TRFS-green

A highly selective off-on fluorescent probe for imaging thioredoxin reductase in living cells, displaying a green fluorescence off-on change induced by the TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore

Mechanism / How it Works

• The probe has a 1,2-dithiolane scaffold and a quenched naphthalimide fluorophore. PubMed

• Activation by TrxR involves two steps:

  1. Disulfide cleavage: TrxR reduces the disulfide bond in the scaffold. PubMed+2Nature+2

  2. Intramolecular cyclization: After reduction, a cyclization reaction occurs to release the masked naphthalimide fluorophore, which then emits fluorescence. PubMed

• Once activated, TRFS-green shows green fluorescence under the right excitation/emission settings. For example, excitation around 377 nm, emission signal around ~480 nm or similar, depending on the exact setup. 

Key Functional Features & Usage

• Selectivity: TRFS-green is selective for TrxR over many related enzymes, and over abundant cellular reducing agents like glutathione (GSH), cysteine (Cys), NADPH, vitamin C, etc.

• Application: It can be used in live cell imaging to see TrxR activity. By treating cells with TRFS-green, one can observe where and how strongly TrxR is active. If you pre-treat with a TrxR inhibitor, the fluorescence is decreased in a dose-dependent manner. PubMed

• Measurement in extracts: It can also be used with cell lysates (protein extracts) to quantify TrxR activity. 

Limitations / Kinetics

• Slow response: One drawback is that TRFS-green responds relatively slowly. It may take 2 hours or more to reach the maximal fluorescence signal. Nature

• Fluorescence fold increase: The magnitude of fluorescence increase is moderate (~30-fold) compared to its background. So while it’s good, it’s not the fastest or brightest among newer probes. Nature+1