Fluorescence Studies of the Bacterial Protease E‐30‐iii

The fluorescence emission of the bacterial protease E‐30‐III, irradiated with 290 nm light, is exclusively due to the tryptophyl residues, which appear to be located in a rather polar environment. One indole side chain, which is fully exposed to the aqueous solvent, gives a minor contribution to the overall emission of the protein. On the other hand, the two strongly emitting indole systems appear to be contained in internal regions having a relatively high dielectric constant; fluorescence quenching studies demonstrate that the efficiency of the fluorescence emission by the two residues is about the same. In particular, one tryptophyl side chain must be spatially adjacent to a glutamyl or aspartyl residue. Exposure of the protein to 280 nm and 290 nm radiation reveals the occurrence of an efficient energy transfer from tyrosinie to tryptophan in the neutral pH region and from tryptophanie to tyrosinate in alkaline solutions; the latter process parallels the ionization of the phenolic side chains, as determined by spectrophotometric titrations. In acid solutions, the electronic energy transfer among aromatic residues is greatly diminished, and clearly resolved emission spectra from the tyrosyl and tryptophyl residues are observed.