| An affinity capillary electrophoresis (ACE) is a clastotype of high performance capillary electrophoresis (HPCE) method, it is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. By studying electrophoretogram of reaction system we can obtain much information about affinity , structure, production . ACE has a great potential of applications for both molecular biological and biochemical study, such as specific dentification about nucleic acid fragments, interaction of protein, competive immunity analysis, drug-receptor and drug-protein study and so on.Dihydrofolate reductase (DHFR) and MTX enantiomer are the main objects of this experiment. ACE was used to analyze DHFR activity and the dynamic parameters of interaction between methotrexate (MTX) enantiomer and DHFR for the first time. The result indicated that reaction between MTX enantiomer and DHFR had three-dimensional selection. The result suggested clinician should consider stereoselectivity between enantiomer and key enzyme in metabolism when used enantiomer drugs.Part I Affinity capillary electrophoresis analyze dihydrofolate reductase activityDHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (FH2) to 5,6,7,8-tetrahydrofolate (FH4). FH4 is important for the synthesis of thymidylate, purine nucleosides, methionine, and other metabolic intermediates, DHFR play a very important role in the metabolism of both prokaryotic and eukaryotic organisms. Studies performed on different human cancers have shown that resistance to chemotherapeutics can arise through different mechanisms, the change of DHFR is one of the key resistant mechanisms. So studying the activity of DHFR from tumor cells has significant meanings.An affinity capillary electrophoresis (ACE) method was developed to monitor activity of dihydrofolate reductase. The enzyme reaction system consisted of 35μl reaction buffer, 10μl DHFR, 5μl of 0.1mg/mL NADPH , 10μl various concentration FH2 (0.005-0.05mg/mL) in 100μl sample tube, then mixed for 30s and incubated for 10 min at room temperature.The optimal separation condition was choosed by adjusting concentration and pH value of running buffer, concentration of surfactant ,voltage and capillary temperature. Using bare fused -silica capillary, the electrophoresis buffer was 50mmol/L sodium tetraborate with 0.2% Brij-35, pH 9.50. The temperature of separation was controlled at 25oC and a voltage of 25kV was applied. The detection wavelength was 254 nm. The difference of peak areas about the product was used to calculate the activity of DHFR .The Km value is 6.05×10-7 mol/L·min. ACE was a rapid, simple and accurate method that applied to study activity of enzyme extensively.Part II Determination of dynamic parameters of interaction between methotrexate enantiomer and dihydrofolate reductase by affinity capillary electrophoresis MTX is a potent inhibitor of the dihydrofolate reductase (DHFR) enzyme whichplays a key role in intracellular folate metabolism and is essential for DNA synthesis and cell growth, The affinity between MTX and DHFR is about 105 times higher than that of the folic acid. So MTX is one of the most important drugs for tumor treatment. Natural MTX is enantiomer , L-(+)-MTX and D-(-)-MTX ,but it has not reported that whether the biologic activity of MTX enantiomer has different or not.An affinity capillary electrophoresis (ACE) method was also developed to monitor the dynamic parameters of interaction between methotrexate (MTX) enantiomer and dihydrofolate reductase (DHFR). The enzyme reaction system consisted of 53μl reaction buffer, 11μl DHFR , 11μl of 1mmol/L NADPH, 15μl of 0.5mmol/L dihydrofolate (FH2) and 10μl of various concentration MTX enantiomer in 100μl sample tube, then mixed for 30s and incubated for 10 min at room temperature. The optimal separation condition was identical with part II. The difference of peak areas about the product was used to calculate the inhibitory rate and IC50 values. The IC50 value of the D-(-)-MTX was 31.67×10-8 mol/L, the L-(+)-MTX was 2.48×10-8 mol/L. The IC50 value of the D-(-)-MTX was about 13 times higher than that of the L-(+)-MTX. The result indicated that reaction between MTX enantiomer and DHFR had three-dimensional selection. ACE could be used to monitor DHFR dynamic reaction, each reaction volum is only 100μl, 2 ml separation reagent was used, within 30 min the reaction mixture could be separated and each reactant or product could be quantitied, ACE had more conspicuous advantage than other ways.Part III Isolation and purification of Dihydrofolate reductase from human NSCLC A549 cell linesCultivate human NSCLC A549 cell lines in vitro, collect 2×108cells in logarithmic growth phase. The cells were utilized immediately or stored frozen at -60 oC. For the preparation of the cell extract, the packed cells were suspended in an equal volume of 10 mmol/L potassium phosphate, pH 8.9, and sonicated and centrifuged at 4 oC for 1 h at 105,000g/min, then the supernatant further was processed by DEAE-Sepharose Fast Flow or stored frozen at -60 oC. By adjusting power , interval time and frequency of sonifier; optimizing pH about balance buffer, eluotropic concentration and flow rate of KCl, we obtain high purity DHFR. Assemble eluotropic protein , protein analysis a was carried out by 12% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) to identity DHFR. All enzymes were vacuum freezing dry and stored under frozen at -60 oC. DEAE-Sepharose Fast Flow bind ion exchange and molecular sieve, we optimize pH of balance buffer by DHFR isoelectric point (PI), it could be used to isolation and purification of DHFR from human NSCLC A549 cell lines.SummaryIn a word, an affinity capillary electrophoresis method was developed to monitor the activity of DHFR and the dynamic parameters of interaction between MTX enantiomer and DHFR. We found inhibitory action that MTX enantiomer inhibit DHFR existed significant disparity. This technique has many advantages: high separative power, short time of analysis, low cost of operation and very low sample consumption. Affinity capillary electrophoresis has a great potential of applications for enzyme kinetics analysis.However, We found in experiment that the main obstacle is the strong adsorption between separated matter and the inner surface of fuesd silica capillaries, resulting in inaccuracy of enzyme kinetics analysis, insufficient separation and low reproducibility.
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