Activity and conformational changes of horseradish peroxidase in trifluoroethanol.
The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5-25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25-40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The alpha-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.
Zhou
Hong-Wei
HW
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, PR China. zhm-dbs@mail.tsinghua.edu.cn
Xu
Yan
Y
Zhou
Hai-Meng
HM
D001665:Binding Sites; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014270:Trifluoroethanol / Q000737:chemistry* / Q000494:pharmacology; D014362:Trypsinogen / Q000737:chemistry; D014508:Urea / Q000737:chemistry
C032591:N-acetyl-trypsinogen; D011506:Proteins; D014270:Trifluoroethanol; D014508:Urea; D014362:Trypsinogen; D006735:Horseradish Peroxidase; D019791:Guanidine