Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory

Abstract

Donor-linker-acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of -0.60 V, much lower than the best thiol reductant reported in literature, the DTT (-0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo' has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.

Activity-Based Sensing for Site-Specific Proteomic Analysis of Cysteine Oxidation

“…Reversibly oxidized cysteine modifications can be indirectly detected by thiol-blocking, reduction of oxidized species, followed by labeling of nascent thiols (Figure a). DTT and TCEP (standard reduction potential = −0.33 and −0.32 V, respectively) are powerful reducing agents that are capable of converting reversible cysteine OxiPTMs to thiols, but they do not effectively reduce −SO 2 H or −SO 3 H. Alternatively, a given OxiPTM may be selectively reduced and tagged for detection. Sodium ascorbate and sodium arsenite were considered as selective reducing agents for −SNO and −SOH, respectively. , Likewise, enzymatic reduction of −SSG by Grxs has been employed to detect S-glutathionylation .…”