QUENCHING OF SINGLET OXYGEN BY HUMAN PLASMA

Direct measurements of the decay of singlet oxygen phosphorescence at 1270 nm were made in human plasma diluted with various amounts of deuterium oxide. The Stern-Volmer plot of the singlet oxygen lifetimes was linear up to 15% plasma concentration (vol/vol). Extrapolation of these measurements to 100% plasma content gave a singlet oxygen lifetime of 1.04 +/- 0.03 microseconds in human plasma. Biological molecules accounted for 77% of the total singlet oxygen quenching while water accounted for 23% of the quenching. The contributions of various types of biological molecules to the total singlet oxygen quenching were calculated from their plasma concentrations and their quenching constants. Plasma proteins quenched most of the singlet oxygen. Uric acid also quenched a significant amount of singlet oxygen (12%). Tocopherols, carotenoids, ascorbic acid and bilirubin made only small contributions to the total singlet oxygen quenching (less than or equal to 4%).     

QUENCHING OF SINGLET MOLECULAR OXYGEN BY PHTHALOCYANINES AND NAPHTHALOCYANINES

Using the direct measurement of the photosensitized luminescence of singlet molecular oxygen (1O2) the rate constants (kq) have been determined for 1O2 quenching by the monomeric molecules of the following phthalocyanines and naphthalocyanines in chloroform: tetra-(4-tert-butyl) phthalocyanine (I); octa-(3,6-butoxy) phthalocyanine (II), tetra-(6-tert-butyl)-2,3 naphthalocyanine (III), aluminium tetra-(1-tert-phenyl)-2,3 naphthalocyanine (IV), tri-(n-hexyl-siloxy) derivatives of silicon- (V), tin- (VI), aluminium- (VII) and gallium- (VIII) 2,3 naphthalocyanine. The following kq values were obtained (kq x 10(-8) M-1 s-1): 2.9 (I), 59 (II), 100 (III), 20 (IV), 3.9 (V), 53 (VI), 33 (VII), 110 (VIII). As most of the quenchers have the low-lying triplet levels, a contribution of the quenching mechanism based on the energy transfer from 1O2 to these levels has been analysed. A formula is proposed describing the relation between kq values caused by this mechanism, and photophysical constants of the quencher triplet state. This formula was applied to phthalocyanines, naphthalocyanines, beta-carotene and bacterochlorophyll a. The data suggest that the energy transfer can fully explain the activity of V and strongly contributes into the activities of II, III and VI-VIII. A charge transfer interaction might be an additional mechanism involved in 1O2 quenching by compounds studied. As some phthalocyanines and naphthalocyanines are strong physical quenchers of singlet oxygen they can be used as efficient inhibitors for photodestructive processes in photochemical systems.     

QUENCHING OF SINGLET OXYGEN BY SENSITIZER IN DYE-SENSITIZED PHOTOOXYGENATION*

Abstract— A general method for the determination of the extent of the singlet oxygen (¹O₂) quenching by sensitizer in the dye-sensitized photooxygenation of olefins is used in the case of rose bengal (RB) in methanol and oil-soluble chlorophyll (M) in benzene with 2-methyl-2-pentene and tetramethylethylene as acceptors. Unlike RB, M which contains only a low percentage of pure chlorophyll (Chi), quenches ¹O₂. It is shown that this very cheap mixture can be used for kinetic studies and that the chemical quenching of ¹O₂ by M is very weak with respect to the physical quenching. The upper limit for the rate constants of physical and chemical quenching of ¹O₂ by Chi is estimated.     

THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS

Abstract— The physical quenching of singlet molecular oxygen (¹Δ_g) by amino acids and proteins in D₂O solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1–06 μm output of a Nd-YAG laser, which absorbs directly in the electronic transition ¹Δ_g+ 1 v →³Σ_g^-. The rate of quenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-blue-photosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in D₂O, pD 8, are (10⁷ℒ mol^-1 s^-1): alanine 0–2, methionine 3, tryptophan 9, histidine 17, carbonic anhydrase 85, lysozyme 150, superoxide dismutase 260, aposuperoxide dismutase 250.     

ON THE QUENCHING OF SINGLET OXYGEN BY α-TOCOPHEROL

Abstract —D-α-tocopherol was found to be an effective quencher of ¹O₂ molecules ( k = 2.5 times 10⁸→mol^-1 s^-1 in pyridine) by measuring its effect on the autosensitized photooxidation of rubrene. The quenching process was shown to be almost entirely 'physical', that is, α-tocopherol deactivated about 120 ¹O₂ molecules before being destroyed. The results suggest that this process may be a mechanism for the protective effect of α - tocopherol in photodynamic action.     

SINGLET MOLECULAR OXYGEN QUENCHING BY SATURATED AND UNSATURATED FATTY-ACIDS AND BY CHOLESTEROL

The rate constants of molecular singlet oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol-membrane critical components - membrane critical components - have been measured by time resolved detection of the 1270 nm phosphorescence of singlet molecular oxygen [O2(1deltag)]. We have determined (i) an increment of 5.7 x 10(2)M(-1)s(-1) per -CH2- in C6D6 and CD3OD for saturated fatty acids between C4 and C20, (ii) an increment of 3 x 10(4)M(-1)s(-1) per non-conjugated cis-double bond for C18 unsaturated fatty acids, identical in C6D6 and DC3OD, (iii) a lower quenching rate constant by a factor of 2.7 for the trans-C16 and trans-C18 as compared to the corresponding cis-monounsaturated fatty acids, (iv) a rate constant of O2x(1deltag) quenching by cholesterol of 5.7 x 10(4)M(-1)s(-1) in benzene. These rate constants are compared to those obtained for other membrane cellular components.     

过渡金属络合物猝灭单重态氧能力的研究

研究了多种不同过渡金属络合物对单重态氧的猝灭问题。结果表明过渡金属络合物猝灭单重态氧的能力主要和络合物分子的几何构型有关。能形成平面四方形结构的络合物由于中心金属原子易于和¹O₂分子相接近因而具有强的猝灭¹O₂的能力,反之形成四面体形结构的络合物则猝灭能力减弱。     

MECHANISM OF PHYSICAL QUENCHING OF SINGLET MOLECULAR OXYGEN BY CHLOROPHYLLS and RELATED COMPOUNDS OF BIOLOGICAL INTEREST

Abstract— The rate constant k₅/ > for physical quenching of singlet oxygen O₂(δ₁;) by the sensitizer in dye-sensitized photooxygenations is determined in the case of chlorophylls a and b (7.3 times 10⁸, 4.2 times 10⁸ M^-1 s^-1 respectively), pheophytins a and b (7.4 times 10⁷, 3.0 times 10⁷ M^-1 s^_1 respectively), tetraphenylporphyrin (4.4 times 10⁷ M^-1 s^_1), magnesium tetraphenylporphyrin (5.0 times 10⁸ M^-1 s^_1), zinc tetraphenylporphyrin (1.5 times 10⁸ M^-1 s^_l) and protoporphyrin IX-dimethylester (9.1 times 10⁷ M ^-1 s^_1) in benzene. These sensitizers show a linear correlation between log k_s^O , and their half-wave oxidation potentials and the value of the slope is similar to that observed for various compounds such as phenols. It is concluded that (i) the interaction between chlorophylls and related compounds with singlet oxygen may involve an exciplex as for phenols, and (ii) physical quenching may be envisaged as a spin-orbit-induced intersystem crossing within the exciplex.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN BY AMINES-III. EVIDENCE FOR A CHARGE-TRANSFER-LIKE COMPLEX

Abstract— A series of amines were found to quench singlet oxygen in the order tertiary > secondary > primary, with a reasonable correlation between the log of their rate constant of quenching and their ionization potential. In addition, a Hammett rho plot gave a rho value of - 1.39 for the quenching of singlet oxygen by a series of substituted N, N-dimethylanilines, in good agreement with the results obtained by a different method. It was found that some of the amines (anilines) quenched the triplet state of the dye-sensitizer (Rose Bengal) used for the production of singlet oxygen. Corrections in the results were made in the calculations of rates of quenching of singlet oxygen to allow for the triplet-state quenching. No extensive quenching of the singlet state of the dye was observed at the concentrations of the amines necessary for singlet-oxygen quenching. In one case (N, N, N', N'-tetramethylphenylenediamine) there was no observable chemical reaction between singlet oxygen and the amine. It was concluded that singlet oxygen undergoes physical quenching by the amines via partial charge-transfer intermediates.     

IS SINGLET OXYGEN FORMATION DOMINANT IN TRIPLET QUENCHING PROCESSES? MEASUREMENT OF THE QUANTUM YIELD OF SINGLET OXYGEN FORMATION BY THE TIME-RESOLVED THERMAL LENS METHOD

The quantum yields of singlet oxygen formation (Ø_Δ) by the quenching of triplet states of organic sensitizers are measured at various concentrations of the sensitizers by using the time-resolved thermal lens method. Above a certain concentration, Ø_Δ is independent of the sensitizer concentration. Below the threshold, Ø_Δ gradually decreases as the concentration of the sensitizer decreases. The extrapolation of Ø_Δ to zero concentration indicates that singlet oxygen formation is not necessarily dominant in the quenching process even for the ³ππ* state in benzene.     

REACTION AND QUENCHING OF SINGLET MOLECULAR OXYGEN WITH ESTERS OF POLYUNSATURATED FATTY ACIDS

Abstract The rate constants for the reactive (k_R) and unreactive (k_Q) interaction of singlet molecular oxygen with three esters of polyunsaturated fatty acids (PUFA: cis-methyl oleate, MO; cis-methyl linoleate, MLA and cis-ethyl linolenate, ELN) are determined. The values of the ratio k_Q/k_R are 0.51, 0.26 and 0.20 for MO, MLA and ELN, respectively. This variation results principally from that of k_R because the values of k_Q are only slightly different (1.24 × 10⁴M^?1 s^?1 for MO and ～1.0 × 10⁴M^?1 s^?1 for MLA and ELN). It is shown that the rate constant k_Q characterizes mainly an interaction with the unreactive part of the molecule rather than with the double bonds (solventlike quenching). Contrary to the already reported case of 1,5-polyenes for which k_Q <<k_R, the present results and those obtained from a number of literature data show that for PUFA and their esters, neither k_R+ k_Q nor k_R are proportional to the total number of double bonds or of methylene groups adjacent to the double bonds. Instead, a linear correlation is observed by plotting k_Rvs the number of methylene groups adjacent to two double bonds. It is deduced that contrary to a common assumption, biallylic hydrogens have a reactivity higher than that of singly allylic hydrogens (reactivity ratio 1.19). The consequence of this result on the estimation of relative contributions of singlet oxygen and radical mechanisms to oxidation processes is discussed. Moreover, the whole of these results allows prediction of the values of k_R and k_Q for all unsaturated fatty acids (and their esters) of similar structure.     

THE ROLE OF SINGLET OXYGEN IN THE PHOTOHEMOLYSIS OF RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINE SULFONATES

Abstract The post-illumination photohemolysis of human red blood cells was used as a model system for studying membrane damage sensitized by various phthalocyanine sulfonates. With a constant fluence and photolysis time at 670 ± 10 nm and with a fixed optical density of the sensitizer, the percentage of hemolysis was measured as a function of time after illumination; and from the usual sigmoidal shaped curves, the times for 50% hemolysis were determined. The most effective central cation was Al, followed by Zn; the metal-free compound and those containing the paramagnetic ions Cu, Fe and Co were inactive. The sensitizer bound to the red blood cells was found to be effective for photohemolysis. Studies of the Al compound in 98.0% deuterated media (as measured by NMR) showed a definite deuterium isotope effect, suggesting the participation of singlet oxygen. This result was supported by the large protective effects of 3.3 raM and 13.3 mM tryptophan present during photolysis. Tryptophan reacts rapidly with singlet oxygen and with OH radicals. The much smaller effects observed with the same concentrations of mannitol and glycerol, OH radical scavengers which do not react with singlet oxygen, are consistent with the predominant role of singlet oxygen in photohemolysis sensitized by Al phthalocyanine sulfonate.     

THE ROLE OF SINGLET OXYGEN IN PHOTOOXIDATION OF EXCITABLE CELL MEMBRANES

Abstract— Giant nerve axons from lobsters were photooxidized by visible light from a Xe arc using eosin Y, rose bengal and methylene blue as sensitizers. The degree of modification was assayed as the rate constant for block of sodium channels during illumination. Protection or enhancement of modification was tested for several reagents known to interact with singlet oxygen. Deuterium oxide enhanced the modification up to 57% while sodium azide protected up to 60%. β -Carotene, triethylamine and DABCO exerted no effect. From a consideration of the probable low polarity environment of the sensitization sites and diffusional restrictions on reagents it is concluded that intramembranously created singlet oxygen plays a significant role in membrane modification.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

QUANTUM YIELD OF SINGLET OXYGEN PRODUCTION BY XANTHENE DERIVATIVES

The singlet oxygen quantum yield (φ₁o₂) of 11 purified fluorescein derivatives was determined by reaction with singlet oxygen acceptors in aqueous and ethanolic solutions; in both solvents φ₁o₂ was enhanced with increasing halogenation. Tryptophan and 2,2,6,6-tetramethylpiperidone were found to be unadapted for the determination of φ₁o₂, in our systems; however, the use of 9.10-dipropionic anthracene acid andp-nitrosodimethylaniline in conjunction with imidazole derivatives was suitable for ¹O₂ detection in water. Both methods lead to results in excellent agreement. As in ethanol. φ₁o₂, was equal to the triplet state quantum yield (φ_T), the comparison between the two solvents showed that φ_T in water was greater than in ethanol. The comparison between our values obtained with polychromatic light with published data obtained with monochromatic light suggests that the triplet quantum yield of fluorescein derivatives is wavelength independent.     

FORMATION OF SINGLET MOLECULAR OXYGEN BY 8-METHOXYPSORALEN*

Abstract— The formation of singlet molecular oxygen (^lO₂) by energy transfer from the excited 8-meth-oxypsoralen (8-MOP) molecule was investigated. This was done in several ways: (a) In the reaction of irradiated 8-MOP with the ¹O₂ acceptor 2-methyl-2-pentene, the characteristic oxidation products were identified. (b) The rate of the 8-MOP sensitized photooxidation of 3, 4-dihydroxy phenylalanine (dopa), which appeared to be also a useful ¹O₂ acceptor, was larger in D₂O than in H₂O. (c) The β-values for reaction of ¹O₂with dopa in the presence of 8-MOP or of methylene blue as ¹O₂ generators were in accordance with each other. The consequences of ¹O₂ formation by 8-MOP sensitization is discussed for the clinical use of this compound.     

PHOTOOXIDATION OF CHLOROPHYLL AND PHEOPHYTIN. QUENCHING OF SINGLET OXYGEN AND INFLUENCE OF THE MICELLAR STRUCTURE

Abstract— When chlorophyll(Chl) and pheophytin(Phn) are irradiated in Triton X-100 water binary solvents, singlet oxygen is formed in the medium in a higher yield for Phn than for Chi. Chlorophyll shows an irreversible photooxidation reaction and a chemical oxidation reaction when 1,3-diphenyliso-benzofuran (DPBF) is added to the solution. During the chemical oxidation, Chi is destroyed by an oxidizing agent that is a reaction product of the endoperoxide formed in the medium by the addition of singlet oxygen to DPBF. This reaction depends on the structure of the medium and has some characteristics of an oxidation by hydroxyl radicals. The highest yield is obtained with the micellar structure. Chlorophyll and Phn are readily oxidized by hydroxyl radicals generated using the Fenton reagent. This suggests that in the presence of Triton X-100, the Mg²⁺ ion of a Chi molecule plays a key role in the irreversible oxidation of the pigment.     

HYDROGEN PEROXIDE-DEPENDENT GENERATION OF SINGLET MOLECULAR OXYGEN BY HUMAN SALIVA: ITS DETECTION BY CHEMILUMINESCENCE FROM A Cypridina LUCIFERIN ANALOG

By the addition of hydrogen peroxide to human saliva, chemiluminescence from Cypridina luciferin analog (CCLA) and oxygen evolution were observed. Chemiluminescence was inhibited by inhibitors of salivary peroxidase, azide and cyanide and by a singlet oxygen quencher, crocin. Deuterium oxide (99.75%) stimulated the initial increase of CCLA by15–50% and the integrated CCLA 2.1-3.6-fold. The result suggest that the generation of singlet oxygen by peroxidase in human saliva depends on hydrogen peroxide.