title: Fluorescence dequenching makes haem-free soluble guanylate cyclase detectable in living cells.
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 increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.
authors.affiliation: Pharma Research Centre, Bayer HealthCare, Wuppertal, Germany.
authors.forename: Linda S
authors.initials: LS
authors.lastname: Hoffmann
authors.forename: Peter M
authors.initials: PM
authors.lastname: Schmidt
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Keim
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Harald H H W
authors.initials: HH
authors.lastname: Schmidt
authors.forename: Johannes-Peter
authors.initials: JP
authors.lastname: Stasch
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006152:Cyclic GMP / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D010078:Oxazines / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D000071756:Soluble Guanylyl Cyclase; D013050:Spectrometry, Fluorescence
chemical_list: D001565:Benzoates; D020536:Enzyme Activators; D005456:Fluorescent Dyes; C111257:NS 2028; D010069:Oxadiazoles; D010078:Oxazines; D018160:Receptors, Cytoplasmic and Nuclear; D012402:Rotenone; D009569:Nitric Oxide; C480588:BAY 58-2667; D006418:Heme; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D003545:Cysteine