Flash photolysis and inactivation of aqueous lysozyme

Abstract— –Flash photolysis of aqueous lysozyme has shown that the initial photochemical products are photo-oxidized tryptophan residues (Λ_max= 500 nm), hydrated electrons (Λ_max= 720 nm), and the cystine residue electron adduct (Λ_max= 420 nm). Comparisons with mixtures of the chromophoric amino acids show that 1 to 2 tryptophan residues provide electrons at a quantum yield of 0.018 (25 per cent). Part of the ejected electrons are captured by cystine residues via a short-range, intramolecular process with essentially unit efficiency. The remainder become hydrated and back react with oxidized tryptophan residues before 10^-4sec. The cystine residue electron adduct decays with 2 msec halftime (25°C) and 1.5 kcal/mole activation energy. The surviving oxidized tryptophan residues decay with a comparable time constant in a hydroxyl ion catalyzed process. In acid solutions the oxidized tryptophan residue and long-lived H atom adduct are observed (Λ_max= 380 nm). The quantum yield of lysozyme inactivation induced by xenon flash irradiation above 250 nm is 0.023 (20 per cent), which is not sensitive to oxygen or pH. Comparison to the primary photochemical reactions indicates that electron ejection from the essential tryptophan residues inactivates the enzyme, irrespective of the electron trap and subsequent reactions. On the basis of the structure and supporting information it is proposed that the tryptophan residues of the active site are involved. Direct disruption of cystine residues does not contribute more than 10 per cent to the inactivation quantum yield in this wavelength region. Lysozyme inactivation may differ from other enzymes because the chromophores include essential residues located in the active center.     

Characterization of transient species in laser photolysis of aromatic amino acids using acetone as photosensitizer

The photochemical processes of aromatic amino acids were investigated in aqueous solution using acetone as photosensitizer by KrF (248 nm) laser flash photolysis. Laser-induced transient species were characterized according to kinetic analysis and quenching experiments. The intermediates recorded were assigned to the excited triplet state of tryptophan, the radicals of tryptophan and tyrosine. The excited triplet state of tryptophan produced via a triplet-triplet excitation transfer and the radicals arising from electron transfer reaction has been identified. Neither electron transfer nor energy transfer between triplet acetone and phenylalanine can occur in photolysis of phenylalanine aqueous solution which contains acetone. Furthermore, triplet acetone-induced radical transformation: Trp/N-Tyr→Trp-Tyr/O was observed directly in photolysis of dipeptide (Trp-Tyr) aqueous solution containing acetone, and the transformation resulting from intramolecular electron transfer was suggested.     

PHOTOLYSIS MECHANISM OF AQUEOUS TYROSINE AND TYROSYL PEPTIDES*

Abstract— Laser flash photolysis at 265 nm has been employed for measuring the initial hydrated electron (e^-_aq) and p-alanylphenoxyl radical (Tyr) in aqueous Tyr, small Tyr peptides and R Nase A. The results indicate that monophotonic photolysis not involving the fluorescent or triplet states is the principal initial process. Equivalent yields of e and Tyr were found in all cases except Tyr, where the Tyr yield was 60% higher than e^-_aq attributed to splitting of the phenolic bond. Computer analysis of e^-_aq and Tyr decays for Tyr indicates the importance of electron-radical recombination in competition with electron scavenging and bimolecular radical-radical reactions. Evidence for intramolecular electron migration has been obtained in cystinyl-bis-Tyr.     

Photochemistry of the iron(III) complex with pyruvic acid in aqueous solutions

Photochemistry of the 1: 1 Fe^pIII complex with pyruvic acid (PyrH) in aqueous solutions was studied by stationary photolysis and nanosecond laser flash photolysis with the excitation by the 3rd harmonics of an Nd:YAG laser. The quantum yield of [FeIIIPyr]2+ under the excitation at 355 nm is 1.0±0.1 and 0.46±0.05 in the absence and in the presence of dissolved oxygen, respectively. In experiments on laser flash photolysis, a weak intermediate absorption in the region 580–720 nm was found. The absorption was ascribed to the [FeII…MeC(O)COO•]^p2+ radical complex. Laser flash photolysis of [FePyr]^p2+ in the presence of methyl viologen dications (MV^p2+) resulted in the formation of the MV•+ radical cations. The proposed reaction mechanism includes the inner-sphere electron transfer in the light-excited complex accompanied by the formation of the [Fe^pII…MeC(O)COO•]^p2+ radical complex followed by its transformation into the reaction products.     

CHLOROPHYLL-QUINONE PHOTOCHEMISTRY IN LIPOSOMES: MECHANISMS OF RADICAL FORMATION AND DECAY*

Abstract— Laser flash photolysis has been used to investigate the mechanism of formation and decay of the radical species generated by light-induced electron transfer from chlorophyll a (Chi) triplet to various quinones in egg phosphatidyl choline bilayer vesicles. Chlorophyll triplet quenching by quinone is controlled by diffusion occurring within the bilayer membrane (kq～ 10⁶M^?1 s^?1. as compared to ～ 10⁹ M^?1 s^?1 in ethanol) and reflects bilayer viscosity. Radical formation via separation of the intermediate ion pair is also inhibited by increased bilayer viscosity. Cooperativity is observed in the radical formation process due to an enhancement of radical separation by electron transfer from semiquinone anion radical to a neighboring quinone molecule. Two modes of radical decay are observed, a rapid (t_1/2= 150μ) recombination between Chi and quinone radicals occurring within the bilayer and a much slower (t_1/2= 1–100 ms) recombination occurring across the bilayer-water interface. The latter is also cooperative, which accounts for a t_1/2 which is dependent upon quinone concentration. The slow decay is only observed with quinones which are not tightly anchored into the bilayer, and is probably the result of electron transfer from semiquinone anion radical formed within the bilayer to a quinone molecule residing at the bilayer-water interface. Direct evidence for such a process has been obtained from experiments in which both ubiquinone and benzoquinone are present simultaneously. With benzo-quinone, approx. 60% of the radical decay occurs via the slow mode. Triplet to radical conversion efficiencies in the bilayer systems are comparable to those obtained in fluid solution (～ 60%). However, radical recombination, at least for the slow decay mechanism, is considerably retarded.     

Photochemical inactivation of trypsin

Abstract —The quantum yield for inactivation of aqueous trypsin fits the expression φ_f=Σ_rf_rφ‘_r, where f_r, is the fraction of incident light absorbed by residues of type r and the φ’_r are constants. The values φ‘_trp= 0.012, φ_tyr= 0.005 and φ’_eys= 0.10, obtained at pH 3 in the wavelength range 240–290 nm, are attributed to independent events by comparing with quantum yields of the initial photochemical products and permanent residue destruction. The proposed inactivating processes are photoionization of one essential tryptophyl residue, photolysis of one essential cystyl residue, and splitting of an essential cystyl residue induced by light absorption in a nearby tyrosyl residue. The initial photochemical process from pH 3–7 identified by flash photolysis is the ejection of electrons from approximately two tryptophyl residues, leading to the formation of the disulfide bridge electron adduct and the hydrated electron. It is proposed that one photoionized tryptophyl residue is permanently disrupted and the other is restored through a back reaction that leads to a damaged, active enzyme form. An enhanced inactivation quantum yield at flash photolysis light intensities is attributed to a biphotonic process. A model based on one-photon photoionization of tryptophan from a short-lived precursor of the fluorescent state and the biphotonic photoionization of tryptophan via the triplet state is consistent with the experimental results.     

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVEN BY TWO PHOTOREACTIONS. I. ETHYLENEDIAMINETETRAACETIC ACID AS AN ELECTRON DONOR via FLAVIN TRIPLET STATE IN THE INNER COMPARTMENT AND CYTOCHROME c AS AN ELECTRON ACCEPTOR FROM CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

Negatively charged vesicle suspensions containing chlorophyll a (chl) dissolved in the lipid bilayer, flavin mononucleotide (FMN) and/or ethylenediaminetetraacetic acid (EDTA) enclosed in the inner compartment as electron sources and oxidized cytochrome c (cyt c[ox]) in the outer compartment as an electron acceptor have been studied using laser flash photolysis and steady-state irradiation methods. Cytochrome c initially quenches the chl triplet state (³chl) generating the chlorophyll cation radical (chi⁺′) in the membrane. Reverse electron transfer from cyt c(red) to chl^+. subsequently occurs in a kinetically biphasic reaction, with rate constants of 430 pT 30 and 21.9 pT 1.7 s^?1 for the fast and slow phases, respectively. In the absence of FMN, reduction of chl⁺′ by EDTA in the inner compartment can be observed during steady-state irradiation but not in a laser flash photolysis experiment. This is due to a low reaction yield, which is probably limited by the repulsive electrostatic interaction between EDTA and the negatively charged membrane. When FMN was enclosed together with EDTA in the inner Compartment, the reaction yield of vectorial electron transfer across the bilayer from EDTA to cyt c(oX) was increased by a factor of six during steadystate white light irradiation. Laser flash photolysis and steady-state irradiation experiments using red and blue light excitation have demonstrated that the enhancement mechanism involves the formation of fully reduced FMN by blue light-sensitized photooxidation of EDTA via the flavin triplet state, occumng simultaneously with red lightsensitized electron transfer to cyt c via the chlorophyll triplet state.     

Comparison of intermediates of tryptophan, tyrosine and their dipeptide induced by UV light and SO. 4 –

The chemical processes of tryptophan (Trp), tyrosine (Tyr) and a dipeptide Trp-Tyr, which are induced by UV radiation and one-electron oxidation of SO^. ₄ ^–, have been investigated in aqueous solution by KrF (248 nm) laser flash photolysis. On the basis of optical studies, the photoionization of Trp and Tyr produces the tryptophan indolyl radical and tyrosine phenoxyl radical, respectively, and these are different from the intermediates resulting from interaction of Trp and Tyr with SO^. ₄ ^–. In the case of Trp, SO^. ₄ ^– would attack the indole moiety to produce a C(2)-yl sulphate radical adduct, and Tyr is oxidized to produce mainly the corresponding one-electron oxidized radical, which deprotonates rapidly to form the phenoxyl radical in neutral solution, and a possible sulphate radical adduct. From transient absorption spectra of photoionization of Trp-Tyr, an intramolecular electron transfer, Trp/N^.-Tyr Trp-Tyr/O^., has been observed, but there was no observation of the process of one-electron oxidation of Trp-Tyr by SO^. ₄ ^–.     

Photochemistry of iron(iii)-lactic acid complex in aqueous solutions

Photochemistry of a 1: 1 Fe^III-lactic acid complex, [Fe(Lact)]⁺, in aqueous solutions was studied by stationary photolysis, nanosecond laser flash photolysis (355 nm, 6 ns), and femtosecond pump-probe spectroscopy (400 nm, 200 fs). The quantum yield of photolysis of [Fe(Lact)]⁺ upon excitation at 355 nm is 0.4 and 0.22 in the deoxygenated and air-saturated solutions, respectively. Weak transient absorption in the range 500–750 nm was observed in the nanosecond experiments. It was assigned to a [Fe^II...-O-CH(Me)-COO^·]⁺ radical complex. The spectral properties of the ligand-to-metal charge transfer excited state and the characteristic time of formation of the radical complex (1.5 ps) were determined in the femtosecond spectroscopy experiments. A reaction mechanism was proposed, which involves inner-sphere electron transfer in the excited complex with the formation of a radical complex [Fe^II...-O-CH(Me)-COO^·]⁺ and its subsequent transformation to the end product of the photochemical reaction.     

Tryptophan photooxidation promoted by new hybrid materials prepared by condensation of naphthalene imides with silicate by the sol–gel process

Three novel hybrid organic/inorganic materials were synthesized from 4-substituted (NO₂, Br, H) 1,8-naphthalene imide-N-propyltriethoxysilane by the sol–gel process. These materials were obtained as a xerogel and partially characterized. The ability to photosensitize the oxidation and degradation of tryptophan indole ring by these materials was studied through photophysical and photochemical techniques. Although the derivatives containing Br and NO₂ as substituent do not cause efficient tryptophan photodamage, the hybrid material obtained from 1,8-naphthalic anhydride is very efficient to promote tryptophan photooxidation. By using laser flash photolysis it was possible to verify the presence of naphthalene imide transient radical species. The presence of oxygen causes an increase of the yield of radical formation. These results suggest that the mechanism of photodegradation of tryptophan occurs by type I, i.e. the transient radical (TrpH⁺) formed by the direct reaction of the triplet state of the naphthalene imide moiety with tryptophan. Thus a inorganic–organic hybrid material that can be used to promote the oxidation of biomolecules was obtained.     

Ultraviolet inactivation of papain.

Abstract— Flash photolysis transient spectra (Λ > 250 nm) of aqueous papain show that the initial products are the neutral tryptophan radical Tip (Λ_max 510 nm), the tryptophan triplet state ³Trp (Λ_m., 460nm), the disulfide bridge electron adduct –?S^-— (Λ_max 420nm) and the hydrated electron e_aq^-. The –?S^-– yield was not altered by nitrous oxide or air, indicating that the formation of this product does not involve electrons in the external medium. The original papain preparation was activated by irradiating under nitrogen. The action spectrum supports previous work attributing the low initial activity to blocking of cysteinyl site 25 with a mixed disulfide. Flash lamp irradiation in nitrogen led to activation at low starting activities and inactivation at higher starting activities, while only inactivation at the same quantum yield was observed with air saturation. The results are consistent with photoionization of an essential tryptophyl residue as the key inactivating step.     

DETECTION OF RADICAL ION INTERMEDIATES IN FLAVIN-PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING

Photosensitized splitting of cis-syn- and trans-syn-l,3-dimethyluracil dimers by 2′,3′,4′,5′-tetraacetylri-boflavin in acetonitrile containing a trace of perchloric acid was studied by laser flash photolysis. Protonation of the flavin prior to excitation resulted in excited singlet and triplet states that abstracted an electron from the dimers and yielded the protonated flavin radical (F1H₂′⁺), which was detected by absorption spectroscopy. Electron abstraction by the excited singlet state predominated over abstraction by the triplet state. Approximately one-third to one-half of the excited states quenched by the trans-syn dimer yielded F1H₂⁺, the balance presumably undergoing back electron transfer within the geminate radical ion pair generated by the initial electron transfer. A covalently linked dimer-flavin exhibited very inefficient flavin radical ion formation, consistent with the known low efficiency of dimer splitting in this system. These results constitute the first identification of a flavin radical ion intermediate in photosensitized pyrimidine dimer splitting.     

Kinetics and mechanism of photolysis of the iron(III) complex with tartaric acid

The formation of MV^•+ radical cations was observed upon the laser flash photolysis of the iron(III) tartrate complex [Fe^IIITart]⁺ (1) in the presence of methyl viologen (MV²⁺). The rate constants of the reactions involving MV^•+ were measured. The intramolecular electron trans-fer to form Fe^II and escape of the organic radical to the solvent bulk upon the photolysis of 1 were proposed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 866–869, May, 2007.     

Characterization of the transient species generated by the photoionization of Berberine: A laser flash photolysis study

Using 266 nm laser flash photolysis it has been demonstrated that Berberine (BBR) in aqueous solution is ionized via a mono-photonic process giving a hydrated electron, anion radical that formed by hydrated electron react with steady state of BBR, and neutral radical that formed from rapid deprotonation of the radical cation of BBR. The quantum yield of photoionization is determined to be 0.03 at room temperature with KI solution used as a reference. Furthermore utilizing pH changing method and the SO₄⁻ radical oxidation method, the assignment of radical cation of BBR was further confirmed, the pK_a value of it was calculated, and the related set up rate constant was also determined.     

266 nm光照下水溶液中氯苯与H2O2的反应机理研究

利用瞬态吸收光谱技术研究了不同条件下C₆H₅Cl与H₂O₂水溶液的激光闪光光解情况, 初步考察了其瞬态物种的生长和衰减等行为. 研究表明, •OH自由基和C₆H₅Cl反应生成C₆H₅Cl-OH adduct, 其反应速率常数在近中性、酸性条件下约为(5.89±0.65)×10⁹和(7.07±0.61)×10⁹ L•mol^－1•s^－1; 其衰减则符合双分子二级反应, 速率常数2k/εl＝1.1×10⁶ s^－1, 而在碱性时则为(4.34±0.51)×10⁹ L•mol^－1•s^－1, 衰减呈准一级反应, 速率常数为2.11×10⁵ s^－1. 在有氧条件下, O₂与C₆H₅Cl-OH adduct反应生成C₆H₅Cl-OHO₂ adduct, 其反应速率常数为6.8×10⁸ L•mol^－1•s^－1.     

PHOTOCHEMISTRY OF 1,2,3-INDANETRIONE*

The photochemistry of 1,2,3-indanetrione (1) has been examined in solution at room temperature by steady state and laser flash photolysis. The triplet state of 1 (T = 6.5 μs, δ_mux, = 360 and 570 nm, in dry acetonitrile) reacts preferentially via an a-cleavage process followed by a considerably slower loss of carbon monoxide. Triplet 1 shows a remarkably fast hydrogen abstraction rate constant when in the presence of 1,4-cyclohexadiene (k_r= 1.4 times 10⁶M^?1s^?1) in spite of its low excitation energy (E_T= 42 kcal/mol). This behavior can be explained by assuming that the vicinal carbonyls coplanar to the ketyl radical play an important role in its stabilization.     

Photochemistry of Fe(III) complex with glyoxalic acid in aqueous solution

Nanosecond laser flash photolysis technique was used to study photochemistry of Fe(III) complex with glioxalic acid. The primary photochemical process was found to be inner-sphere electron transfer in the excited complex leading to formation of the long-lived radical complex [Fe^II … ^·OOC-C(O)H]²⁺. A number of important spectral-kinetic parameters of this species were determined and mechanism of photolysis of Fe(III)-glioxalate complex was proposed. 1 The article was translated by the authors.     

Photoinduced Electron Transfer between N,N, Nr,N' ‐Tetra‐(p‐methylphenyl)‐4, 4‐diamino‐1, 1'‐diphenyl Ether (TPDAE) and Fullerenes (C60/C70) by Laser Flash Photolysis

The photoinduced electron‐transfer reaction of N, N, N', N'‐tetra‐(p‐methylphenyl)‐4,4'‐diamino‐1,1'‐diphenyl ether (TPDAE) and fullerenes (C₆₀/C₇₀) by nanosecond laser flash photolysis occurred in benzonitrile. Transient absorption spectral measurements were carried out during 532 nm laser flash photolysis of a mixture of the fullerenes (C₆₀/C₇₀) and TPDAE. The electron transfer from the TPDAE to excited triplet state of the fullerenes (C₆₀/C₇₀) quantum yields and rate constants of electron transfer from TPDAE to excited triplet state of fullerenes (C₆₀/C₇₀) in benzonitrile have been evaluated by observing the transient absorption bands in the near‐IR region where the excited triplet state, radical anion of fullerenes (C₆₀/C₇₀) and radical cations of TPDAE are expected to appear.     

One electron oxidation of Ni(II)-iminodiacetate by carbonate radical

Reactions of carbonate radical (CO₃ ^–), generated by photolysis or by radiolysis of a carbonate solution with nickel(II)-iminodiacetate (Ni(II)IDA) were studied at pH 10.5 and ionic strength (I)==0.2 mol·dm^–3. The stable product arising from the ligand degradation in the complex is mainly glyxalic acid. Time-resolved spectroscopy and transient kinetics were studied using flash photolysis. From the kinetic data it was suggested that the carbonate radical initially reacts with Ni(III)IDA with a rate constant (2.4±0.4)·10⁶ dm³·mol^–1·s^–1 to form a Ni(II)IDA species which, however, undergoes a first-order transformation (k=2.7·10²·s^–1) to give a radical intermediate of the type Ni(II)RNHCHCO ₂ ^– ) which rapidly forms an adduct containing a Ni–C bond. This adduct decays very slowly to give rise to glyoxalic acid. From a consideration of equilibrium between Ni(II)IDA and Ni(III)IDA, the one electron reduction potential for the Ni(III)IDA/Ni(II)IDA couple was determined to be 1.467 V.     

An indirect measurement of the absolute rate constant of the self-reaction of tert-butoxy radicals

No reliable rate constant is available for the self-reaction of tert-;butoxy radicals. We have set up a competition between hydrogen abstraction and self-reaction of tert-butoxy radicals in a flash photolysis electron spin resonance study to extract this information. Experimental values of hydrogen abstraction product radical concentrations under various hydrogen donor concentrations were then compared with theoretically calculated values with different values of 2k₄ to obtain the best fit. Hydrogen donors such as cyclopentane, anisole, methyl tert-butyl ether, and methanol were chosen for the study. A value of (1.3 ± 0.5) × 10⁹M^?1 sec^?1 for the rate constant of the self-reaction of tert-butoxy radicals has been determined at 293°K.