Fluorometric Detection of Histidine‐Tryptophan Complexes in Peptides and Proteins

A study of the fluorescence properties of cyclo(l-histidine-l-tryptophan), cyclo(l-histidine-d-tryptophan), α,N-acetyl-l-histidine-l-tryptophan methyl ester, α,N-acetyl-L-histidine-L-tryptophan and (l-histidine-l-tryptophan)3 has revealed an intramolecular interaction between the indole and the protonated imidazole side chains which results in marked quenching of the fluores-cence 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 80%. 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 pH 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. In systems where a histidine-tryptophan interaction was observed, the pK value of the interacting histidine was evaluated from the fluorometric titration curve.