Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug-HSA complex, and the effective quenching constants (K-a) were 2.852 x 10(4), 2.765 x 10(4), 2.638 x 10(4) and 2.542 x 10(4) M-1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Delta H) and entropy change (Delta S) for the reaction were calculated to be -3.634 kJ mol(-1) and 72.66J mol(-1) K-1 according to van't Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Forster's theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug-HSA complex was also discussed. Crown Copyright (c) 2009 Published by Elsevier B.V. All rights reserved.