Studies on the interaction between benzidine and bovine serum albumin by spectroscopic methods
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The interaction between bovine serum albumin (BSA) and benzidine (BD) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra and UV–Vis spectroscopy, as well as resonance light scattering spectroscopy (RLS). It was proved from fluorescence spectra that the fluorescence quenching of BSA by BD was a result of the formation of BD–BSA complex, and the binding constants (K a) were determined according to the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be −34.11 kJ mol−1 and −25.89 J mol−1 K−1, respectively, which implied that van der Waals force and hydrogen bond played predominant roles in the binding process. The addition of increasing BD to BSA solution caused the gradual enhancement in RLS intensity, exhibiting the forming of the aggregate. Moreover, the competitive experiments of site markers suggested that the binding site of BD to BSA was located in the region of subdomain IIA (sudlow site I). The distance (r) between the donor (BSA) and the acceptor (BD) was 4.44 nm based on the Förster theory of non–radioactive energy transfer. The results of synchronous fluorescence and CD spectra demonstrated the microenvironment and the secondary conformation of BSA were changed.
KeywordsBenzidine Bovine serum albumin Fluorescence quenching Binding site Circular dichroism spectra
We gratefully acknowledge the financial support of Chinese 863 Program (2007AA06Z407); National Natural Science Foundation of China (Grant Nos. 30570015, 20621502); Natural Science Foundation of Hubei Province (2005ABC002); and the Research Foundation of Chinese Ministry of Education (8-IRT0543).
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