UNAMBIGUOUS EVIDENCE FOR THE PARTICIPATION OF SINGLET OXYGEN (1δ) IN PHOTODYNAMIC OXIDATION OF AMINO ACIDS

Abstract

A variety of experimental tests have been applied to the methylene-blue-sensitized photooxidation of amino acids to distinguish between singlet oxygen and non-singlet oxidation mechanisms. Conventional flash photolysis and laser photolysis were used to measure the rate constants for the quenching of excited triplet sensitizer and singlet oxygen by the amino acids histidine. tryptophan and methionine and the nucleotide guanosine-5'-monophosphate. In the case of histidine, the rate constants alone rule out an oxidation mechanism involving direct reaction with excited dye. With the other amino acids, and with guanosine monophosphate, the oxidation rates might be accounted for by either mechanism. The inhibition of the photooxidation of both tryptophan and methionine as well as histidine by the singlet-oxygen quenchers N3-and tetramethylethylene suggests that these reactions occur via a singlet-oxygen mechanism. A newly developed test of singlet oxygen reactions involving a comparison of photooxidation rates in normal and perdeuterated solvents has been used to establish that the photooxidation of tryptophan proceeds primarily by a singlet-oxygen mechanism. These experiments appear to constitute the first proof that singlet oxygen is involved in the photooxidation of the three amino acids tryptophan, methionine and histidine. I N T R O D U C T I O NINSIGHT into the mechanism of photodynamic action has been seriously hampered by the lack of knowledge concerning the primary events in the dye-sensitized photooxidations of biologically important compounds in the presence of oxygen[ 1,2].Although it has been amply demonstrated that oxygen is involved[ 1,2], the nature of the primary oxidizing species still remains to be identified. In trying to deduce the mechanism of any dye-sensitized photooxidation, there are at least two major pathways to be considered, including: (a) direct reaction between sensitizer triplets and the oxidizable substrate, followed eventually by reaction with oxygen, and (b) reaction between oxidizable substrates and singlet oxygen which has been generated by energy transfer from triplet-state sensitizers.In pathway (a) there are many possible mechanisms whereby oxygen can participate in the processes following the initial reaction between dye triplet, 3 S , and substrate. For example, molecular oxygen may reoxidize semireduced dye intermediates. Alternatively, it may act as an electron acceptor and cause irreversible formation of oxidized products by electron transfer from half-oxidized substrate molecules (X') as follows:3s+x-+ s -+ xx*+o, + x+ + 0,-2 0 , -+ 2 H + + 02+H20,

Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

“…The same questions can be raised regarding the possible role of -OH in chemiluminescence. Although superoxide dismutase and catalase inhibited luminescence, the only effective -OH scavenger was tryptophan, and it also has been reported to quench '02 (25,26). Neither mannitol nor benzoate had an effect, whereas formate, although inhibitory, is also a known trap for H202 (36).…”

“…In control experiments, neither enzyme inhibited significantly when inactivated by autoclaving, and 100 ,ug each of two other proteins, bovine serum albumin and lysozyme, had no effect on activity. (23)(24)(25)(26), either stimulated chemiluminescence or had no effect on activity. We examined the action of these agents on the chemiluminescence of the xanthine oxidase-acetaldehyde system, which is mediated by oxygen radicals (19 (13,31), which has been attributed to removal of the direct oxidative effects of O2 (31).…”

Investigation of the Effects of Different Carotenoids on the Absorption and CD Signals of Light Harvesting 1 Complexes

“…Carotenoids are also known to be important photoprotectors for the organism by virtue of their ability to quench the BChl excited triplet states and prevent the formation of singlet oxygen that is highly reactive and harmful for the complex. 14,15 In the purple bacteria, BChls and carotenoids associate very effectively with the LH polypeptides to form LH complexes that capture sunlight and transfer the electronic excitation very efficiently to the reaction center (RC), 16,17 where a charge separation takes place. Nowadays, structures and/or structural models are available for many of the complexes involved in the process described above, although the resolution is not always to the atomic level.…”

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

“…Such carbonyl groups are normally rare in tissues, enabling site-specific labeling of oxidized amino acids on proximal proteins. For example, tryptophan is converted into ketonecontaining kynurenine or N-formylkynurenine (17)(18)(19). Biotinconjugated hydrazides or hydrazines (BHs) have specific reactivity for ketones and aldehydes (15,20).…”