Enzyme activity inhibition and secondary structure disruption of nano-TiO2 on pepsin.
In this study, the binding and enzyme activity inhibitory effect of nano-TiO(2) on pepsin was explored compared with micro-TiO(2). Nano-TiO(2) was about 60 nm and micro-TiO(2) was about 200 nm, both round in shape. The activity of pepsin was depressed significantly by nano-TiO(2) comparing to micro-ones. The results of UV spectrometry, HPLC, SDS-PAGE and CD assay proved that micro-TiO(2) has only physical absorption effect on pepsin, but no impairment on primary sequences or secondary structure. However, nano-TiO(2) had coordination interaction with pepsin besides physical binding effect. The secondary structure of pepsin was unfolded with the treatment of nano-TiO(2) at pH 6.5 and pH 3.53, which might consequently affect the beta-hairpin loop that protects the active center of pepsin, and then reduce the enzyme activity. Furthermore, the thermodynamic mechanisms of interaction between nano-TiO(2) and pepsin were explored by fluorescence spectrum and ITC analysis. According to the results of thermodynamic analysis, the K value was 3.64x10(6), stoichiometry (N(pepsin:nano-TiO2)) was 3.04x10(3), the total DeltaH was -2277 cal/mol, DeltaS was 22.7 cal/(K mol), therefore the nano-TiO(2)-pepsin interaction is spontaneous. The depression of activity and the unfolding of secondary structure of pepsin were resulted from non-covalent reactions, including electrostatic force and hydrophobic binding. This work studied the different inhibitory effects and revealed mechanisms of the interaction between micro/nano-TiO(2) and pepsin, and provided a useful approach for evaluating the health risk of nano-materials on level of proteins.
Zhu
Rong-Rong
RR
Shanghai Key Laboratory of Cell Signaling and Diseases, School of Life Science and Technology, Tongji University, Shanghai, PR China.
Wang
Wen-Rui
WR
Sun
Xiao-Yu
XY
Liu
Hui
H
Wang
Shi-Long
SL
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000633:toxicity*; D008564:Membrane Potentials / Q000187:drug effects; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D010434:Pepsin A / Q000037:antagonists & inhibitors* / Q000737:chemistry; D017433:Protein Structure, Secondary; D013552:Swine; D014025:Titanium / Q000737:chemistry / Q000633:toxicity*
D004791:Enzyme Inhibitors; C009495:titanium dioxide; D014025:Titanium; D010434:Pepsin A