Capillary Electrophoresis As Analysis Technique For Histone Deacetylases Activity Assay

Histone deacetylases(HDACs)is a class of enzyme responsible for catalytic deacetylation histone,it is closely related to a variety of cell processes and become an important target for anticancer drug research,the establishment of accurate and efficient screening methods for HDACs inhibitors is of great practical significance for the development of new drugs.Capillary electrophoresis(CE),as an efficient and rapid microanalysis technology,it has an outstanding advantages of high separation efficiency,short analysis time,less sample consumption se well as high degree of automation.Therefore,this paper focuses on the construction and application of the CE methods,the main parts are as followings:(1)This paper has established the CE-IMER method for determining HD AC kinetic parameters and its inhibitors screening.Taking advantages of the characteristic that polydopamine(PDA)is easily deposited onto solid surfaces such as capillary wall,The inner wall of the capillary was coated with polydopamine and characterized by SEM,proving the successful establishment of IMER,HDACs are immobilized on the inner wall of the capillary through the electrostatic attraction,we optimized the conditions of dopamine concentration,the modification time of dopamine,the time of enzyme immobilization and incubation time of substrate and enzyme,the enzyme activity still maintained 90%of the initial activity after repeated running more than 40 times,it shows the excellent performance of this method.The kinetic parameters(Km and Vmax)of the enzyme and the IC50 values of its inhibitors were determined under the optimal conditions,which further confirmed the reliability of the method.In addition,we have successfully found potential inhibitors from more than 10 chemical compounds.(2)In order to further screen selective inhibitors from compounds working to HDACs targets,EMMA method was developed for determination of HD AC 8 kinetic parameters and inhibitor screening.We investigated the optimal conditions for electrophoresis analysis.The final experimental conditions were as follows:beckman electrophoresis apparatus equipped with diode array detector,detection wavelength of 327 nm,column temperature of 37℃,fused quartz capillary(inner diameter of 75 μm,effective length of 50 cm),The separation buffer was chosen as Tris-HCl(100 mM,pH=8.0),separation voltage was 15 kV,The incubation buffer solution was Tris-HCl(25 mM,pH=8.0,137 mM NaCl,2.7 mM KCl,1 mM MgCl2,1 mg/mL BSA)solution.The substrate and enzyme were mixed for 0.2 min at the mixing voltage of 3 kV,and the incubation time was 5 min.After the enzymatic hydrolysis effect,the product and unreacted substrate were separated and detected.The kinetic parameters(Km and Vmax)of HD AC 8 and the IC50 value of its two representative inhibitors,Vorinostat(SAHA)and PCI3405 1,were determined by the established method.The feasibility of this method is proved and the superiority of this method is compared and discussed.In conclusion,this study developed two methods based on capillary electrophoresis for the determination of kinetic parameters and screening of inhibitors of HDACs.Among them,the CE-IMER method has good stability,and the enzyme still retains good activity after repeated operation,which can greatly save the enzyme usage,it has very important practical significance for the expensive and scarce enzyme samples.EMMA method have the prominent advantage of online inhibitors screening and enzyme activity detection,which the put the place of enzymatic reaction and production detection together,reducing the manual mistakes greatly,the highly automated method fully meets the need of high-speed development of modern pharmacology.