In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320-360 nm range with additional soret peaks around 400-410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology