CN100399017C - Screening method for medicine with electrophoresis in capillary zone - Google Patents
Screening method for medicine with electrophoresis in capillary zone Download PDFInfo
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- CN100399017C CN100399017C CNB031111653A CN03111165A CN100399017C CN 100399017 C CN100399017 C CN 100399017C CN B031111653 A CNB031111653 A CN B031111653A CN 03111165 A CN03111165 A CN 03111165A CN 100399017 C CN100399017 C CN 100399017C
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Abstract
The present invention provides a method for sieving medicaments by capillary zone electrophoresis, which has the following concrete steps: 1). a receptor, or a matching body is prepared to form a series of samples of standard concentration, an electrophoresis signal spectrogram is obtained through pressurized running and detection, peak height h is measured, each concentration corresponds to one peak height, and thus, a linear correlation equation is worked out to be used as a working curve; 2). a receptor, or a matching body having the concentration with the receptor, or the matching body in the step 1) is mixed with matching bodies, or receptors of different concentration for sampling, an electrophoresis signal spectrogram is obtained through pressurized running and detection under the same process condition with the condition in the step 1), peak height h is measured, and the h is substituted into the working curve to work the free concentration C[f] of the corresponding receptor, or matching body out; 3). if the receptor is used as an exploration object, and the C[f] is the concentration of the free receptor, then, gamma=(C[o]-C[f])/C[t], wherein the C[o] is the total concentration of the added receptor, and the C[t] is the total concentration of the added matching body; and 4). an equation that gamma/C[f]=-Kgamma+nK is adopted, and the binding constant K and the binding bit counting n of thermodynamics are quantitatively worked out according to the regression of the equation.
Description
Technical field:
The present invention relates to a kind of medicine be carried out method for screening, a kind ofly medicine is screened, be particularly useful for bioengineering with the capillary zone electrophoresis method.
Background technology:
In drug screening, adopt the affinity capillary electrophoresis method at present, as at pertinent literature:
1)D.Chen，L.Clohs，J.Chapman，“Capillary?Electrophoresis?and?Its?Applicationsin?Pharmaceutical?Industry”，the?educational?short?course?of?Canadian?Societyfor?Chemistry?Conference?and?Exhibition，2002，Vancouver，Canada.
2) J.N.Little, J.L.WATERS, D.E.Hughes; " Affinity capillary electro-phoresis forrapid assay development and Screening of newly discovered targets "; Societyfor Biomolecular Screening; 2001; among the USA; affinity capillary electrophoresis changes to come quantitatively by the migration rate of monitoring solute; reappearance is better, but acceptor or part need be joined in the running buffer, and the sample consumption rate is bigger, can only be used for the weak interaction system, and be used to weigh aspect the marker that electroosmotic flow changes also relatively harsher in searching.
Summary of the invention:
The purpose of this invention is to provide and a kind ofly more effectively medicine is carried out method for screening with capillary zone electrophoresis, this method separating effect is relatively good, and reagent and sample consumption are few, be applicable to strong combination and weak articulated system, can be used for the drug screening of natural products (as Chinese medicine), synthetic drug or biotech medicine product.
Capillary zone electrophoresis provided by the invention is carried out method for screening to medicine, and its concrete steps are as follows:
1) acceptor or part are mixed with series of standards concentration sample introduction, pressurization operation, detection obtain the electrophoresis signal spectrogram, measure peak height h, and the corresponding peak height of each concentration is tried to achieve the linear dependence equation, thus as working curve;
2) will be with 1) acceptor of same concentrations or part respectively with the part or the acceptor mixing sample introduction of variable concentrations; With 1) pressurization operation under the identical process conditions, detect, obtain the electrophoresis signal spectrogram, measure peak height h ', with h ' substitution working curve, try to achieve the Cf C of corresponding acceptor or part
f
3) be when investigating object with the acceptor, C
fBe the acceptor density that dissociates, γ=(C then
0-C
f)/C
t, wherein, C
0Be the acceptor total concentration that adds, C
tIt is the part total concentration that adds;
4) adopt equation γ/C
f=-K γ+nK,
In the formula: K is the thermodynamics binding constant,
N is a binding site number,
γ is the acceptor density that combines with part and the ratio of part total concentration the ratio of acceptor body total concentration (the part bulk concentration that combines with the acceptor body with).
Return by above-mentioned equation, quantitatively try to achieve thermodynamics binding constant K, binding site number n.
Find through experiment: the peak height when the receptor signal peak height when having only the acceptor sample introduction and acceptor, ligand mixture sample introduction (acceptor density under two kinds of situations is the same) has significant difference.The present invention utilizes the linear dependence of concentration and peak height just, by measuring the peak height of spectral line simply, then by step 1)-4) tried to achieve thermodynamics binding constant K, binding site number n quantitatively.
Capillary zone electrophoresis provided by the invention carries out can adopting the capillary column that is coated with stain in the method for screening to medicine, can be coated with the stain acrylamide on the capillary column, to obtain post effect and peak value preferably.
Capillary zone electrophoresis provided by the invention is carried out method for screening to medicine, and its acceptor can be an albumen, and part is a medicine; Acceptor can be an enzyme, and part is an enzyme inhibitor; Perhaps acceptor can be a nucleic acid, and part is a medicine.
Capillary zone electrophoresis provided by the invention is carried out method for screening to medicine, and its damping fluid is trishydroxymethylaminomethane-acetate (containing certain density sodium chloride) solution.
Capillary zone electrophoresis provided by the invention is carried out method for screening to medicine, and its detection method can be a ultraviolet, also can be fluorescence.
Capillary zone electrophoresis provided by the invention is carried out method for screening to medicine, wants incubation a period of time sample introduction more in case of necessity after mixing.
Quantitatively asking the binding constant of calculating medicine (part) and acceptor interaction with the capillary zone method mostly is medicine and albumen system, and draw binding constant and metering simultaneously in conjunction with very lacking, the report that can provide this information aspect two is simultaneously arranged at more not seeing on of nucleic acid and medicine than the example of (binding site number); Our method is suitable for receptor-ligand systems such as albumen-medicine, enzyme-inhibitor, nucleic acid-medicine, and coverage rate is wider, and can provide simultaneously binding constant and the metering in conjunction with than.
Effect such as when our capillary electrophoresis method has low cost, efficient, joint, and can adopt the interaction of capillary zone technology quantitative expedition medicine (part) and acceptor flexibly, and can compare its rationality, be undoubtedly a kind of very attractive method in the drug screening of this method in bioengineering.
Description of drawings:
Fig. 1 is 11.1 μ M 14-merDNA and variable concentrations netropsin results of interaction
Line is 0 μ M netropsin a),
Line b) 3 μ M netropsins,
Line c) 19.8 μ M netropsins,
Line d) 23.8 μ M netropsins,
Line e) 39.9 μ M netropsins;
Fig. 2 is that curve according to Fig. 1 is by Scatchard equation regression results.
Embodiment:
Reaction system with netropsin and double-stranded DNA is an example: adopt 20mM trishydroxymethylaminomethane-acetate (Tris-acetate) on the technology, 10mM sodium chloride (pH7.0-7.2) is running buffer, use ultraviolet detection (260nm), acrylamide layer capillary column (the column length 31.2cm of internal diameter 50 μ m, effective long 21cm), sample introduction is 4 seconds under pressure 0.5psi, applies the reverse voltage operation of 8kV; Acceptor is 11.1 μ M 14-mer DNA, and part is respectively 0 μ M netropsin, 3 μ M netropsins, 19.8 μ M netropsins, 23.8 μ M netropsins, 39.9 μ M netropsins; Detect signal (peak shape and post the are imitated all relatively good) (see figure 1) of DNA easily, and through pressing equation γ/C
f=-K γ+nK returns, and reflects the variation that adds DNA signal behind the netropsin: γ/C well
f=0.125-0.107 γ, i.e. K=0.107, nK=0.125, n=1.168.
Claims (10)
1. a capillary zone electrophoresis is carried out method for screening to medicine, it is characterized in that concrete steps are as follows:
1) acceptor is mixed with series of standards concentration sample introduction, pressurization operation, detection obtain the electrophoresis signal spectrogram, measure peak height h, and the corresponding peak height of each concentration is tried to achieve the linear dependence equation, thus as working curve;
2) will be with 1) acceptor of same concentrations respectively with the part mixing sample introduction of variable concentrations; With 1) pressurization operation under the identical process conditions, detect, obtain the electrophoresis signal spectrogram, measure peak height h ', with h ' substitution working curve, try to achieve the Cf C of corresponding acceptor
f
3) be when investigating object with the acceptor, C
fBe the acceptor density that dissociates, γ=(C then
0-C
f)/C
t, wherein, C
0Be the acceptor total concentration that adds, C
tIt is the part total concentration that adds;
4) adopt equation γ/C
f=-K γ+nK,
In the formula: K is the thermodynamics binding constant,
N is a binding site number,
γ is the acceptor density that combines with part and the ratio of part total concentration,
Return by above-mentioned equation, quantitatively try to achieve thermodynamics binding constant K, binding site number n.
2. according to the described capillary zone electrophoresis of claim 1 medicine is carried out method for screening, adopt the capillary column that is coated with stain when it is characterized in that moving.
3. according to the described capillary zone electrophoresis of claim 2 medicine is carried out method for screening, it is characterized in that being coated with on the capillary column stain acrylamide.
4. according to the described capillary zone electrophoresis of one of claim 1～3 medicine is carried out method for screening, it is characterized in that acceptor is albumen or nucleic acid, part is to treat screening of medicaments.
5. according to the described capillary zone electrophoresis of one of claim 1～3 medicine is carried out method for screening, it is characterized in that damping fluid is the solution that trishydroxymethylaminomethane-acetate contains certain density sodium chloride.
6. according to the described capillary zone electrophoresis of claim 4 medicine is carried out method for screening, it is characterized in that damping fluid is the solution that trishydroxymethylaminomethane-acetate contains certain density sodium chloride.
7. according to the described capillary zone electrophoresis of one of claim 1～3 medicine is carried out method for screening, it is characterized in that its detection method be ultraviolet or fluorescence.
8. according to the described capillary zone electrophoresis of claim 4 medicine is carried out method for screening, it is characterized in that its detection method be ultraviolet or fluorescence.
9. according to the described capillary zone electrophoresis of claim 5 medicine is carried out method for screening, it is characterized in that its detection method be ultraviolet or fluorescence.
10. according to the described capillary zone electrophoresis of claim 6 medicine is carried out method for screening, it is characterized in that its detection method be ultraviolet or fluorescence.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB031111653A CN100399017C (en) | 2003-03-14 | 2003-03-14 | Screening method for medicine with electrophoresis in capillary zone |
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| CNB031111653A CN100399017C (en) | 2003-03-14 | 2003-03-14 | Screening method for medicine with electrophoresis in capillary zone |
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| CN1530648A CN1530648A (en) | 2004-09-22 |
| CN100399017C true CN100399017C (en) | 2008-07-02 |
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| CNB031111653A Expired - Fee Related CN100399017C (en) | 2003-03-14 | 2003-03-14 | Screening method for medicine with electrophoresis in capillary zone |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06118059A (en) * | 1992-10-02 | 1994-04-28 | Otsuka Denshi Kk | Measuring method for coupling constant by capillary electrophoresis |
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2003
- 2003-03-14 CN CNB031111653A patent/CN100399017C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06118059A (en) * | 1992-10-02 | 1994-04-28 | Otsuka Denshi Kk | Measuring method for coupling constant by capillary electrophoresis |
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Granted publication date: 20080702 Termination date: 20110314 |