titration data.xlsx

SYPRO^® Orange (SyO) is a zwitterionic dye that is used for protein gel staining, for thermal melt assays of proteins, and as a marker for misfolded proteins. However, while widely utilized, much of SyOs’ photophysics remains unexplored. We studied the effect of pH on the photophysical properties of SyO in aqueous solution and found two well-defined transitions in the 0 to 10 pH range between three SyO species with distinct absorption and fluorescence properties. The first transition occurs around pH 1.5 and appears to be a coupled deprotonation–aggregation event. The second transition occurs between pH 4 and 5, and its pK_a depends on the concentration of SyO. A link between the concentration dependence of the pK_a of the second pH transition and the aggregation behavior of SyO at neutral pH is discussed, and aggregation equilibrium titrations are presented that suggest that SyO forms multimeric aggregates at neutral pH containing ten or more SyO molecules.