Guido Galiazzo

Dye-sensitized selective photooxidation of methioxine

Abstract The photooxidation, sensitized by rose bengal and methylene blue, of cystine, methionine, histidine, tyrosine, tryptophan and of some related peptides was studied in acid media. Rose bengal in formic acid solution, and methylene blue or rose bengal in aqueous acetic acid solution sensitized a selective oxidation of methionine, which was quantitatively converted to methionine sulphoxide. Irradiation of ribonuclease A (ribonucleate pyrimidinenucleotide-2′-transferase (cyclizing), EC 2.7.7.16) under the same conditions caused the modification of the four methionyl residues and a concomitant 87% decrease of the enzymatic activity, which was correlated to a conformational change of the protein. Chemical reduction of the photooxidized ribonuclease A by thioglycolic acid resulted in the full recovery of the enzymatic activity.

Heavy atom effect on the luminescence of phenanthrene

Abstract The internal heavy atom effect on the radiative and radiationless transitions from the lowest excited states of phenanthrene was investigated. Fluorescence and phosphorescence quantum yields and triplet lifetimes were determined for phenanthrene and its methyl, chlorine and bromine derivatives, and approximate rate parameters to and from the lowest triplet level were calculated. The halogen substitution induces a larger increase of the intersystem crossing S 1 -T 1 rate than of the T 1 -S 0 one, resulting in a higher overall triplet formation quantum yield and, also, higher radiative decay quantum efficiency from the triplet. An effect of the halogen position on the luminescence properties was also observed.

Photo-oxidation of horse heart cytochrome c. Evidence for methionine-80 as a heme ligand

In a previous paper [I] , we showed that the irradiation of a protein containing a photosensitizer covalently linked to known positions in the molecule, causes the selective modification of only those potentially photo-oxidizable amino acid residues which are in close proximity to the sensitizer. Therefore, once the modified side chains have been identified, their location in the three-dimensional network of the protein molecule can be deduced. In principle, it should be possible to extend this method to proteins naturally possessing a chromophoric group which can act as a photosensitizer. As a first approach we attempted to explore the environment of the porphyrin prosthetic group in horse heart cytochrome c. Since porphyrins efficiently mediate the photo-oxidation of amino acids [2], it was expected that the porphyrin group of this protein would likewise serve as such a photosensitizer. Cytochrome c was chosen also because its primary structure is known, and its biological, physical and chemical properties have been studied in detail [3] . The results presented in this communication show that only those susceptible amino acids which are adjacent to the heme group are photo-oxidized upon irradiation of ferricytochrome c. Furthermore, our data provide compelling evidence that Met-80 serves as a protein ligand for the F

Selective and quantitative photochemical conversion of the tryptophyl residues to kynurenine in lysozyme

+ ion in ferricytochrome c. 2. Materials and methods

Evidence of adiabatic channels in the singlet photoisomerization of cis-1,2-diarylethenes: a fluorimetric study

Abstract Dye-sensitized photooxidation is increasingly used as a tool for the elucidation of the structure-activity relationship in proteins. However, the similarity in the relative rates of oxidation of the susceptible amino acids is a serious limitation on the method. In a recent paper (Benassi et al., 1967), we showed that, in formic acid solution, only tryptophan and methionine are photoreactive, if proflavine is used as the sensitizer: the former is converted to kynurenine, the latter to methionine sulphoxide. Since methionine sulphoxide is easily reverted to methionine by reaction with mercaptans (Hofmann et al., 1966; Jori et al., 1968), the possibility exists to perform selective photooxidation of the tryptophyl residues. The method was successfully applied to model peptides (Benassi et al., 1967), however, its feasibility in the case of proteins could only be evaluated through actual application. This report details our findings about the photodynamic action of proflavine on lysozyme (N-acetylmuramide glycanohydrolase, EC 3.2.1.17). Lysozyme was chosen since its threedimensional configuration has been elucidated (Blake et al., 1967), and the chemistry of the enzyme has been thoroughly investigated.

Decay pathways of the first excited singlet state of cis-1-styrylpyrene

Abstract The deactivation of the first excited singlet state (S1) of some cis isomers of 1,2-diarylethenes was studied by fluorimetric and photochemical measurements. When at least one of the aryl groups is a large polycyclic group of low S1 energy (as in the case of pyrenyl and anthryl derivatives) the minimum at 90° (typical of stilbene) in the potential energy curve as a function of the twisting coordinate may disappear. This leads to an unusual fluorescence emission at room temperature and favours the adiabatic 1cis* → 1trans* isomerization mechanism instead of the well-known diabatic pathway, which implies internal conversion to the ground state from the 1perp* configuration.

Solvent effects on the emissive properties of trans-3-styrylquinoline in neutral and acidic solutions

Abstract The cis isomer of 1-styrylpyrene displays an unusual fluorescence emission in fluid media at room temperature. The photophysical properties of the fluorescent state were studied in non-polar solvents in a large temperature range. The fluorimetric and photochemical results evidenced a substantial contribution of an adiabatic singlet mechanism to the cis→trans photoisomerization.

3-styrylquinoline conformers: A photophysical and photochemical study

Abstract The wavelength dependence of the fluorescence quantum yields of trans-3-styrylquinoline and of its protonated form has been studied in solvents of different polarity and hydrogen-bonding characteristics. The results obtained show that in all the examined solvents trans-3-styrylquinoline, in both neutral and protonated forms, exists as a mixture of two conformational isomers. The fluorescence lifetimes, emission and trans → cis photoisomerization quantum yields strongly depend on the solvent but they show no unequivocal correlation with its properties.

Paramagnetic metal ions as protectors of selected regions of protein molecules from photodynamic action.

Abstract The emission spectrum of a 10−6 M cyclohexane solution of trans-3-styrylquinoline varies with the excitation wavelength. Fluorescence excitation spectra at room temperature and at 77 K indicate the presence of two emitting species which are assumed to be conformational isomers arising from rotation about the single bond linking the styryl part of the molecule to the quinoline ring. The dependence of the photophysical and photochemical properties of trans-3-styrylquinoline in various solvents on the excitation wavelength are reported. The influence of pH on the composition of the conformers and the dependence on the excitation wavelength of the percentage of molecules which attain the prototropic equilibrium in the excited state were studied.

Probing the topography of proteins in solution by photosensitized oxidation. The heme environment in horse heart ferrocytochrome c.

Abstract The paramagnetic ion Co2+ and the diamagnetic ion Zn2+ were coordinated with the glutamyl-35 and aspartyl-52 residue at the active site of lysozyme. Upon irradiation with visible light in the presence of proflavin, Zn2+-lysozyme underwent total loss of enzymic activity as well as of the tryptophyl and methionyl side chains; the kinetics of photoinactivation and of tryptophan photooxidation were coincident with those determined for native lysozyme. On the contrary, irradiation of Co2+ lysozyme caused the specific modification of the peripheral tryptophan-63 and tryptophan-123, and a decrease of the enzymic activity to 84%. The photooxidized sample showed no appreciable distortion in the spatial conformation with respect to the unirradiated protein, thus explaining the retainment of a high catalytic efficiency. The strong inhibitory power of paramagnetic ions against the photodynamic action of dyes on proteins was also evidenced by our studies with ribonuclease A, whose complexes with Zn2+ or Cu2+ were subjected to proflavin-sensitized photooxidation over the pH range 5.8–8.2. At all pH values Zn2+-ribonuclease A displayed a marked photolability, whereas the presence of Cu2+ minimized or completely prevented both the impairment of the biological activity and the damage of the amino acid side chains. In particular, after irradiation at pH 6.9, we isolated a 72% active sample which was modified at methionine-79 and histidine-105. Conformational studies on the photooxidized derivatives of lysozyme and ribonuclease A, and the analysis of our results on the basis of the X-ray models of the native proteins demonstrated that the inhibitory action of Co2+ and Cu2+ occurs throughout almost the entire protein molecule, the efficiency of the protection being related to the distance of a given amino acid from the paramagnetic ion and the symmetry of the magnetic field. Besides providing a valuable tool for protecting enzymes from photodynamic inactivation, the aforesaid technique opens new prospects in the field of dye-sensitized photooxidation of proteins.