title: Spectroscopic study on structure of horseradish peroxidase in water and dimethyl sulfoxide mixture.
abstract: The structure of horseradish peroxidase (HRP) in phosphate buffered saline (PBS)/dimethyl sulfoxide (DMSO) mixed solvents at different compositions is investigated by IR, electronic absorption, and fluorescence spectroscopies. The fluorescence spectra and the amide I spectra of ferric HRP [HRP(Fe3+)] show that overall structural changes are relatively small up to 60% DMSO. Although the amide I band of HRP(Fe3+) shows a gradual change in the secondary structure and a decrease in the contents of a helices, its fluorescence spectra indicate that the distance between the heme and Trp173 is almost constant. In contrast, the changes in the positions of the Soret bands for resting HRP(Fe3+) and catalytic intermediates (compounds I and II) and the IR spectra at the C-O stretching vibration mode of carbonyl ferrous HRP [HRP(Fe2+)-CO] show that the microenvironment in the distal heme pocket is altered, even with low DMSO contents. The large reduction of the catalytic activity of HRP even at low DMSO contents can be attributed to the structural transition in the distal heme pocket. In PBS/DMSO mixtures containing more than 70 vol % DMSO, HRP undergoes large structural changes, including a large loss of the secondary structure and a dissociation of the heme from the apoprotein. The presence of the components of the amide I band that can be assigned to strongly hydrogen bonding amide C=O groups at 1616 and 1684 cm(-1) suggests that the denatured HRP may aggregate through strong hydrogen bonds.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Fukui University, Japan. y_maeda@acbio.fukui-u.ac.jp
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Maeda
authors.forename: Maki
authors.initials: M
authors.lastname: Fujihara
authors.forename: Isao
authors.initials: I
authors.lastname: Ikeda
mesh_terms: D004121:Dimethyl Sulfoxide; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D014867:Water
chemical_list: D014867:Water; D006735:Horseradish Peroxidase; D004121:Dimethyl Sulfoxide