Biophysical Studies on the Interactions of a Classic Mitochondrial Uncoupler with Bovine Serum Albumin by Spectroscopic,Isothermal Titration Calorimetric and Molecular Modeling Methods |
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Authors: | Yue Zhang Jia-Han Li Yu-Shu Ge Xiao-Rong Liu Feng-Lei Jiang Yi Liu |
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Institution: | (1) State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) & College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, People’s Repulic of China; |
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Abstract: | The interaction between a classic uncoupler (2,4-dinitrophenol, DNP) and bovine serum albumin (BSA) was investigated by fluorescence
spectroscopy under the physiological conditions. The fluorescence quenching constants were calculated by the Stern-Volmer
equation, and based upon the temperature dependence of quenching constants, it was proved that DNP caused a static quenching
of the intrinsic fluorescence of BSA. Owing to the static quenching mechanism, different associative binding constants at
various temperatures were determined and thus the thermodynamic parameters, namely enthalpy (ΔH = −21.12 kJ mol−1) and entropy changes (ΔS = 23.51 J mol−1 K−1) could be calculated based on the binding constants. Moreover, the enthalpy and entropy changes are consistent with the “Enthalpy-Entropy
Compensation” equation obtained from our previous work. The negative enthalpy and positive entropy indicated that the electrostatic
interactions played a major role in DNP-BSA binding process. Site marker competitive displacement experiments were carried
out by using fluorescence and isothermal titration calorimetry (ITC) methods. These results showed that DNP bound with high
affinity to Sudlow’s site I (subdomain IIA) of BSA. The distance (r = 3.78 nm) between donor (BSA) and acceptor (DNP) was obtained according to the mechanism of fluorescence resonance energy
transfer (FRET). Furthermore, the results of synchronous fluorescence and circular dichroism (CD) spectroscopic studies indicated
that the microenvironment and the secondary conformation of BSA were altered. The above results were supported by theoretical
molecular modeling methods. |
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