RESONANCE ENERGY TRANSFER IN PEPSIN CONJUGATES

A series of pepsin derivatives was prepared in which a substrate analogue containing a chromophore selected to be a resonance energy acceptor from tryptophan was covalently attached to the active site of the enzyme. The decreases in pepsin fluorescence intensity and lifetime produced by chromophores differing in absorption spectrum and intensity could be attributed to resonance energy transfer, and showed that the tryptophan groups emitted independently and had different probabilities of transfer to the surface chromophore. The efficiency of sensitization of fluorescent chromophores by tryptophan, and the depolarization of the chromophore fluorescence sensitized by tryptophan absorption, indicated similar quantum yields and random orientation for these residues. The observed efficiencies of energy transfer were very much higher than those calculated for model systems having a randomly distributed and oriented array of either fixed or rapidly rotating dipoles transferring energy to one acceptor placed in the surface of the volume containing the dipoles. The reason for this discrepancy in pepsin and two other proteins has been considered in terms of non-uniform distributions, intertryptophan energy transfer and the low polarizability of the macromolecular interior. © 1969.