Fluorescence anisotropy of tyrosine using one-and two-photon excitation

We examined the emission spectra and steady-state anisotropy of tyrosyl fluorescence with two-photon excitation from 565 to 578 nm. The emission spectra of phenol and N-acetyl-L-tyrosinamide (NATyrA) were all the same for one-photon excitation (OPE) and two-photon excitation (TPE), and the tyrosine emission from ribonuclease A showed 10-nm shift to longer wavelengths with TPE. Surprisingly, the anisotropy of tyrosine, NATyrA and Leu5-enkephalin in frozen solution were near zero for TPE as compared to near 0.3 for OPE. Low values of the anisotropy near 0.05 were also found for phenol and ribonuclease A. A low anisotropy appears to be a basic characteristic of tyrosine or tyrosyl residues with two-photon excitation.