Abstract
The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the SβS Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the SβS bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one SβS bridge of BSA, a mean Gibbs free energy of β7 kJ mol^β1^ is derived by studying the reaction equilibrium. The corresponding value for the HSA SβS bridge reduction is β2 kJ mol^β1^. The reaction kinetics observed via the SβS or amide I Raman bands are identical giving a reaction rate constant of (1.02 Β± 0.11) M^β1^ s^β1^ for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations. Β© 2008 Wiley Periodicals, Inc. Biopolymers 89: 623β634, 2008.
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