Study On Supramolecular Tandem Enzyme Assay

As a kind of biologic catalysts that widely exists in nature and organisms,enzyme plays an important role in the life activities of cells.Enzyme assays are of vital significance to disease diagnosis,drug development and industrial production.However,traditional methods of enzymatic assay are complex,insensitive,costly,and limited by instruments or technologies,which hardly meet the growing demand for high-throughput assays.Nowadays,the newly developed supramolecular tandem assay(STA)provides a new method for enzymatic assay.This method is simple,fast,highsensitivity and low-cost,which holds broad application potential in disease diagnosis,treatment and drug development.In this thesis,we constructed STA systems for the detection and inhibitors evaluation of three different types of enzymes.First,for the Methionine Decarboxylase(MetDC),which is greatly related with homocystinuria and hypermethioninemia,the binding constants of seven macrocyclic hosts with their fluorescent indicators,L-methionine and 3-methylthiopropylamine were determined by fluorescence titration experiments.After screening,the STA system based on cucurbit[7]uril(CB7)and acridine orange(AO)was constructed,using Lmethionine as the substrate.The binding forces of CB7 with 3-MTP were elucidated by NMR and computational simulations as hydrogen bonding,electrostatic interactions and hydrophobic effects.The continuous real-time assays of MetDC activities and determination of kinetic parameter were achieved based on the constructed system,its substrate specificity was demonstrated to be high,and hydroxylamine has a low semiinhibitory concentration(IC50=38±4.4 μM)with favorable inhibition,and methionine methyl ester has a high inhibitory rate.Secondly,for DOPA Decarboxylase(DDC),which is associated with Parkinson’s disease and other neurodegenerative diseases,the STA system was constructed based on CB7 and AO with L-DOPA as the substrate,then the real-time continuous assays of the activity of DDC were achieved,the kinetic parameter of DDC were determined and its substrate specificity was proved to be high.This method has a low detection limit(LOD=0.659 μg/mL).The potential inhibitors 5-bromosalicylic acid and 5iodosalicylic acid have high inhibitory rate,hydroxylamine has low half-inhibitory concentration of(IC50=41.0±5.9 μM)with good inhibitory effect.Finally,for the industrial enzyme Aspartase(AspA),with L-aspartate as the substrate,the STA system was constructed based on the quaternary ammonium calix[4]arene derivative and sulfonated cyanine 5,realizing the rapid assays of AspA.the results were consistent with ultraviolet-visible spectrophotometry(UV-Vis).The inhibitory effects of various inhibitors on AspA were determined,among which Nethylmaleimide was found significantly more effective.Compared with UV-Vis,this method has stronger anti-interference.This thesis has 67 figures,3 tables,and 178 references.