Development Of Online Enzyme Assay And Enzyme Inhibitor Screening By Capillary Electrophoresis

Capillary electrophoresis(CE)has the advantages of high degree of automation,small sample consumption,short analysis time,and suitable for connection with a variety of detectors,which make it a powerful tool in enzyme assay.The overexpression of many enzymes plays a driving role in the occurrence and progression of diseases.Cathepsin B and carbonic anhydrase Ⅸ are enzymes closely related to diseases such as cancer.This paper focused on the development of capillary electrophoresis based immobilized enzyme microreactor(IMER)and electrophoretically mediated microanalysis(EMMA).The two methods were used to online screening of cathepsin B and carbonic anhydrase Ⅸ inhibitors,and to cathepsin B online detection.The main contents of the investigations can be described as follows:In the first part,cathepsin B was immobilized on the inner wall of the capillary using glutaraldehyde method.An immobilized enzyme microreactor was developed for screening cathepsin B inhibitors.Scanning electron microscopy results showed that cathepsin B was successfully immobilized on the capillary.After the optimization of online separation conditions and enzyme reaction conditions,substrate and product could be separated within 3 min,and the effect of inhibitors was reflected by the change of product yield.The Michaelis constant(K_m)of cathepsin B was measured to be 0.85 mM.The raised method was then applied to discover the inhibitory potential of 17 standard compounds from traditional Chinese medicines.Five natural products,including kaempferol,rutaecarpine,evodiamine,theophylline,lycobetaine showed potential inhibition for cathepsin B.In addition,molecular docking study was carried out to further explore the interactions between inhibitors and cathepsin B.In the second part,electrophoresis mediated microanalysis combined with transverse diffusion method was used for the determination of cathepsin B in real samples.After optimizing the enzymatic reaction and online separation conditions,the separation of substrate and product was achieved.The linear equation of enzyme concentration-product peak area was successfully established,the correlation coefficient is higher than 0.9961,the limit of quantification(LOQ)can reach 0.1 U/mL,which meet the needs of real sample detection.The characteristic absorption wavelength of the product is 345 nm and at this detection wavelength,the interference of most heterogeneous peaks in the real sample can be eliminated.Under the optimal reaction conditions,quantitative detection of cathepsin B in synovial fluid of osteoarthritis patients was achieved.In the third part,a screening method for carbonic anhydrase Ⅸ inhibitor in traditional Chinese medicine based on electrophoresis mediated microanalysis was established,combined with ttransverse diffusion method,the injection,enzymatic reaction,separation,and detection were performed in the same capillary.In this experiment,p-nitrophenyl acetate was used as the substrate for the enzyme reaction.The online reaction and separation conditions were investigated,and the detection of product can be achieved within 3 min of separation time.The The Michaelis constant of carbonic anhydrase Ⅸ was determined to be 1.2 mM,and the feasibility of this method for screening carbonic anhydrase Ⅸ inhibitors was evaluated using a known inhibitor acetazolamide.In the end,4 natural compounds of 15 Chinese medicine standards were discovered to exhibit enzyme inhibitory activity,including polydatin,matrine,etc,providing a new method for screening carbonic anhydrase Ⅸ inhibitors.