Characterization of the indole triplet excited state in proteins utilizing laser flash photolysis

The triplet-triplet absorption spectrum of the sole indole side chain of human serum albumin and its decay kinetics were previously characterized, at room temperature, by using a conventional flash photolysis method [(1978) Proc. Natl. Acad. Sci. USA 75, 1172-1175]. Exploitation of this potentially useful long lived reporter group in protein studies was limited by the excessively large sample size required by that apparatus. The 265 nm laser flash instrument used in the present work avoids this problem at the price of a loss in photo-selectivity. We report that the latter concern can be mitigated. Melittin was studied first because this polypeptide contains a single aromatic residue (W-19), and because its monomeric and tetrameric forms are good models for solvent exposed and buried indole side chains of proteins. For both forms, the indole triplet and neutral radical absorption spectra could be readily time resolved and identified on the basis of shape and differential dioxygen sensitivity. The single tryptophan containing protein human serum albumin was studied next because it contains a large number of other 265 nm absorbing moieties whose transient spectra might complicate the detection of the indole triplet. These transients were shown to not interfere significantly in the wavelength region 450 nm to 600 nm, and, in contrast to the indole triplet, they were relatively dioxygen insensitive. Thus, a facile means is available by which the indole triplet of proteins may be characterized. Subsequently the question of whether this species could be detected in the presence of nuclei acid components was investigated by flashing the phage fd. The putative nucleic acid transients were shown not to interfere and the absorbance of the indole triplet was readily time resolved. The spectral assignment was persuasively confirmed by showing that the indole triplet absorption and phosphorescence emission spectra decay with the same lifetime. The present work thus provides additional evidence for the general applicability of the indole triplet excited state as a long lived intrinsic protein reporter group.