GUANIDINE-INDUCED DENATURATION OF BETA-GLYCOSIDASE FROM SULFOLOBUS-SOLFATARICUS EXPRESSED IN ESCHERICHIA-COLI

Citation
F. Catanzano et al., GUANIDINE-INDUCED DENATURATION OF BETA-GLYCOSIDASE FROM SULFOLOBUS-SOLFATARICUS EXPRESSED IN ESCHERICHIA-COLI, Biochemistry (Easton), 37(41), 1998, pp. 14484-14490
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0006-2960
Volume
37
Issue
41
Year of publication
1998
Pages
14484 - 14490
Database
ISI
SICI code
0006-2960(1998)37:41<14484:GDOBFS>2.0.ZU;2-H
Abstract
Guanidine-induced denaturation of Sulfolobus solfataricus P-glycosidas e expressed in Escherichia coli, S beta gly, was investigated at pH 6. 5 and 25 degrees C by means of circular dichroism and fluorescence mea surements. The process proved reversible when the protein concentratio n was lower than 0.01 mg mL(-1). Moreover, the transition cut-yes dete rmined by fluorescence did not coincide with those determined by circu lar dichroism, and the GuHCl concentration corresponding at half-compl etion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL(-1) Gel filtration chromatography experi ments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, uneq uivocally, indicated that the guanidine-induced denaturation of S beta gly was not a two-state transition with concomitant unfolding and dis sociation of the four subunits. A mechanism involving a dimeric interm ediate species was proposed and was able to fit the experimental fluor escence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degrees C and pH 6.5. Thi s figure, when normalized for the number of residues, showed that, at room temperature, S beta gly has a stability similar to that of mesoph ilic proteins.