Phenolic Chromophores Research Articles

Pulsefluorimetry of tyrosyl peptides.

Abstract The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x1 which determines the conformation of the tyrosyl residue around its Cα-Cβ bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.

Block oligopeptides (L‐lysyl)m‐(L‐alanyl)n‐L‐Tyrosyl‐(L‐Alanyl)n‐(L‐Lysyl)m. II. Circular dichroism and pulsefluorimetry conformational studies

AbstractThe conformation of chromatographically pure block oligopeptides (L‐lysyl)m‐(L‐alanyl)n‐ L‐tyrosyl‐(L‐alanyl)n‐(L ‐lysyl)m with n = 3 and m = 6 or 3 is investigated. By circular dichroism it is shown that these peptides may exhibit a partially α‐helical structure depending upon pH, ionic strength, solvent, and temprerature. An attempt is made to describe the helical content of these small peptides by utilizing the data obtained on high‐molecular‐weight poly(L‐lysine). By measurement of the quantum yield and the decays of the peptides fluorescence, it is shown that, in aqueous solution, at neutral pH, the fluorescence of the peptides is quenched by interactions with the peptide carbonyl groups. The decays are multiexponential, which shows the presence of several conformations of the phenolic chromophore relative to the peptide chain. The addition of methanol, which induced the helix formation, decreases the quenching of the fluorescence and the multiexponential character of the decays. In presence of sodium hydroxide, which further increases the helical content of the peptides, a dynamic quenching occured that can be attributed to interactions between the phenol hydroxyl group of tyrosine (ith residue) and the ε‐amino groups of the (i+4)th and (i ‐4)th lysyl residues.

Thermal perturbation difference spectra and D 2O vs H 2O isothermal difference spectra of protein-model aromatic chromophores

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D 2O and H 2O spectra of these chromophores. For phenols approximately equal Δϵ values are obtained in both types of spectra, but for their methyl ethers Δϵ values of D 2O vs H 2O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D 2O or H 2O. For phenols in D 2O vs H 2O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D 2O and H 2O. The refractive index difference between D 2O and H 2O was shown to be a minor contribution by means of experiments in which D 2O was at 5 dgC and H 2O at 47 dgC, conditions of equal refractive index (Na D). D 2O vs H 2O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D 2O vs H 2O spectra of proteins are discussed.

On the luminescence of estrogens.

Abstract—The absorption, fluorescence, and phosphorescence of a series of estrogens is reported. The behavior of estrogens without a keto‐group is compared with that of the isolated phenylic, phenolic and naphtholic chromophores. In the latter two, attachment to the steroidal frame is inconsequential as regards their luminescent behavior. In all keto‐estrogens with the exception of equilenin there is a very strong transfer of excitation energy from the A ring chromophore to the keto group. The fluorescence of 6‐keto‐estradiol, which has a conjugated aromatic carbonyl chromophore, is apparently due to the rigidity of the steroid frame and exhibits a rather perculiar excitation spectrum.

Fluorescence of neurotoxins from middle-asian cobra venom

The shape and position of the tryptophanyl fluorescence spectra, quantum yields and parameters of quenching have been studied for Neurotoxins I and II from middleasian cobra venom in aqueous solutions at pH values from 2 to 12. The data show that the single tryptophan residue in both neurotoxin molecules is located on the surface of the macromolecule and is accessible for freely relaxing water. In both toxins there is a lysyl ε-amino group positioned at the distance of kinetic contact from the indole chromophore. This group in its deprotonated state (pH 8.5) can result in the fluorescence quenching. One or more additional positive groups are located in the tryptophan environment of Toxin I. They are probably arginine residues because they are able to quench the fluorescence and are not titratable in the pH range from 2 to 12. The tyrosine residues of both toxins have a very low fluorescence yield that is intrinsic for buried phenolic chromophores.

Intramolecular Interaction between the Phenol and the Indole Chromophores

Abstract The singlet-energy transfer from the phenol to the indole chromophores occurs intramolecularly, with a high efficiency, in the compounds containing these chromophores joined by the methylene and the amide groups. The total emission spectra of the compounds consisted only of those from the indole chromophore, neither the fluorescence nor the phosphorescence of the phenol chromophore contributing to them, and the excitation spectra of the fluorescence were similar to the absorption spectra. The quantitative analysis of the fluorescence-polarization spectra suggested that the 1Lb transition of the indole chromophore was excited in the energy transfer. The intramolecular interactions between these chromophores in the ground and excited states are discussed.

Changes in Ultraviolet Absorption Produced by Alteration of Protein Conformation

Transfer of an aromatic chromophore from the interior of a protein into the water solvent upon denaturation of the protein produces an absorption change throughout the ultraviolet region, 215 to 320 mµ, approximately 6 times the magnitude of that produced by transfer of the same chromophore from 20% ethylene glycol into water. In the 230 mµ wave length region, the transfer of an indole chromophore produces an absorption change approximately 7 times that produced by the transfer of a phenolic chromophore; the chromophores of the amino acids cystine, histidine, and phenylalanine produce only slight changes in absorption. Difference spectra for protein denaturation were calculated by summation of the 20% ethylene glycol perturbation difference spectra of the amino acids, and multiplication by the factor of 6. Between 215 and 320 mµ, difference spectra produced by acid, autolysis, or urea denaturation closely resemble calculated ones, but are red shifted 2 to 3 mµ near 280 mµ and 4 to 10 mµ near 230 mµ. Thus, the absorption changes observed near 230 mµ for globular proteins result primarily from changes in the environment of the aromatic chromophores indole and phenol. The helix to coil transition and the denaturation-produced solvent perturbation of the amide group must contribute less than 10% of the absorption changes observed near 230 mµ.