Fluorescence of tyrosine and tryptophan in proteins using one- and two-photon excitation.

We examined the emission spectra of tyrosine- and tryptophan-containing proteins using one-photon (270-310 nm) and two-photon (565-610 nm) excitation. Emission spectra for two-photon excitation of native and denatured human serum albumin and of three purine nucleoside phosphorylases indicated an absence of the tyrosine emission normally seen for one-photon excitation below 290 nm. We examined the one-photon and two-photon excitation spectra of tyrosine-tryptophan mixtures to determine the origin of selective excitation of the tryptophan residues. These results confirmed a short-wavelength shift of the tyrosine two-photon excitation spectrum relative to that of tryptophan, as recently reported by Rehms and Callis (1993) Chem. Phys. Lett. 208, 276-282.