The Preparation Of Thiolated Polypseudorotaxane And The Interactions With Chymotrypsin And Urease

Polypseudorotaxanes is a linear supramolecular self-assembled system widely used in nanotechenology devices,sensors,molecular converters,drug delivery systems,gene release vectors and tissue engineering scafforlds.In this study,a redox controllable polypseudorotaxanes hydrogel was prepared by modifying mercapto group at the end of PEG.The PEG?SH/?-CyD polypseudorotaxanes was used as carriers to entrap chymotrypsin and urease.The activity and the structure of chymotrypsin and urease in the presence of polypseudorotaxanes were determined.The interaction between the ternary system and the nucleation mechanism during phase-transition of the polypseudorotaxanes were investigated.Bis-thiolated PEG was prepared by esterification method and three-step-synthesis method.Characterized by FTIR,Raman and 1H NMR spectra,the results showed that the esterification method was proper to synthesize bis-thiolated PEG,and the yield was more than 70%.The thiolated polypseudorotaxanes was prepared by fabrication of bis-thiolated PEG and ?-CyD.The XRD analysis revealed that the crystallographic pattern of polypseudorotaxanes belonged to the hexagonal system.In order to explore whether the thiolated polypseudorotaxanes could act as a carrier to soft-immobilize enzyme,we investigated the activity and structure of chymotrypsin in PEG?SH/?-CyD polypseudorotaxanes.The results showed that the activity of chymotrypsin increased 19% in the PEG ? SH/ ?-CyD polypseudorotaxanes system,and the stability of chymotrypsin was better than the native chymotrypsin.The results of fluorescence spectroscopy,synchronous fluorescence spectroscopy,1H NMR,13 C NMR,COSY,NOESY and circular dichroism showed that this was mainly due to the hydrogen bond,van der waals force and hydrophobic interaction between chymotrypsin and thiolated polypseudorotaxanes.So the structure of the chymotrypsin was slightly changed,resulting in the increase of the catalytic efficiency of the enzyme.In addition,thiolation could improve the activity of chymotrypsin.The interaction of urease and thiolated polypseudorotaxanes were studied as a comparison.The results showed that thiolated polypseudorotaxanes had nearly no effect on urease activity and stability.But in thioglycolic acid solution,urease rapidly inactivated.The fluorescence spectra of urease indicated that the fluorescence intensity decreased when it was mixed with thiolated polypseudorotaxanes.The microenvironment of Trp and Tyr residues were also changed with the addition of thiolated polypseudorotaxanes.Furthermore,a preliminary study of the nucleation mechanism during phase-transition of polypseudorotaxanes was carried out.In 0.1 g/mL ?-CyD solution,the formation rate of polypseudorotaxanes increased with the increase of PEG concentration,and finally stabilized at 5×10-4 mg/(mL?s)during the fabrication and phase-transition process,and the resulting polypseudorotaxanes had a particle size in the range of about 1.5 to 3 ?m.The zeta-potential measurement indicated that once the coagulate nucleus appeared,they would aggregate rapidly and form an unstable system,and phase-transition further occurred.