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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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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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Acknowledgments

We thank Prof. Pierre D. Harvey of Université de Sherbrooke for reading the manuscript. The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 20873096 and 20921062) and the Research Foundation of Wuhan University (Grant No. 20082030201000013)

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  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

    Yue Zhang, Jia-Han Li, Yu-Shu Ge, Xiao-Rong Liu, Feng-Lei Jiang & Yi Liu

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  1. Yue Zhang
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Correspondence to Yi Liu.

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Zhang, Y., Li, JH., Ge, YS. et al. Biophysical Studies on the Interactions of a Classic Mitochondrial Uncoupler with Bovine Serum Albumin by Spectroscopic, Isothermal Titration Calorimetric and Molecular Modeling Methods. J Fluoresc 21, 475–485 (2011). https://doi.org/10.1007/s10895-010-0733-y

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