Assisting the reactivation of guanidine hydrochloride-denatured aminoacylase by hydroxypropyl cyclodextrins.

Academic Article

Abstract

  • Cyclodextrin is a water-soluble circular oligosaccharide with a cylinder shape characterized by exterior hydrophilic rims and an interior hydrophobic cavity, which makes it an ideal additive to prevent proteins from aggregating during refolding. In this research, three hydroxypropyl cyclodextrins (HPCDs), HP-alpha-, beta-, and gamma-CD, were used to investigate the molecular mechanism of their effects on assisting aminoacylase refolding. The aggregation and reactivation experiments suggested that at moderate concentrations, HPCDs could suppress aggregation and assist aminoacylase refolding in a concentration-dependent manner, and HP-beta-CD was the most efficient of the three HPCDs. Low concentrations of HP-alpha-CD and high concentrations of HP-gamma-CD promoted off-pathway aggregation. Spectroscopic studies indicated that the hydrophobic exposure of the unstructured species in the refolded solutions was gradually reduced by the three HPCDs with the efficiency HP-beta-CD > HP-gamma-CD > HP-alpha-CD. Furthermore, the fast phase of aminoacylase reactivation was slowed down by the addition of 75 mM HP-beta- and gamma-CD, but no significant effect was observed for HP-alpha-CD. The dissimilarity in the effects of the three HPCDs suggested that the internal cavity size played a crucial role in their antiaggregation ability. Further analysis suggested that the observations might be much more complicated than expected because of the various types of interactions between cyclodextrins and proteins in addition to their ability to bind to protein aromatic residues.

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    Keywords

  • 2-Hydroxypropyl-beta-cyclodextrin, Amidohydrolases, Enzyme Activation, Enzyme Reactivators, Guanidine, Hydrophobic and Hydrophilic Interactions, Protein Denaturation, Protein Folding, Spectrometry, Fluorescence, alpha-Cyclodextrins, beta-Cyclodextrins, gamma-Cyclodextrins

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    Author List

  • Kim S-H; Zhang J; Jiang Y; Zhou H-M; Yan Y-B

    • Start Page

  • 686

    • End Page

  • 693

    • Volume

  • 91

    • Issue

  • 2