Ribonuclease A

• CAS: 9001-99-4
• Catalog Number: ACM9001994
• Category: Main Products
• Molecular Weight: 226.23246
• Molecular Formula: C9H14N4O3

Case Study

Oxidant-Induced Electrophilic Modification of Ribonuclease A for Detection via Fluorescein-Labeled Glutathione

Lopez A. C, et al. Redox Biochemistry and Chemistry, 2025, 11, 00048.

In this case, Ribonuclease A was exposed to several oxidative systems-singlet oxygen, AAPH-derived peroxyl radicals, ferryl species from Fe(II):EDTA/H₂O₂, and peroxynitrite-under physiologically relevant conditions. The resulting modifications were probed using fluorescein-labeled glutathione (Fl-GSH), a nucleophilic marker that forms covalent adducts with electrophilic protein residues.
Experimental conditions involved reacting 50 μM native or oxidized RNase A with 2 mM Fl-GSH in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mM DTPA. The mixture was incubated in the dark at room temperature for 2 hours. For quantification, a fluorescein-labeled RNase A standard was prepared using 3 mM NHS-fluorescein in 100 mM sodium borate buffer (pH 8.5), followed by purification via PD-10 gel filtration. The extent of labeling was quantified spectrophotometrically at 495 nm (ε = 70,000 M⁻¹cm⁻¹). This protocol highlights the utility of RNase A as a model system for identifying oxidative electrophilic modifications, with LC-MS/MS confirming tyrosyl and histidyl oxidation sites. These findings underscore a mechanistic pathway for protein damage in oxidative stress.