Purification and Characterization of High-Molecular-Weight Products Derived from Photofrin®-Mediated Photodynamic Treatment of Recombinant Procaspase-3

• Main Authors: Shu Yao Lin, 林淑瑤
• Other Authors: K.Y. Chien
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/03519876094248802434

碩士 === 長庚大學 === 生物醫學研究所 === 98 === Photodynamic therapy (PDT) is also known as photo-radiation therapy (PRT) for the treatment of a variety of diseases including cancer via photochemical reaction. Photosensitization using the tumor-localizing photosensitizer and appropriate visible light produces reactive oxygen species and triggers a series of signaling pathway, leading to apoptosis or necrosis in target cells. Although the mechanism and its consequences have been widely investigated, the exact mechanism of modification of cellular proteins remains unclear. Our previous studies found that Photofrin®-mediated PDT induced modification of endogenous procaspase-3 in cell lines and recombinant procaspase-3-D3A in vitro to form unusual high-molecular-weight (HMW) products. Caspase-3 activity decreased following the increase of HMW products after Photofrin®-PDT treatment. In the present study, we further study the characteristics and modification sites of the HMW products. We found that the isoelectric points of recombinant procaspase-3-D3A and HMW products were more acidic after PDT treatment by 2-D gel electrophoresis analysis. In addition, the substrate-binding ability of PDT-treated procaspase-3 and HMW products was lower than untreated procaspase-3, and similar results couls also be observed by the gel-based activity assay. The PDT-treated procaspase-3 and HMW products were separated and purified using reverse phase hydrophobic interaction chromatography (C4 column), digested by trypsin, and the resulting peptides from PDT-treated procaspase-3 and HMW products were separately analyzed by LC-MS/MS (MicrOTOF-Q). After comparing the MS spectra using Metabolite tools (a software for MS spectrum comparison), 42 probably modified/cross-linked peptides were detected. So far, we have analyzed three candidate peptides by de novo sequencing, but we didn’t find cross-linked peptides. Instead, these peptides are post translational modified by PDT treatment which might result from the unstable cross-linking bonds of the HMW products. The actual cross-linking site(s) in the HMW products of procaspase-3 need further analysis.