Unfolding and refolding study of a large dimeric protein β-glucosidase from almond monitored by fluorescence spectroscopy

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 t1/2 (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. © 2015 bentham science publishers.