Using high-performance liquid chromatography to measure the effects of protein-stabilizing cosolvents on a model protein and fluorescent probes.

The effect of cosolvents on the fluorescence of solutes was measured manually and in an automated high-performance liquid chromatography (HPLC) system that eliminates fluorescent contaminants on-line. The HPLC system was used to show that the effect of cosolvents on the fluorescence spectrum of heated chymotrypsin (a measure of unfolding) correlates with the effect of the solutes on the heat stabilization of catalytic activity; r2=0.73 with 12 example cosolvents. Changes in the fluorescence of model probes showed that known counteracting solutes slightly decrease the polarity of the solvent. Different cosolvents affect the proton transfer indicator, 2-naphthol (a model for tyrosinyl residues) differently, polyhydric alcohols enhance the protonated naphthol emission whereas zwitterionic solutes enhance naphthoxide fluorescence. The results with the automated system are consistent with the known stabilizing effects of the cosolvents and validate it as a tool to explore the development of novel cosolvents and their effects on multiple biological systems.