Unusual emission properties of the tryptophans at the surface of short ragweed allergen Ra5.

The fluorescence and low-temperature phosphorescence of short ragweed pollen allergen Ra5 have been examined. Both the fluorescence of Ra5 in aqueous solution at room temperature and the phosphorescence of Ra5 in rigid glycol-water solvents at low temperature result entirely from the tryptophan residues in the molecule. The heterogeneity in the low-temperature phosphorescence spectra indicates that the two tryptophans in Ra5 are located in distinct environments and can be studied independently. The fluorescence wave-length maximum and large solvent-relaxation shift in the fluorescence of the allergen in 1:1 ethylene glycol-water reveal that at least one of the residues is exposed to the polar solvent. Studies of the iodide and cesium ion quenching of the fluorescence of Ra5 are consistent with this assignment but suggest that one of the tryptophans may be shielded by a negative charge in the protein. Phosphorescence data obtained in the presence of iodide and with increasing temperature confirm that both aromatic residues are readily subject to perturbation but identify the more susceptible one with one of the two spectral components that manifest themselves in the phosphorescence of Ra5. The phosphorescence wavelength maxima and the triplet-state lifetimes of the distinct tryptophans reveal that both residues lie in unusual local environment at the protein surface. While one aromatic side chain appears to be stabilized by an intramolecular interaction with a polar function, the other is perturbed quite possibly through an interaction with a disulfide linkage in the molecule.