataluminescence bottom liquid reaction generation excitation state luminol anion L*；
After C, enzymic catalytic reaction are stable, apply electric field, make the excitation state luminol anion L* and hydrogen ion H in anode pool generated in cathode pool+MRB is formed, the MRB of generation shifts to anode；
D, the functional relation existed according to MRB rate travels and peroxidase activity, measure peroxidase activity index.
Preferably, in step A, the gel is Ago-Gel, and mass fraction is 1%~2%.
Preferably, in step A, background electrolyte is contained in the gel.
Preferably, in step A, the background electrolyte is the sodium chloride or Klorvess Liquid that ionic strength is 20mmol/L~250mmol/L.
Preferably, the ionic strength of the background electrolyte in the gel is identical with mixed solution a ionic strengths.
Preferably, the ionic strength of the background electrolyte in the gel is identical with the ionic strength of background electrolyte in cathode pool.
In step A, the electrolyte in anode pool is that the ionic strength of the electrolyte in the sodium chloride or potassium chloride mixed solution that hydrochloric acid and ionic strength are 20mmol/L~250mmol/L, the anode pool is identical with the ionic strength of the background electrolyte in gel；In the step B, the electrolyte in cathode pool is that the ionic strength of the electrolyte in the sodium chloride or Klorvess Liquid that ionic strength is 20mmol/L~250mmol/L, the cathode pool is identical with the ionic strength of the background electrolyte in the gel.
Preferably, in step C, the electric field of the application is 10V/mm~15V/mm.
Preferably, in step D, the peroxidase activity index includes enzymatic activity, enzyme's reaction speeding, maximum reaction rate, catalytic constant.
Preferably, the calculation equation of the enzymatic activity is：
The calculation equation of the specific activity of the enzyme is：
The enzyme's reaction speeding calculation equation is：
The calculation equation of the maximum reaction rate and Michaelis constant is：
Adjust substrate H2O2Concentration, according to measuring v in the case of different concentration of substrateMRB, v can be obtainedmaxWith Km。
The catalytic constant calculation equation is：
Wherein, in aforesaid equation, cL*And vL*It is the concentration (mol/L) and migration rate (m/s) of excitation state luminol anion, c respectivelyH+And vH+It is hydrionic concentration (mol/L) and migration rate (m/s) respectively；VMRB, the rate travel for being MRB (m/s)；VenWith Δ tenIt is the liquor capacity (mL) and time of enzymatic reacting (S) of enzymic catalytic reaction system respectively；cenIt is that enzyme concentration (mg/mL) Δ t in cathode pool in enzymatic reaction pond is to apply the electric field time (S)；cL,cuIt is substrate and instantaneous concentration (mg/mL)；[ET] be enzyme concentration (mg/mL).
Function derivation is as follows in the MRBET：
MRB rate travels are：
L1It is t1The distance of time MRB movements, L2It is t2The distance of time MRB movements.t1To apply the time of electric field, t for the first time2For the time of second of application electric field.
It is by the excitation state luminol anion L* MRB rate travels formed with hydrogen ion H+：
Wherein cL*and vL*It is the concentration (mol/L) and migration rate (m/S) of excitation state luminol anion, c respectivelyH+And vH+It is hydrionic concentration (mol/L) and migration rate (m/S) respectively.So, the concentration of excitation state luminol anion can be simply expressed as：
Assuming that product (excitation state luminol anion L*) has all been converted into by all substrates of enzymatic reaction (luminol), then concentration of substrate can be expressed as in enzymatic reaction pond：
cL,inAnd cL,cuIt is the initial concentration and instantaneous concentration of substrate (luminol) respectively.
According to the definition (1U=1 μm of ol/min) of enzymatic activity, following enzymatic activity expression formula can be obtained：
Expression formula (4) is substituted into (5), obtained：
VenWith Δ tenIt is the liquor capacity and time of enzymatic reacting of enzymic catalytic reaction system in cathode pool respectively.Therefore, the specific activity of enzyme can be expressed as：
Wherein, cenRefer to the enzyme concentration (mg/mL) in cathode pool in enzymatic reaction pond.
Enzyme's reaction speeding vrateIt is defined as
Δ t is enzymic catalytic reaction time (S)；Δ c is concentration of substrate change.
Expression formula (3) is substituted into expression formula (8), the c in formula (3)L*Δ c in corresponding (8), can be obtained
In expression formula (9), MRB rate travels can be obtained from expression formula (1).
In enzyme kinetic analysis, Michaelis-Menten equation is as follows：
vrateIt is enzyme's reaction speeding, Km is Michaelis constant, vmaxIt is maximum enzyme's reaction speeding, [H2O2] it is substrate H2O2Concentration by expression formula (9) substitute into expression formula (10), can obtain：
Due in the reaction,Concentration with luminol and luminol* is that difference is equal, and the H in related experiment2O2Concentration and luminol concentration very close to therefore, expression formula (11) can be written as：
In enzyme reaction system, Michaelis-Menten equation can be expressed as：
By expression formula (9) and [H2O2]=2cL,cuExpression formula (13) is substituted into, can be obtained
Adjust substrate H2O2Concentration, according to measuring v in the case of different concentration of substrateMRB, v can be obtainedmaxWith Km。
And according to：
vmax=Kcat[ET] (15)
Herein, [ET] is the total concentration (mg/mL) of enzyme in reaction system, with reference to expression formula (14) and (15), can be obtained：
Expression formula (16) is the expression formula that enzymatic constant is calculated by moving reaction boundary.
Compared with prior art, the present invention has following beneficial effect：
1st, this method can effectively detect the activity of various peroxidase；
2nd, this method speed is fast, and titration process can be completed in 20 seconds；
3rd, this method only needs to visualize under dark room conditions, without complicated optical detecting instrument equipment.
Brief description of the drawings
By reading the detailed description made with reference to the following drawings to non-limiting example, other features, objects and advantages of the invention will become more apparent upon：
Fig. 1 is MRBET schematic diagrames of the invention.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art and further understand the present invention, but the invention is not limited in any way.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to protection scope of the present invention.
Embodiment
1
In the present embodiment, horseradish peroxidase activity is determined using electrophoresis titration method, described electrophoresis titration method is as shown in Figure 1.The channel part of chip is filled to injection chemiluminescence bottom liquid and the mixed solution b with background electrolyte in the mixed solution a that hydrochloric acid solution and background electrolyte are injected in gel, anode pool, cathode pool first；HRPO solution is injected in cathode pool immediately after, the excitation state luminol anion L* that the liquid reaction generation of cataluminescence bottom sends blue-fluorescence is allowed to；Enzymic catalytic reaction after a period of stabilisation after, apply voltage so that the excitation state luminol anion L* generated in cathode pool face south Ghandler motion it is dynamic and with hydrogen ion H from anode pool to cathode direction that moved in+MRB is formed, MRB shifts to anode；During titration, blue-fluorescence is the position for denoting interface, can carry out visual inspection；Afterwards, using the relation between MRB Interface Movings speed and luminol concentration, according to a series of derivation formulas, enzyme's reaction speeding, the enzymatic activity of horseradish peroxidase, Michaelis constant, maximum enzyme's reaction speeding, catalytic constant can be calculated.
Relation between the MRB Interface Movings speed and luminol concentration is
l1It is t1The distance of time MRB movements, l2It is t2The distance of time MRB movements.t1To apply the time of electric field, t for the first time2For the time of second of application electric field.
The calculation formula of the enzymatic activity is：
Ven andΔtenIt is the liquor capacity and time of enzymatic reacting of enzymic catalytic reaction system respectively.
The specific activity calculation formula of the enzyme is：
cenRefer to the enzyme concentration (mg/mL) in enzymatic reaction pond.
The enzyme's reaction speeding calculation formula is：
Change substrate concentration of hydrogen peroxide, draw MRB Interface Movings speed under different condition.
The concentration of excitation state luminol under the conditions of different concentration of substrate is calculated according to expression formula (3).
The concentration of excitation state luminol under the conditions of the different concentration of substrate calculated is brought into expression formula (13), the relational expression of maximum enzyme's reaction speeding and Michaelis constant can be obtained：
Adjust substrate H2O2Concentration, according to measuring v in the case of different concentration of substrateMRB, v can be obtainedmaxWith Km。
Again by below equation,
vmax=Kcat[ET] (15)
[ET] represent enzyme concentration.Therefore, with reference to expression formula (14) and (15), the catalytic constant K of enzyme is obtainedcat,
The gel used in the MRBET titration is Ago-Gel, and weight fraction is 1%~2%.
Contain background electrolyte in the gel.
The background electrolyte is sodium chloride or Klorvess Liquid of the ionic strength from 20mmol/L to 250mmol/L.
The ionic strength of background electrolyte in the gel is identical with mixed solution a ionic strengths.
The ionic strength of background electrolyte in the gel is identical with the ionic strength of background electrolyte in cathode pool.
The horseradish peroxidase activity index result that the present embodiment is measured is：Maximum reaction rate：3.1×10-4mM/s；Michaelis constant：3.15×10-4, catalytic constant：619.
The horseradish peroxidase activity of the present embodiment is measured using traditional fluorescence microplate reader method, and its result is：Maximum reaction rate：3.15×10-4mM/s；Michaelis constant：3.09×10-4, catalytic constant：615.It is consistent with the result that the present embodiment is determined, illustrate the reliability of the inventive method.
In summary, the electrophoresis titration method of the present invention for determining peroxidase activity, can effectively detect the activity of various peroxidase；This method speed is fast, and titration process can be completed in 20 seconds；This method only needs to visualize under dark room conditions, without complicated optical detecting instrument equipment.
The specific embodiment of the present invention is described above.It is to be appreciated that the present invention is not limited to the above specific embodiments, those skilled in the art can make various deformations or amendments within the scope of the claims, and this has no effect on the substantive content of the present invention.
Claims (10)
1. a kind of electrophoresis titration method for determining peroxidase activity, it is characterised in that the described method comprises the following steps：
A, the mixed solution b that the channel part of chip is filled to injection chemiluminescence bottom liquid and background electrolyte in the mixed solution a that hydrochloric acid solution and background electrolyte are injected in gel, anode pool, cathode pool；
B, Peroxidase Solution injected in cathode pool, be allowed to cataluminescence bottom liquid reaction generation excitation state luminol anion L*；
After C, enzymic catalytic reaction are stable, apply electric field, make the excitation state luminol anion L* and hydrogen ion H in anode pool generated in cathode pool+MRB is formed, the MRB of generation shifts to anode；
D, the functional relation existed according to MRB rate travels and peroxidase activity, measure peroxidase activity index.
2. the electrophoresis titration method according to claim 1 for determining peroxidase activity, it is characterised in that in step A, the gel is Ago-Gel, mass fraction is 1%~2%.
3. the electrophoresis titration method according to claim 1 for determining peroxidase activity, it is characterised in that in step A, contain background electrolyte in the gel.
4. the electrophoresis titration method of the measure peroxidase activity according to claim 1 or 3, it is characterised in that the background electrolyte is the sodium chloride or Klorvess Liquid that ionic strength is 20mmol/L~250mmol/L.
5. the electrophoresis titration method according to claim 3 for determining peroxidase activity, it is characterised in that the ionic strength of the background electrolyte in the gel is identical with mixed solution a ionic strengths.
6. the electrophoresis titration method according to claim 3 for determining peroxidase activity, it is characterised in that the ionic strength of the background electrolyte in the gel is identical with the ionic strength of background electrolyte in cathode pool.
7. the electrophoresis titration method according to claim 1 for determining peroxidase activity, it is characterised in that in step C, the electric field of the application is 10V/mm~15V/mm.
8. the electrophoresis titration method according to claim 1 for determining peroxidase activity, it is characterised in that in step D, the peroxidase activity index includes：Enzymatic activity, the specific activity of enzyme, enzyme's reaction speeding, maximum reaction rate, Michaelis constant, catalytic constant.
9. the electrophoresis titration method according to claim 8 for determining peroxidase activity, it is characterised in that
The calculation equation of the enzymatic activity is：
The calculation equation of the specific activity of the enzyme is：
The enzyme's reaction speeding calculation equation is：
Wherein, in aforesaid equation, cL*And vL*It is the concentration and migration rate of excitation state luminol anion, c respectivelyH+And vH+It is hydrionic concentration and migration rate respectively；VMRBFor MRB rate travel；VenWith Δ tenIt is the liquor capacity and time of enzymatic reacting of enzymic catalytic reaction system respectively；cenIt is the enzyme concentration in cathode pool in enzymatic reaction pond.
10. the electrophoresis titration method according to claim 8 for determining peroxidase activity, it is characterised in that
The calculation equation of the maximum reaction rate and Michaelis constant is：
The calculation equation of the catalytic constant is：
Wherein, in aforesaid equation, vL*It is the migration rate of excitation state luminol anion, cH+And vH+It is hydrionic concentration and migration rate respectively；VMRBFor MRB rate travel；ΔtenIt is the time of enzymatic reacting of enzymic catalytic reaction system；cL,cuIt is the instantaneous concentration of substrate；[ET] be enzyme concentration.
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Cited By (4)
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| CN109541005A (en) * | 2018-12-12 | 2019-03-29 | 上海交通大学 | Uric acid visualization measurement method based on moving reaction boundary electrophoresis titration chip |
| CN110082419A (en) * | 2019-04-30 | 2019-08-02 | 上海交通大学 | A kind of method of quick detection nano grain surface ligand content |
| CN110361440A (en) * | 2018-04-09 | 2019-10-22 | 上海交通大学 | The portable electrophoresis of alkaline phosphatase activities titrates detection method |
| CN112029816A (en) * | 2020-09-11 | 2020-12-04 | 南京医科大学 | Method for rapidly detecting activity of single biomacromolecule |
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| CN104713933A (en) * | 2015-03-27 | 2015-06-17 | 上海交通大学 | Electrophoresis titration method for quantitatively determining adulteration degree of dairy product |
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| US20030003598A1 (en) * | 2001-05-10 | 2003-01-02 | Invitrogen Corporation | Method, system and computer program product for measuring unit activity of an enzyme |
| CN103175885A (en) * | 2013-03-20 | 2013-06-26 | 上海交通大学 | High-flux protein titration method based on moving reaction boundary electrophoresis |
| CN104713933A (en) * | 2015-03-27 | 2015-06-17 | 上海交通大学 | Electrophoresis titration method for quantitatively determining adulteration degree of dairy product |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110361440A (en) * | 2018-04-09 | 2019-10-22 | 上海交通大学 | The portable electrophoresis of alkaline phosphatase activities titrates detection method |
| CN110361440B (en) * | 2018-04-09 | 2021-08-06 | 上海交通大学 | Portable electrophoresis titration detection method for activity of alkaline phosphatase |
| CN109541005A (en) * | 2018-12-12 | 2019-03-29 | 上海交通大学 | Uric acid visualization measurement method based on moving reaction boundary electrophoresis titration chip |
| CN110082419A (en) * | 2019-04-30 | 2019-08-02 | 上海交通大学 | A kind of method of quick detection nano grain surface ligand content |
| CN112029816A (en) * | 2020-09-11 | 2020-12-04 | 南京医科大学 | Method for rapidly detecting activity of single biomacromolecule |
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