Studies on chemiluminescence in the enzymatic and autoxidative transformation of 3,4-dihydroxyphenylalanine into eumelanins

The catechol oxidase-catalysed and autoxidative transformation of 3,4-dihydroxyphenylalanine (DOPA) to eumelanin have been studied by oxygen consumption, energy transfer, absorption and fluorescence spectroscopy. Formation of transient dopachrome (λ_max = 480 nm) and dopalutin (λ_ex = 423 nm, λ_em = 491 nm) have been found in the enzymatic and autoxidative reaction. In the enzymatic reaction, neither a photon emission with quantum yield Φ > 10^?13 nor energy transfer to triplet and singlet energy acceptors (sensitizers such as anthracene derivatives, xanthene dyes and chlorophyll-a) in water and micellar solutions have been found. The autoxidative reaction is chemiluminescent (Φ = 10^?9), the emission occurring in the 400-600 nm range. The excitation energy is not transferred to sensitizers. The effect of various enzymes and traps of active oxygen species as well as the spectral distribution of chemiluminescence indicate that there is no emission from oxygen dimoles. Carbonates and active species of oxygen are shown to participate in the chemiexcitation reaction.     

Spatially resolved two-photon irradiation of an intracellular singlet oxygen photosensitizer: Correlating cell response to the site of localized irradiation

Abstract

The response of HeLa cells to subcellular spatially localized two-photon irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. Upon irradiation under these conditions, a localized population of PpIX excited states can be produced with meaningful intracellular spatial resolution; the dimensions of the domain where the incident light flux is high enough for PpIX two-photon absorption are defined by the microscope optics and by the diffraction of light (spot diameter at beam waist of ?0.5–1.0 μm). In turn, the dimensions of the intracellular domain containing cytotoxic PpIX-sensitized singlet oxygen will likewise be confined. Most importantly, cell response (e.g., morphological signs of cell death) correlates with the light dose delivered and the intracellular domain irradiated. Thus, controlling light delivery can complement other techniques used to impart intracellular spatial localization in mechanistic studies of photoinitiated reactive oxygen species. Such controlled light delivery is also expected to be a particularly useful tool to study the so-called bystander effect in which a selectively-perturbed cell can influence a neighboring cell through intercellular signaling mechanisms.     

Slow reversible bleaching and fluorescence quenching caused by carotenoid absorption in chromatophores and cells of Chromatium vinosum

Light absorbed by carotenoids in Chromatium can result in photobleaching of bacteriocholorophyll and quenching of B 890 fluorescence, whereas light of the same intensity absorbed by bacteriochlorophyll has no such effect. Photobleching and fluorescence quenching are partly and slowly reversible in the dark. They are prevented by removal of oxygen or by addition of various reductants and decreased by addition of NaN₃₋ histidine or tryptophane. This suggests the participants of singlet excited oxygen in the measured phenomena. As change in temperature between 30° and –30°C and addition of gramicidin S, which changes the permeability of the membranes, does not affect photobleaching or fluorescence quenching markedly, enzymatic or structural properties do not seem to be involved. The results suggest that light absorbed by carotenoids is partly transferred to bacteriochlorophyll and partly used to excite carotenoids to triplet states. The latter process will counteract the function that carotenoids have in protecting chromatophore components against photobleaching.     

Excitation transfer between photosynthetic units: the 1964 experiment

We review here the background and the experiments that led to the concept of excitation energy transfer among photosystem (PS) II units. On the basis of a kinetic analysis of oxygen evolution and chlorophyll a fluorescence yield, the authors showed, in 1964, that the PS II photochemical reaction involved in the formation of oxygen is not a first-order process. We concluded that excitation energy localized in a `photosynthetic unit' including a reduced primary acceptor is transferred with a high probability to neighboring PS II units. Here, the beginnings and the original data of this topic are presented. This revised version was published online in August 2006 with corrections to the Cover Date.     

No or little production of singlet molecular oxygen in HOCl or HOClH2O2 a model system for myeloperoxidase/H2O2/Cl−

The HOCl in chlorine-water oxidizes DPF to cis-DBE in parallel to the HOCl concentration. The addition of H₂O₂ produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis-DBE is initiated by a 1,2-position attack of OH^? and Cl⁺, followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H₂O₂ solution, on the pH range 6.0 to 8.0 where both HOCl and OCl^? are present.     

三种藻胆蛋白在复合累积LB膜中的能量传递

制备了R-藻红蛋白(R-PE)、C-藻蓝蛋白(C-PC)及变藻蓝蛋白(APC)的单组分累积LB膜及三组分复合累积LB膜. 吸收及荧光光谱测定表明三种蛋白的LB膜的光谱重叠性质与他们在水溶液中的性质相同. 结构测定表明, 三种蛋白在其LB膜中排列有序, 且由它们组装的复合累积LB膜结构类似于藻类植物中的藻胆体结构. 通过稳态荧光光谱及其光谱解叠, 观察到了三种蛋白在复合累积LB膜中的激发能量传递现象. 根据给体荧光峰的猝灭, 计算出在复合累积LB膜中从给体R-PE经C-PC到受体APC的能量传递效率为51%.     

Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the O? O bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H₂O₂ by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl-hydroxo complexes, as that formed during the oxidation of metmyoglobin by H₂O₂, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.     

Activation of Molecular Oxygen by Infrared Laser Radiation in Pigment-Free Aerobic Systems

With the goal of mimicking the mechanisms of the biological effects of low energy laser irradiation, we have shown that infrared low intensity laser radiation causes oxygenation of the chemical traps of singlet oxygen dissolved in organic media and water saturated by air at normal atmospheric pressure. The photooxygenation rate was directly proportional to the oxygen concentration and strongly inhibited by the singlet oxygen quenchers. The maximum of the photooxygenation action spectrum coincided with the maximum of the oxygen absorption band at 1270 nm. The data provide unambiguous evidence that photooxygenation is determined by the reactive singlet ¹_g state formed as a result of direct laser excitation of molecular oxygen. Hence, activation of oxygen caused by its direct photoexcitation may occur in natural systems.     

Earlier researches on the mechanism of oxygen evolution: A personal account

A brief overview is given of the research which led to the discovery of the period-4 oscillations of the flash-induced oxygen production and which is the basis of the generally accepted Kok's model for water splitting and oxygen evolution.In this paper I discuss the earlier work of the groups of Robert Emerson, James Franck, C. P. Whittingham, and myself in relation to the development of new techniques for the detection of photosynthetic oxygen evolution. Also discussed are various hypotheses and speculations related to the concept of a priming photoreaction which is required for oxygen evolution. Finally, I discuss my long scientific collaboration with Bessel Kok which led to the elaboration by Kok of the classical model in which the formation of oxygen requires the sequential accumulation of four positive charges on the same photochemical center.Written at the invitation of Govindjee and Gernot Renger.     

Acquirement and characterization of a carotenoid mutant (GM309) of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> 601

A green mutant was obtained among the chemically induced mutants of Rhodobacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular dichroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.     

Are dioxetanes chemiluminescent intermediates in lipoperoxidation?

Ultraweak chemiluminescence arising from lipoperoxidation has been attributed by several authors to the radiative deactivation of singlet oxygen and triplet carbonyl products. The latter emitters have been suggested to come from annihilation of RO. and ROO. radicals as well as from the thermolysis of dioxetane intermediates formed by (2 + 2) cycloaddition of 1O2 to polyunsaturated fatty acids. This article questions possible dioxetane intermediacy in lipoperoxidation, as the literature clearly states that addition of 1O2 to alpha-hydrogen-containing alyphatic olefins yields only the corresponding allylic hydroperoxides. These compounds may undergo dark thermal or Lewis acid-assisted decomposition to the same product obtained from dioxetane cleavage. Here, reexamining the chemiluminescence properties of dioxygenated tetramethylethylene and linoleic acid and comparing them with those of tetraethyldioxetane, a hindered dioxetane, we corroborate the literature information that only steric hindrance leads to dioxetane formation upon singlet oxygen addition to electron-poor olefins, albeit in very low yields. Proton nuclear magnetic resonance (1H-NMR) analysis, quenching by dioxygen and energy transfer studies to 9,10-dibromoanthracene, as well as gas chromatography (GC) analysis of triphenylphosphine-treated and untreated photo- and chemically dioxygenated olefins support our final conclusion that dioxetane formation during lipoperoxidation can be safely excluded on the basis of the data presently available.     

基于light-oxygen-voltage（LOV）结构域光敏剂的细胞毒性研究

目的 光敏剂被特定波长的光激发后,产生的活性氧类能破坏细胞组织,介导细胞死亡,对微生物感染、肿瘤等相关疾病的治疗具有重要意义。方法 基于粳稻类向光素1B（Oryza sativa japonica phototropin-1B-like）的LOV（light-oxygen-voltage）结构域,设计得到光敏剂LovPSO2及其突变体LovPRO2。在445 nm、70 μmol·m^-2·s^-1蓝光照射下,每隔 2 min测量LovPSO2和LovPRO2的单线态氧产量,持续10 min,每隔1 min测量其超氧阴离子产量,持续5 min,并研究温度、光照对其稳定性的影响,最后将其转入E. coli BL21（DE3）和HeLa细胞中表达并分析光毒性效果。结果 在445 nm、70 μmol·m^-2·s^-1蓝光照射下,LovPSO2是一种能产生大量单线态氧的II型光敏剂（Φ_Δ=0.61）,LovPRO2是一种能够同时产生单线态氧和超氧阴离子的光敏剂。蛋白质稳定性分析结果表明,LovPSO2和LovPRO2具有较好的温度稳定性,其中LovPRO2的光稳定性更好。蛋白质的光毒性分析结果表明,445 nm、30 mW/cm2蓝光照射30 min后,LovPSO2和LovPRO2对E. coli BL21（DE3）菌株有较好的光毒性,致死率高达90%。结论 LovPSO2和LovPRO2可作为抗菌光敏剂,在食品和医疗等方面均有较为广阔的应用前景。     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.