Gas-Binding Studies of Class 1 Sugar Beet Phytoglobin and C86A Mutant Using Isothermal Spectral Shifts in High-Precision Microliter Assay

Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein-gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts upon ligand binding. Phosphate-buffered saline with Tween20 (PBS-T20) ensures gas stability and minimizes nonspecific adsorption. Using this approach, we characterized CO and NO binding to the recombinant wildtype (rWT) of <i>Beta vulgaris</i> Pgb 1.2 (BvPgb 1.2) and its C86A mutant. CO titrations revealed biphasic binding, with EC₅₀ ~400 nM and ~700 μM (rWT) and ~500 nM and ~400 μM (C86A). NO binding showed K_D values of ~1600 nM (rWT) and ~400 nM (C86A), implicating Cys86 in ligand affinity. This assay provides a robust, low-volume method for high-affinity protein-gas studies and shows biphasic dynamics in BvPgbs.