Isolation and Partial Characterization of a Novel Psoralen-Tyrosine Photoconjugate from a Photoreaction of Psoralen with a Natural Protein

Abstract

The photoreaction of psoralens with DNA is a well-characterized reaction. However, the photoreactions of psoralens with proteins is not very well understood. Our objective was to isolate an amino acid-psoralen photochemical adduct. We photoreacted 8-methoxypsoralen (8-MOP) with T7 RNA polymerase, a protein that carries out the fundamental biological process of transcription. Amino acid composition analysis of the photoreacted polymerase revealed that tyrosines quantitatively reacted with 8-MOP. From the acid hydrolysates of the photoconjugated T7 RNA polymerase, an 8-MOP-tyr adduct was partially purified by HPLC. The purified 8-MOP-tyr adduct and related parent compounds were analyzed by UV-visible absorption, fluorescence and mass spectroscopy. Excitation/absorption spectra suggested that the pyrone of the original 8-MOP was modified in the isolated photoadduct, and that the adduct probably contained a benzofuran. Chemical ionization mass spectrometry was consistent with the photoaddition of tyr to 8-MOP with a conservation of the overall mass (±1 atomic mass units). As far as we know, this work represents the first instance of isolation and partial characterization of an amino acid-psoralen photoadduct.