The Species Difference Of Toxicant Metabolizing Enzymes CE2 Fluorescent Probe And Its Ligand Binding Sites

Carboxylesterases 2（CE2）can hydrolyze environmental toxins and cancerogens containing carboxylic ester bond,amide bond,thioester bond and so on,and it has been widely used as the biomarkers of pyrethroid pesticides.Using small molecular probe substrate to determine the activity of biomarkers can reflect the concentration and toxicity of contaminants in environmental specimens and organisms quickly and sensitively.With the new-type of ratio-dependent two-photon fluorescence probe NCEN and near-infrared DDAB of hCE2 as the research objects,this thesis analyzes the species differences and the ligand binding site of toxicant metabolizing enzyme CE2 fluorescent probe.（1）After the experiment of metabolic profiles of the probe and inhibitory effects of various esterase inhibitors on probe hydrolysis in liver microsomes from different animal species,we learned that Carboxylesterases has played a predominant role in the hydrolysis of DDAB and NCEN livers microsomes of various animal species.According to the comparison of amino acid sequences between CE1 and CE2.In addition,DDAB and NCEN themselves having the structural characteristics of small acyl and big alcohol radical tend to be metabolized by CE2.It is thus concluded that the two fluorescent probes NCEN and DDAB are primarily hydrolyzed by CE2 in the liver microsomes of humans,rats,mice,monkeys,dogs and pigs.（2）Modeling the hCE2 monomer and using Molecular Docking finds that the ligand binding sites of probes DDAB,NCEN and FD are different.In addition,Inhibition kinetic models among NCEN,DDAB and FD are all noncompetitive inhibitions.It is concluded that the probes DDAB,NCEN and FD have different ligand binding sites with hCE2.From the above researches,the author obtained the metabolic types of NCEN and DDAB from different species,and with kinetic parameters K_m and V_max,it is concluded that the two probes can be used as CE2 fluorescence probes for mammal species.The ligand binding sites of DDAB,NCEN and FD（fluorescence probe of hCE2）are also detected,laying a theoretical foundation for using DDAB and NCEN to determine the activity of CE2 in mammal species,thus providing a powerful means for determining the concentration and the toxicity of the contaminants in environmental specimens and organisms in a quick,sensitive and high-throughput way for further researches.