中国生物工程杂志

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the only antioxidant enzyme which could reduce membrane-bound hydroperoxy lipids. In this report, Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were employed to investigate the effect of temperature on enzymatic activity and the conformational change of a recombinant Oryza sativa PHGPx (OsPHGPx). PHGPx activity was increased slowly from 10 to 27.5°C and reached the maximum at 27.5°C, then descended rapidly from 27.5°C to 45°C. At temperatures exceeding 45°C, the enzyme activity was completely lost. A classic two phase thermal unfolding of OsPHGPx was found with temperature increasing. First, from 20 to 40°C, Far UV CD, intrinsic fluorescence and DSC spectra did not change obviously with increased temperature, indicating the whole structure nearly maintained integrity. Second, from 40 to 55°C, an abrupt secondary level change was found in the far UV CD spectra, indicating the secondary structure started unfolding; the change of intrinsic fluorescence spectra revealed that the tertiary structures began partial unfolding and the region near active site was buried into the unfolding structure. The DSC indicated OsPHGPx exhibited thermal transitions at around 42 °C. Third, at over 55°C, the whole conformation of the protein kept unchanged.