Influences of temperature and threshold effect of NaCl concentration on Alpias vulpinus OCT.
Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0-3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus. Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 degrees C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV-vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.
Department of Organic and Biological Chemistry, University of Messina, Salita Sperone, 31 (S. Agata), 98166 Messina, Italy. bellocco@isengard.unime.it
Ersila
E
Bellocco
Davide
D
Barreca
Giuseppina
G
Laganà
Ester
E
Tellone
Silvana
S
Ficarra
Federica
F
Migliardo
Ugo
U
Leuzzi
Salvatore
S
Magazù
Antonio
A
Galtieri
D000818:Animals; D002942:Circular Dichroism; D006358:Hot Temperature; D007700:Kinetics; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D012754:Sharks / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D012965:Sodium Chloride; D009954:Ornithine Carbamoyltransferase