| In this work, combined flow injection (FI) technique with chemiluminescence (CL) analysis, a luminol-α-chymotrypsin (ChT) CL system was developed and the mechanism of ChT enhanced luminol CL signal was discussed; utilizied luminol as a luminescence probe, the interaction of ChT with flavonoids (rutin、luteolin、chrysin、quercetin、genistein) was studied by FI-CL and molecular docking methods. Based on the FI-CL model for protein-drug interaction previously constructed, the binding parameters (binding constant KD and the number of binding sites n) and the thermodynamic parameters (△H、△S、△G) of flavonoids to ChT were calculated, the relationship of structure-affinity for ChT/flavonoids interaction was also investigated. It was found that the concentrations of flavonoid drugs and clarithromycin (CLA) were linear to the CL intensity, a novel method for flavonoid drugs and CLA determination was proposed and successfully applied the analytes in real samples. This work contained two parts:Part 1 IntroductionChapter 1 112 references were cited. As one of the special serine pancreas, ChT was introduced. Recent situations and techniques on investigation for ChT-small molecules interactions were summarized. It gave a brief description about some frequently used FI-CL systems. In the meanwhile, the application of nanoparties on FI-CL analysis was reviewed; the content of this work were outlined.Part 2:Research reportsChapter 2 Luminol-ChT CL SystemIt was found that ChT could accelerate the electron transferring rate of excited 3-aminophthalate and greatly enhance the CL intensity of luminol-dissolved oxygen reaction, luminol—ChT CL system was developed. FI-CL, fluorescence spectroscopy coupled with molecular docking methods were used for the study of luminol—ChT CL behavior, the results revealed that luminol could enter into the S1 hydrophobic pocket with a 2:1 luminol/CT complex formed. By fluorescence spectroscopy, it was suggested that the quenching of luminol fluorescence was caused by the formation of a 1:2 ground—state complex between ChT and luminol, with the binding constant and the number of binding site were given. Molecular docking further proved that two luminol molecules were entered into the S1 hydrophobic pocket, giving the possible CL mechanism of luminol—ChT reaction.Chapter 3 Study on the Interaction of ChT with FlavnoidsIt was found that flavonoid drugs could apparently quench the CL intensity of luminol-ChT system. Using luminol as luminescence probe, based on FI-CL model (lg[(I0-I)/I]=lgKD+nlg[D]) constructed for protein-small molecules interaction, the binding constant of rutin、luteolin、quercetin、chrysin and genistein with ChT was obtained, the KD values were at 105～106 L mol-1 level and following the order:rutin< chrysin< genistein< quercetin< luteolin, the number of binding sites was about to 1; The calculated theromodynamic parameters suggested that the flavonoid drugs binding to ChT was a spontaneous process, with entropy increased and free enthalpy decreaed. Hydrophobic interaction was the major force in ChT/flavonoids interaction. By analyzing the structure-affinity relationship of flavonoids with ChT, it was revealed that the glocosylation of 3-hydroxyl and the amplified of substituent group might weaken the affinity of flavonoids to ChT, the numbers of hydroxyl in B ring made a great effect on ChT/flavonoids interaction. The docking results exported by softwares pymol and ligplus provided direct proof that the five flavonoids entered into the S1 hydropobic pocket of ChT, the binding site contained MET192, TRP215, SER195, GLY216, GLY226, and the 3’,4’-hydroxyls apparently took part in ChT/flavonoids interaction, suggesting the 3’,4’-hydroxyls in B ring played a signinificant role in reaction; Hydrogen bond force also worked in ChT/flavonoids interaction except rutin. In addition, it was proved that the result from FI-CL experiment was nearly in accordance with that obtained by molecular docking.Chapter 4 Continous monitoring CLA in human saliva Utilized the FI-CL model previously constructed, the ChT-CLA interaction was investigated. Molecular docking revealed that CLA could enter into the S1 hydrophobic pocket mainly via hydrophobic force, with KD of 6.63x 105 L mol-1 and n of 0.71. The approach for CLA determination was proposed based on the quenching effect of CLA on luminol-ChT CL intensity, the decreased CL intensity was linear to the logarithm of CLA’s concentrations varied from 7.0 x 10-2～70.0 ng mL-1. The methods was successfully applied to detecting CLA in spiked human serum samples, with recoveries from 92.1%～110.3%, RSD< 2.7% for seven determinaiton. Human saliva CLA during 24 h after oral intake of 250 mg CLA tablets was contiously monitored by this method, with pharmacokinetic parameters including ka, ke, t1/2 were derived.Chapter 5 Continous monitoring rutin in human urineIt was found that rutin could inhibit the CL intensity of luminol-ChT system; a method using luminol-ChT system for rutin assay was developed. This proposed method was successfully applied to the quantitative monitoring of rutin levels in spiked human serum samples, with recoveries from 93.9%～106.3%, RSD< 4.0% for seven determinaiton. Continously monitoring rutin in human urine during 8 h after a single oral dose of 30/60 mg intake, the results showed that rutin reached its maximum concentration Cmax of 1.37 ± 0.02 mg mL-1 at approximately 2.5 h, and the total elimination ratio was 85.7%. The pharmacokinetic parameters ka,ke and t1/2 of rutin were calculated to be1.124± 0.005/1.203 ± 0.007 h-1,0.715 ± 0.002/0.681 ± 0.006 h-1 and 0.99 ± 0.01/1.10 ± 0.02 h, respectively. |
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