Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

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.在心血管疾病中，保护性一氧化氮（nitric oxide, NO）/可溶性鸟苷酸环化酶（soluble guanylyl cyclase, sGC）/环磷酸鸟苷（cyclic guanosine monophosphate, cGMP）信号通路受损，原因在于一氧化氮敏感型含血红素（haem）可溶性鸟苷酸环化酶的储备量减少，同时非一氧化氮敏感型无血红素可溶性鸟苷酸环化酶的数量出现反向升高。然而，目前除了通过新型治疗类血红素模拟剂（如BAY 58-2667）激活该酶之外，尚无直接检测细胞内无血红素可溶性鸟苷酸环化酶的方法。本研究证实，基于光学活性血红素辅基与连接的双砷荧光染料FlAsH（biarsenical fluorophor FlAsH）之间的相互作用的荧光去淬灭技术，可用于检测细胞内可溶性鸟苷酸环化酶的血红素状态变化。血红素与FlAsH的发射光谱存在部分重叠，这使得荧光基团可将能量转移至血红素，从而降低FlAsH的荧光强度。当血红素辅基丢失时（例如由氧化应激或被血红素模拟剂BAY 58-2667取代所致），能量转移过程会被阻断，进而导致荧光强度升高。研究通过观测到的一氧化氮诱导的可溶性鸟苷酸环化酶活性下降、可溶性鸟苷酸环化酶蛋白水平降低，以及BAY 58-2667的作用增强，验证了血红素的丢失。使用无血红素可溶性鸟苷酸环化酶突变体以及不被血红素淬灭的双砷染料作为对照，进一步证实了荧光强度升高是由血红素辅基的丢失所导致。本方法基于工程化改造的可溶性鸟苷酸环化酶变体的细胞表达，因此其应用范围仅限于重组表达系统。尽管如此，该方法仍可用于监测活细胞内可溶性鸟苷酸环化酶的氧化还原调控过程，未来的优化或许能够将该技术拓展至体内环境中。