Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry, spectroscopic, and molecular modeling methods.
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.
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. 1842457577@qq.com.
Xiangrong
X
Li
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.
Tianjun
T
Ni
D000818:Animals; D002151:Calorimetry; D002942:Circular Dichroism; D062105:Molecular Docking Simulation; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D024502:alpha-Tocopherol / Q000378:metabolism* / Q000494:pharmacology
D014361:Trypsin Inhibitors; D014357:Trypsin; D010434:Pepsin A; D024502:alpha-Tocopherol
Isothermal titration calorimetry; Molecular modeling; Pepsin; Spectroscopy; Trypsin; α-Tocopherol