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Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods

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Abstract

α-Tocopherol is a required nutrient for a variety of biological functions. In this study, the binding of α-tocopherol to trypsin and pepsin was investigated using isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements, circular dichroism (CD) spectroscopy, and molecular modeling methods. Thermodynamic investigations reveal that α-tocopherol binds to trypsin/pepsin is synergistically driven by enthalpy and entropy. The fluorescence experimental results indicate that α-tocopherol can quench the fluorescence of trypsin/pepsin through a static quenching mechanism. The binding ability of α-tocopherol with trypsin/pepsin is in the intermediate range, and one molecule of α-tocopherol combines with one molecule of trypsin/pepsin. As shown by circular dichroism (CD) spectroscopy, α-tocopherol may induce conformational changes of trypsin/pepsin. Molecular modeling displays the specific binding site and gives information about binding forces and α-tocopherol-tryptophan (Trp)/tyrosine (Tyr) distances. In addition, the inhibition rate of α-tocopherol on trypsin and pepsin was studied. The study provides a basic data set for clarifying the binding mechanisms of α-tocopherol with trypsin and pepsin and is helpful for understanding its biological activity in vivo.

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Acknowledgments

This work was supported by the Key Research Project of Colleges and Universities of Henan Province (15A150004), the Doctoral Startup Fund of Xinxiang Medical University (505078), the Foundation for Fostering of Xinxiang Medical University (2014QN122) and the National Natural Science Foundation (81401470).

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  1. Department of Chemistry, School of Basic Medicine, Xinxiang Medical University, 601 Jin-sui Road, Hong Qi District, Xinxiang, Henan, 453003, People’s Republic of China

    Xiangrong Li & Tianjun Ni

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Correspondence to Xiangrong Li.

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Li, X., Ni, T. Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods. J Biol Phys 42, 415–434 (2016). https://doi.org/10.1007/s10867-016-9415-6

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  • DOI: https://doi.org/10.1007/s10867-016-9415-6

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