fluorescence dequenching makes haem-free soluble guanylate cyclase detectable in living cells荧光dequenching使haem-free可溶性鸟苷酸环化酶检测活细胞.pdfVIP

Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells 1¤ 2 1 3 4 Linda S. Hoffmann , Peter M. Schmidt , Yvonne Keim , Carsten Hoffmann , Harald H. H. W. Schmidt , Johannes-Peter Stasch1,5* 1 Pharma Research Centre, Bayer HealthCare, Wuppertal, Germany, 2 CSIRO Materials, Science and Engineering, Parkville, Victoria, Australia, 3 Julius-Maximilians-University of Wuerzburg, School of Pharmacology and Toxicology, Wuerzburg, Germany, 4 Department of Pharmacology, Maastricht University, Maastricht, The Netherlands, 5 Martin-Luther-University, School of Pharmacy, Halle, Germany Abstract In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO- sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increas