Unfolding and Refolding Study of a Large Dimeric Protein β-Glucosidase from Almond Monitored by Fluorescence Spectroscopy

Title:Unfolding and Refolding Study of a Large Dimeric Protein β-Glucosidase from Almond Monitored by Fluorescence Spectroscopy

Volume: 22 Issue: 7

Author(s): Kanti K. Yadav and Subhankar Paul

Affiliation:

Keywords: ANS fluorescence, β-glucosidase, fluorescence quenching, refolding kinetics, surface hydrophobicity, tryptophan fluorescence.

Abstract: In our present investigation, the unfolding and refolding of β-glucosidase (BGL-Al) from sweet almond was investigated using tryptophan (Trp) fluorescence spectroscopy. When the unfolding of BGL-Al was induced by guanidium chloride (GdnHCl) and monitored using biological activity as well as Trp fluorescence spectroscopic measurement, we observed that the denaturation of BGL-Al could be easily induced by low concentration of GdnHCl and the enzyme was completely inactivated at 1.0 M GdnHCl. Higher unfolding in the presence of reducing agent revealed that the protein perhaps containing multiple di-sulfide bonds indicating a reason of high stability against unfolding by GdnHCl. Refolding results suggested that the protein refolded with high yield from 1 M GdnHCl denatured state, however, refolded with negligible yield from completely unfolded state.

The kinetic studies of BGL-Al refolding unravel a two phase refolding process with calculated t_1/2 (refolding half time) of 1.8 and 33 min, respectively. When 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as extrinsic fluorophore, we found that the surface hydrophobicity of BGL-Al was continuously decreased during GdnHCl-mediated unfolding. The surface hydrophobicity of the protein was calculated to be as high as 128.32. Acrylamide quenching study demonstrated that Trp residues of BGL-Al are mostly and hence they must be located either on the surface or in the crevices accessible by quenchers.

Cite this article as:

Yadav K. Kanti and Paul Subhankar, Unfolding and Refolding Study of a Large Dimeric Protein β-Glucosidase from Almond Monitored by Fluorescence Spectroscopy, Protein & Peptide Letters 2015; 22 (7) . https://dx.doi.org/10.2174/0929866522666150511151818