Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces.

Nile red is an uncharged hydrophobic molecule whose fluorescence is strongly influenced by the polarity of its environment. It interacts with many, but not all, native proteins, including beta-lactoglobulin, kappa-casein, and albumin, with a wide range of spectral changes for different proteins. It detects the exposure or formation of new hydrophobic surfaces induced by ligand binding to calmodulin, oligomerization of melittin, or unfolding of ovalbumin during early thermal denaturation. The dye is photostable, the working wavelength range is broad and removed from those at which many biomolecules absorb, the fluorescence is unaffected by pH between 4.5 and 8.5, the quantum yield is high, and hydrophobic sites on proteins may be investigated in dilute solutions.