The heme oxygenase‐1 protein is susceptible to oxidative modification through protein carbonyl formation

• Published in: The FASEB journal 2007, Vol.21 (6), p.A816-A816
• Main Authors: Yang, Guang, Hu, Angela, Bordner, Jessica, Dennery, Phyllis A
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.21.6.A816

Heme oxygenase‐1 (HO‐1) is an integral membrane of the smooth endoplasmic reticulum. This hydrophobic membrane‐binding domain is located at the c‐terminus adjacent to a PEST sequence that can be cleaved by proteases. Recently we have found that an HO‐1 protein lacking the C‐terminus localized to the nucleus under oxidative stress. Protein carbonyl formation is a hallmark of oxidative stress and carbonylation at specific sites can alter protein function. Because HO‐1 is a stress response protein we wanted to know whether HO‐1 protein was susceptible to carbonyl formation and whether carbonylation at specific sites explained HO‐1 nuclear localization. Cultured fibroblasts were incubated with hemin at 0–60 μM for 8 h, with 0–200 μM H2O2 or with a mixture of 100 μM FeCl3 + 25 mM ascorbic acid (MCO) for 4 h. Cell lysates were immunoprecipitated with HO‐1 antibodies and were subjected to protein carbonyl detection using a commercially available kit. In other experiments, lysates were incubated with 5 mM biotin hydrazide and then immunoprecipitated with streptavidin and loaded onto an acrylamide gel for HO‐1 detection using Western analysis. Protein carbonyl formation on HO‐1 was observed with all oxidant incubations. Because hydrazide labels specific carbonyls from the side chains of lysine or arginine, this suggests that carbonyls on HO‐1 are more likely to target lysine or arginine residues.