Giuseppe Cilento

PEROXIDASE CATALYZED GENERATION OF TRIPLET ACETONE

Abstract— Triplet acetone generated in the isobutanal/horseradish peroxidase/O, system is quenched by collisional agents of the diene type. A Stern‐Volmer analysis indicates that collision is considerably impeded, the barrier being largely entropic. Triplet‐triplet energy transfer to biacetyl is also observed. Quenching of triplet acetone by energy transfer to 9,10‐dibromoanthracene‐2‐sulfonate (DBAS) is a much faster process than energy transfer to these collisional agents, in accordance with the earlier inference that transfer to DBAS is a long‐range triplet‐singlet transfer process. The fraction of the triplet acetone which undergoes reduction to isopropanol is not interceptable by quenchers.

FROM FREE RADICALS TO ELECTRONICALLY EXCITED SPECIES

Biologically/medically important compounds, when metabolized, can generate free radicals from which electrically excited products--often in the triplet state--are generated. Peroxidases are particularly apt to catalyze such processes, which usually entail oxidations by electron transfer. In the latter case, the chemiluminescence may derive from peroxyl and alkoxyl radicals or excited states derived from dioxetanes. Besides peroxidases, prostaglandin-H synthase and lipoxygenase may catalyze the formation of excited carbonyls. The pronounced similarity in the chemical behavior and reactivity of radicals and excited species derives in part from the biradical nature of the latter. Usually in analyzing the biological effects of xenobiotics, only radicals and/or reactive ground state products have been considered. However, in such processes the generation of excited species is possible, which should be tested for by direct and/or sensitized emission or by photochemical transformation.

PHOTOCHEMISTRY and PHOTOBIOLOGY WITHOUT LIGHT

Abstract— This review covers the literature since 1980 on chemically and enzymatically generated electronically excited species. The emphasis lies on triplet states of carbonyl products that are derived from dioxetanes and dioxetanones as precursors or from suitable enzymatic oxygenations. Singlet oxygen, an important excited state species in biological processes, is not explicitly treated. The utilization of triplet excited carbonyl products to promote photochemical and photobiological transformations by energy transfer are of primordial interest and not the photomechanistic behavior, photophysical properties and inherent photochemical reactions of such excited state species. Thus, the coverage concentrates on photodamage of DNA and RNA, the photochemistry of flavins, vitamin D, tryptophan, arachidonic acid, chlorophyll, lipid peroxidation, urocanase activation, excitation of chlorophlasts, and the aerobic oxidation of Schiff bases derived from amino acids and proteins. The potential perspectives of employing authentic dioxetanes and enzymatically generated dioxetane intermediates as effective photon equivalents in photochemotherapy, phototoxicity, photoaffinity labeling and photogenotoxicity are pointed out, in the hope of stimulating more intensive activity in this emerging and novel bioorganic and photobiological field.

ENERGY TRANSFER FROM ENZYMICALLY‐GENERATED TRIPLET SPECIES TO ACCEPTORS IN MICELLES

Abstract— Electronically excited triplet species generated during the peroxidase catalyzed aerobic oxidation of appropriate substrates efficiently elicit fluorescence from acceptors in micelles, as shown with 9, 10‐dibromoanthracene and chlorophyll solubilized by various surfactants. In the case of 9, 10‐dibromoanthracene excited by triplet acetone, phosphorescence also can be detected near O2 depletion.

Photobiochemistry in the Dark

The generation of electronically excited states in biological processes not readily classifiable as bioluminescent has been under consideration for a long time (Szent-Gyorgyi, 1941; Anderson, 1947; Steele, 1963; Cilento, 1965, 1973; Forster, 1967; White and Wei, 1970; White et al., 1970, 1971, 1974). Clearly, if the cell were endowed with the potential of photochemistry in the absence of light, it would have at hand an extremely useful, although potentially detrimental, tool. One may suspect that nonemissive energy may also be generated, or possibly triggered and subsequently transferred. It is inherently difficult to believe that electronic energy should be generated in vivo exclusively for emission purposes.

Generation of electronic energy in the peroxidase catalyzed oxidation of indole-3-acetic acid

Summary The horseradish peroxidase-catalyzed aerobic oxidation of indol-3-acetate results in the conversion of the enzyme to a green form. The spectral changes observed after the oxidation of small amounts of substrate may be reproduced approximately by irradiation of the enzyme at 290 nm. The possibility that electronic energy is generated in the indoleacetate/peroxidase/0 2 system, and is responsible for the change is supported by the chemiluminescence, albeit very weak, observed in the presence of sodium 9,10-dibromoanthracene-2-sulfonate, and of eosin. It appears that the chemienergized species is indol-3-aldehyde in its triplet state.

Chlorophyll: an efficient detector of electronically excited species in biochemical systems

Micelle-solubilized chlorophyll efficiently detects electronically excited species generated in enzymatic systems. In most, if not all, systems the chemiexcited species is formed in the triplet state; chlorophyll fluorescence is observed as result of energy transfer. Red emission can also be elicited from chlorophyll in chloroplasts or bound to microsomes.

CHEMIENERGIZED SPECIES IN PEROXIDASE SYSTEMS

Abstract— Several hemeprotein‐catalyzed reactions generate products of the type expected from the cleavage of a high energy intermediate. For some systems, the formation, in high yield, of a carbonyl compound in its excited triplet state has been firmly established on the basis of (i) equivalence of the chemiluminescence and phosphorescence spectra of the expected products; (ii) energy transfer to sensitizers containing heavy atoms and (iii) occurrence of photoproducts. The excited species appears to be generated within the enzyme and shielded from quenching by oxygen. It may be quenched, however, via long‐range triplet‐singlet energy transfer.

ELECTRONICALLY EXCITED SPECIES IN THE PEROXIDASE CATALYZED OXIDATION OF INDOLEACETIC ACID. EFFECT UPON DNA AND RNA

Abstract— The electronically excited species generated in the peroxidase (oxidase) catalyzed oxidation of the plant hormone indole‐3‐acetic acid is an excited state of indole‐3‐carboxaldehyde.

FREE RADICALS AND EXCITED SPECIES IN THE METABOLISM OF INDOLE‐3‐ACETIC ACID AND ITS ETHYL ESTER BY HORSERADISH PEROXIDASE AND BY NEUTROPHILS

Abstract— The peroxidative metabolization of indole‐3‐acetic acid, a biologically important process, has been followed by EPR spectroscopy with the aim of obtaining information on the mechanism of generation of electronically excited species.