Premium
A FLUORESCENCE STUDY OF TRYPTOPHAN‐HISTIDINE INTERACTIONS IN THE PEPTIDE ANANTIN AND IN SOLUTION
Author(s) -
Vos Rita,
Engelborghs* Yves
Publication year - 1994
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1994.tb03938.x
Subject(s) - chemistry , tryptophan , quantum yield , histidine , fluorescence , imidazole , indole test , peptide , phenylalanine , residue (chemistry) , photochemistry , quenching (fluorescence) , stereochemistry , amino acid , organic chemistry , quantum mechanics , biochemistry , physics
—Anantin is a heptadecapeptide in which the C‐terminal peptide chain pierces the covalently cyclized peptide ring formed by an amide link between the α‐NH 2 end group and the β‐carboxyl group of Asp(8). It contains a tryptophan and a histidine at positions 5 and 12 , respectively. Des‐Phe(17)‐anantin lacks the C‐terminal phenylalanine. Fluorescence emission intensity as a function of pH follows the ionization of a single residue. The pK a amounts to 7.23 ± 0.03 for anantin and is attributed to His(12). At pH 9 the quantum yield is 0.12 ± 0.01 for anantin, whereas at pH 4.5 the quantum yield decreases more than two‐fold (0.05 2 0.01). Practically identical parameters are observed for des‐Phe(17)‐anantin. This pH dependency reveals intramolecular quenching of the excited indole ring of Trp(5) by the imidazole of His(12), which results in a marked decrease of the tryptophan fluorescence at low pH. In a multifrequency phase fluorometric study the fluorescence lifetimes for both peptides at pH 4.5 and pH 9 are determined. At both, pH fluorescence decay is well described by a sum of two exponentials. For anantin at pH 4.5 the lifetimes are 0.72 ± 0.07 ns and 1.67 ± 0.07 ns. At pH 9 the lifetimes are 1.11 ±0.12 ns and 2.55 ± 0.03 ns. In methanol we find two lifetimes for anantin: 0.68 ± 0.01 ns and 2.57 ± 0.01 ns. The lifetimes are found to be slightly dependent upon emission wavelength. For des‐Phe(17)‐anantin practically the same values are observed. The quenching of tryptophan fluorescence by histidine is further studied in solution using N ‐acetyl‐tryptophanamide in the presence of increasing concentrations of imidazole in the protonated (pH 4.5) and unprotonated (pH 9) state and in methanol. At both pH values and in methanol, a linear increase in both the inverse of the steady‐state fluorescence F o /F and the inverse of the lifetime 1/τ with increasing imidazole concentration indicates that a collisional mechanism is at the root of the observed quenching. The quenching efficiency values, γ, are calculated and amount to about 0.32 at pH 4. 5 , 0.02 at pH 9 and 0.002 in methanol, showing that protonated imidazole is a better quencher than the unprotonated form, and that the nature of the solvent is involved even in the quenching by unprotonated imidazole. Tryptophan‐histidine interactions in solution and in the peptide are compared.