Spectroscopic and mechanistic analysis of the interaction between Jack bean urease and polypseudorotaxane fabricated with bis-thiolated poly(ethylene glycol) and α-cyclodextrin.

Self-assembled polypseudorotaxanes (PPRXs) fabricated with α-cyclodextrin and poly(ethylene glycol) (PEG) or its thiolated derivatives were candidate functional materials for enzyme soft-immobilization, encapsulation and controlled-release. The study of their interaction with Jack bean urease (JBU) indicated that they inconspicuously influenced the activity and stability of JBU during long storage, up to 30 days. The macro-species were inaccessible to JBU's active site and the steric effect might play a significant role in the stabilization of JBU, when compared with the small-molecular sulfhydryl inhibitor thioglycolic acid. Circular dichroism and fluorescence spectra analyses revealed that thiolated PEG ₄₀₀ -(SH) ₂ and its assembly PPRX ₄₀₀ (SH) brought in perturbations to certain α-helical or β-sheet domains of JBU, making JBU's conformation more flexible. The resulting partial unfolding of domains exposed several hydrophobic clusters and varied JBU's surface hydrophobicity. It also rendered the chromophores more hydrophilic and more bared to the polar environment, leading to the typical bathochromic-shift and quenching in intrinsic and synchronous fluorescence spectra. Moreover, the surface hydrophobicity profile of JBU was depicted by fluorescent probe monitoring and the unique "hydrophobic cave" motif was proposed by analyzing JBU's structural data from the Protein Data Bank. It should be pointed out that conformational variations mainly occurred at the surface region of JBU, while the buried active bi-nickel center was not markedly influenced by the macro-species. The results demonstrated that the PPRXs might act as a proper carrier for JBU encapsulation or soft-immobilization.
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