A study of the fluorescence properties of cyclo(L-histidine-L-tryptophan), cyc$o(L-histidine-Dtryptophan), a,N-acetyl-L-histidine-L-tryptophan methyl ester, a,N-acetyl-L-histidine-L-tryptophan and (L-histidine-L-tryptophan), has revealed an intramolecular interaction between the indole and the protonated imidazole side chains which results in marked quenching of the fluorescence intensity of the indole moiety. All five model compounds were found to possess a characteristic fluorometric titration curve which practically coincides with the potentiometric titration curve of the imidazole side chain of the histidine residue. Theoretical derivations of the pK from both titration curves are given. The extent of quenching by the protonated imidazole moiety, however, is different for each compound and varies from 40 to 8001,.To detect the presence of intramolecular histidine-tryptophan complexes in natural peptides and proteins, fluorometric titrations in aqueous solutions were carried out in the range of pH 4-9. Marked changes in fluorescence intensity with pH in the range of p H 5 to 8.5, suggesting the existence of well defined complexes, were observed with papain, activated papain, chymopapain, ficin and bromelain. Glucagon, pepsinogen, pepsin and goose egg white lysozyme show no change in fluorescence in this pH range. I n systems where a histidine-tryptophan interaction was observed, the pK value of the interacting histidine was evaluated from the fluorometric titration curve.The formation of complexes of the charge-transfer type in aqueous solution between compounds containing an indole ring and compounds containing a protonated imidazole ring has been recently demonstrated [1,2]. Protonatedimidazole and histidine derivatives were also shown to quench the fluorescence of the indole ring [ 1, 21. Since the fluorescence spectra of proteins are due almost exclusively to emission from the indole side chains of the tryptophan residues [3], intramolecular interaction between tryptophan and a protonated histidine side chain should reduce the total quantum yield of the protein molecule. Moreover, since only the ionized form of the imidazole is involved in this type of quenching, the deprotonation of the ionized imidazole should be followed by an increase in the fluorescence intensity. It might therefore be expected that a fluorometric titration (i,e., a determination of the change in fluorescence intensity with pH) of various systems containing interacting histidine and tryptophan side chains, will enable the evaluation of the pK of the imidazole moiety participating in the complex. It should be born in mind, however, that proteins usually contain other titrable quenchers, such as the unionized E-amine of lysine [4,5], the unionized carboxyl group [6,7], and the ionized a-amine [5-71.I n this study, we describe the fluorometric titration of a scries of model compounds containing histidyl and tryptophyl residues as well as of several proteins. From these titration curves, we have derived the extent of quenching and the ba...