Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0–3.0M) 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 1.5Mconcentration. The presence of 3.0MNaCl completely avoids OCT unfolding at 60, 64 and 66 ◦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 showthat high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.

Influences of temperature, threshold effect of NaCl concentration on Alopias vulpinus OCT.

BELLOCCO, Ersilia Santa;BARRECA, Davide;LAGANA', Giuseppina;TELLONE, Ester;FICARRA, Silvana;MIGLIARDO, Federica;LEUZZI, Ugo;MAGAZU', Salvatore;GALTIERI, Antonio
2008-01-01

Abstract

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0–3.0M) 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 1.5Mconcentration. The presence of 3.0MNaCl completely avoids OCT unfolding at 60, 64 and 66 ◦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 showthat high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.
2008
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11570/1869469
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