It has been recently demonstrated that α-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process. The kinetics of aggregation was found to obey a second-order reaction with respect to protein concentration. The aggregation-prone A-state and aggregation-deficient TFE- or T-state of CT were found to be induced at lower TFE concentrations in the presence of salts. Use of acidic and alkaline conditions and lysine modification also promoted the formation of the T-state. Results presented suggest a role for electrostatic interactions in the aggregation process. © 2006 Elsevier Inc. All rights reserved.
Role of electrostatic interactions in 2,2,2-trifluoroethanol-induced structural changes and aggregation of α-chymotrypsin
Rezaei-Ghaleh N.;
2007-01-01
Abstract
It has been recently demonstrated that α-chymotrypsin (CT) can be driven toward amyloid aggregation by addition of 2,2,2-trifluoroethanol (TFE), at intermediate concentrations. In the present article, the process of TFE-induced CT aggregation was investigated in more detailed kinetic terms where the effects of medium conditions, such as temperature, presence of kosmotropic and chaotropic salts, pH and chemical modification of lysine residues were examined. Various techniques, including light scattering, fluorescence and circular dichroism spectroscopy, were used to follow and characterize this process. The kinetics of aggregation was found to obey a second-order reaction with respect to protein concentration. The aggregation-prone A-state and aggregation-deficient TFE- or T-state of CT were found to be induced at lower TFE concentrations in the presence of salts. Use of acidic and alkaline conditions and lysine modification also promoted the formation of the T-state. Results presented suggest a role for electrostatic interactions in the aggregation process. © 2006 Elsevier Inc. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
