LASER FLASH PHOTOLYSIS AND PHOTOINACTIVATION OF SUBTILISIN BPN'

Abstract— Laser flash photolysis of subtilisin BPN'at 265 nm has shown that photoionization of tryptophanyl (Trp) and tyrosinyl (Tyr) residues are the principal initial photochemical reactions. The initial products are the corresponding oxidized radicals. Trp and Tyr, and hydrated electrons (e_aq) which react with the enzyme at: k (e_aq+ subt. BPN') = 2.1 × 10¹⁰ M⁻¹ s⁻¹. The photoionization quantum yield was 0.032 ± 0.005 at 265 nm, which was enhanced 3.5-fold by simultaneous excitation at 265 and 530 nm. The photoionization yields were unchanged by 3 M bromide ion and 8 M urea. which did affect the enzyme fluorescence excited at 265 and 295 nm. A similar lack of correlation between the effects of perturbants on the photionization yields and fluorescence yields was found for subtilisin Carlsherg. The results indicate that the monophotonic and biphotonic ionization of the Trp residues does not involve the thermally-equilibrated. lowest excited singlet state and that singlet energy transfer from Tyr to Trp does not contribute to Trp photoionization. The photoinactivation quantum yield was 0.014 for 265 nm laser excitation. which was not changed by simultaneous 530 nm excitation. The corresponding quantum yield was 0.009 for low intensity 254 nm radiation, indicative of a biphotonic contribution to photoinactivation. The results are explained by postulating that photolysis of Trp-113 leads to disruption of hydrogen bonding to Asn-117 and a shift in the primary chain sequence associated with the aromatic substrate binding sites. The photoionization quantum yields in subtilisin BPN'and subtilisin Carlsberg agree with a model based on the assumption that exposed Trp and Tyr residues contribute independently at intrinsic photoionization efficiencies characteristic of the chromophores.     

Hydrogen Atoms Are Produced When Tryptophan within a Protein Is Irradiated with Ultraviolet Light

Abstract— The UV photolysis of the aromatic amino acid, tryptophan (Trp), in the Ca²⁺-binding protein, cod paralbumin, type III, was studied using electron paramagnetic resonance (EPR) spectroscopy in the temperature range 4–80 K. For the Ca²⁺-bound protein, irradiation with UV light (250–400 nm) resulted in the generation of atomic hydrogen with a hyperfine splitting of 50.9 mT, whereas in the Ca²⁺-free form, where the Trp is exposed to solvent, the trapped atomic hydrogen was not in evidence. In the same spectra, the radical signal in the g = 2.00 region could be detected. The line shape of the Ca²⁺-bound form is similar to the EPR line shape obtained for Trp in micellar systems. In contrast, the EPR line shape for the Ca²⁺-free form is essentially featureless up to 80 K. The EPR spectra of the photoproducts of Trp and the nature of the photoreactions are therefore sensitive to the environment of Trp within the protein.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

PHOTOLYSIS OF THE SINGLE TRYPTOPHAN RESIDUE OF EEL TROPONIN C

Abstract— The tryptophan (TRP) residue of eel troponin C was selectively degraded by direct UV irradiation, at 302 nm, in Ar-saturated solution. Depending on the absence or presence of calcium ions, this TRP residue is exposed to aqueous medium or buried in a hydrophobic environment. Tryptophan loss was determined by both absorption and fluorescence spectroscopy and by amino acid analysis. The photodegradation yield was significantly higher for the exposed TRP residue than for the buried ones. These results give more detail on previous observations on several other proteins and corroborate the predominant influence of the polarity on the photosensitivity of a TRP residue in polypeptidic structures.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.     

THE PHOTOLYSIS RATES OF SOME DI- AND TRIPEPTIDES OF TRYPTOPHAN

We have measured the relative rates of photolysis of free tryptophan (trp), the dipeptides Gly-Trp, Trp-Gly, Leu-Trp, and Trp-Leu, and the tripeptides Gly-Trp-Gly and Leu-Trp-Leu. The photolyses were performed in neutral 0.1 mM aqueous solutions at 25°C using monochromatic 290 nm Xe arc radiation. Tryptophan loss was monitored by absorption, fluorescence and phosphorescence spectroscopy. The rate of tryptophan fluorescence loss was found to be different in the di-and tripeptides than in tryptophan monomer. These rate differences depended on both the identity of the neighboring amino acid (gly or leu) and on the nature of the linkage, e.g., the rate of Gly-Trp photolysis was more than 10 times greater than the rate of Trp-Gly photolysis. Degassing was found to markedly reduce (factor of 8) the photolysis rates of Trp, Trp-Gly, and Trp-Leu, but degassing only slightly reduced (less than a factor of 2) the photolysis rates of the other di-and tri-peptides. Photochemical product structures were not determined, but absorption and fluorescence spectra were obtained and products could be inferred in some cases by comparison with data of previous workers. The products appeared to differ greatly among the various peptides studied; Trp, Trp-Gly, and Trp-Leu gave oxidation products, while Gly-Trp and Leu-Trp apparently gave ring closure products, not requiring oxygen.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—II. SPIN TRAPPING OF PHOTOLYSIS PRODUCTS FROM SULFANILAMIDE AND 4-AMINOBENZOIC ACID USING 5,5-DIMETHYL-1-PYRROLINE-1-OXIDE

Abstract— The photodecomposition of sulfanilamide, 4-aminobenzoic acid and related analogs in aqueous solution has been studied with the aid of spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and CH₃NO₂ as well as by direct electron spin resonance techniques. The NH₂ radical was trapped by DMPO during the photolysis of aqueous solutions of sulfanilamide with a Xe arc lamp. Studies with [¹⁵N¹]-sulfanilamide indicated that the NH₂ radical was generated by homolytic fission of the sulfur-nitrogen bond. Under the same conditions DMPO trapped the H and SO⁻₃ radicals during photolysis of sulfanic acid. Direct photolysis of sulfanilamide, sulfanilic acid and Na₂SO₃ in the absence of any spin trap yielded the SO⁻³ radical. Photolysis of 4-aminobenzoic acid at pH 7 gave the H radical which was trapped by DMPO. At low pH values OH and C₆H₄COOH radicals were generated during the photolysis of 4-aminobenzoic acid. No e⁻_aq were trapped by CH₃NO₂ when acid (pH 4) and neutral aqueous solutions of sulfanilamide or 4-aminobenzoic acid were photoirradiated. The mechanism of formation of known photoproducts from the free radicals generated by sulfanilamide and 4-aminobenzoic acid during irradiation are discussed. The free radicals generated by these agents may play an important role in their phototoxic and photoallergic effects.     

Tryptophan Modification and Fluorescence Spectrum of Hyaluronidase

Tryptophan residues in hyaluronidase （HAase） were modified by N-bromosuccinimide （NBS）, the results indicated that there were eleven tryptophan residues in HAase, one of which was exposed and essential for the activity of the enzyme. The study on fluorescence quenching showed that KI could not quench all of the fluorescence from Trp residues in HAase, while acrylamide （Acr） could quench almost all of the fluorescence from Trp residues in HAase. The collisional quenching constants （KD） of HAase at different concentrations of Acr were calculated in terms of Stern-Volmer equation. The results implied that some of the Trp residues were buried in the interior of HAase and the Trp residue on the surface of HAase was not located in the hydrophobic pocket.     

QUANTUM YIELDS OF TRIPLET FORMATION OF SOME DERIVATIVES OF ANTHRAQUINONE

Abstract— Quantum yields of triplet formation for five amino substituted anthraquinones have been determined by the comparative actinometer method using laser flash photolysis. Substitution reduces the yields to values of ˜10^--². and the requirement of low laser intensities required high sensitivity of detection of triplet absorption. The ø_T values are compared with the quantum efficiencies of fluorescence and decomposition for the compounds, and the criteria for light stability discussed.     

在不同激发波长下的单壁碳纳米管的拉曼光谱研究

采用直流电弧法制备单壁碳纳米管样品,用457.5和632.8nm两种不同的激发光分别测得单壁碳纳米管的正常拉曼光谱和共振拉曼光谱.通过理论分析得到了单壁碳纳米管的直径分布,进一步推测了其类型及结构参数;对单壁碳纳米管的正切拉伸模的成分进行了归属.在632.8nm激发波长下得到了IG/ID值随激光功率变化的曲线,认为在2.5mW时,单壁碳纳米管缺陷的结构可能发生了改变.在用457.5nm波长激发的单壁碳纳米管的拉曼光谱中,首次发现了1421cm-1的拉曼谱峰.     

透明质酸酶的色氨酸残基修饰与荧光光谱研究

北五味子[Schisandra chinensis（Turcz．）Baill．]属广义木兰科植物,主产于我国东北,故又称“辽五味”,中药五味子的主要药材为北五味子的干燥果实,作为一种传统中药,五味子具有收敛固涩,益气生津,补肾宁心的功效,用于肺喘虚咳,心悸失眠诸病。     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF 2-AMINO-4 PTERIDINONE: A NATURAL PIGMENT

Abstract— The characteristics of the fluorescence and phosphorescence emission of 2-amino-4 (3H) pteridinone (or pterin) in aqueous solutions are pH dependent. The room temperature fluorescence quantum yield is low and is maximum at pH = 10 (φ_F∼ 0.057). The 77K phosphorescence emission consists of two overlapping emissions originating from τ* triplet states. In agreement with low temperature results, the 353nm laser flash photolysis makes it possible to detect at pH 9.2, two transient triplet absorptions (τ₁∼ 0.3 μs and τ₂∼ 2.3 μs). The longer lived triplet is characterized by φ_TM∼ 0.20 and ∼ (550nm) = 2000 M ⁻¹ cm⁻¹. It reacts with the solvent forming the semireduced pterin with a quantum yield φ_R∼ 0.06. The photosensitizing properties of pterin have been studied by laser flash spectroscopy and steady state irradiations. Photoreactions implying singlet oxygen formation are shown to occur. Laser flash spectroscopy indicates that the pterin triplet is reduced by amino acids and nucleic acid bases. Corresponding bimolecular reaction rate constants have been measured.     

Ultraviolet-B-induced inactivation of human OGG1, the repair enzyme for removal of 8-oxoguanine in DNA

The OGG1 proteins are DNA N-glycosylases-apurinic-apyrimidinic lyases that are responsible for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) base in DNA. The human enzyme (hOGG1) is a monomer of 345 amino acids containing 10 buried tryptophan (Trp) residues that are very sensitive to UVB irradiation. The photolysis quantum yield of these Trp residues is about 0.3 and 0.1 in argon- and air-saturated solutions, respectively. Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry shows that several cleavage sites are identical under aerobic and anaerobic photolysis of Trp residues; one of them includes the active site. Western blots and polyacrylamide gel electrophoresis indicate that fragments of high molecular size are also formed. In addition to common photochemical paths with argon-saturated solutions, specific reactions occur in air-saturated solutions of hOGG1. The photolysis rate is inhibited by more than 50% on binding of hOGG1 to a 34mer oligonucleotide containing a single 8-oxoG-C base pair. Binding to the oligonucleotide with 8-oxoG-C induced a 20% quenching of the hOGG1 fluorescence, suggesting interaction of nucleic acid bases with the Trp residue(s) responsible for the photolysis. Using 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) and 8-oxoG as substrates, it is shown that protein photolysis induces photoinactivation of the DNA N-glycosylase activities. The excision of 8-oxoG is more affected than that of Me-FapyG at the same dose of UVB irradiation under both air and argon conditions. Besides the role of Trp residues, the possible involvement of Cys 253 in the photoinactivation process of hOGG1 is discussed.     

TRANSIENT PHOTOCHEMISTRY OF SAFRANIN-O

Abstract— We have characterized the spectra, acidity constants and decay kinetics of the triplet and semireduced radical species of Safranin-O. Between pH 3.0 and 10.6, there are three triplet species denoted ³DH^{2 +}₂, ³DH⁺ and ³D, the p K _as being 7.5 and 9.2. All three triplet species exhibit first order decay, the rate constant for ³DH⁺ being ca. 5-fold lower than the rate constants of ³DH⁺ and ³D. Ascorbic acid and ethylenediaminetetraacetic acid (EDTA) quench the triplet state under appropriate pH conditions and the pH dependencies of the yield of semireduced dye indicate that ³DH⁺ is more reactive than ³DH⁺ or ³D. With EDTA as the reducing agent, there is the additional requirement that at least one of the amino nitrogens be deprotonated to obtain a significant yield of semireduced dye. In these reactions, ascorbic acid is oxidized reversibly, but EDTA is oxidized irreversibly, so that with the latter reducing agent photolysis causes buildup of the leucodye, which on subsequent photolysis can reduce triplet state dye. With ascorbic acid as the reducing agent, the regeneration of the ground state dye is reversible, the decay of the semireduced radical being second order. In general, the transient photochemistry of Safranin-O resembles that of Thionine, the major difference being that the lifetimes of ³DH^{2 +}₂ and ³DH⁺ are much longer for Safranin-O than for Thionine.     

ALKALINE QUENCHING OF BACTERIORHODOPSIN TRYPTOPHANYL FLUORESCENCE: EVIDENCE FOR AQUEOUS ACCESSIBILITY OR A HYDROGEN-BONDED CHAIN

Abstract— Comparison between Trp fluorescence yields of membrane-bound bacteriorhodopsin (BR) and retinylidene-free bacterioopsin (BO) is consistent with a model in which all eight Trp residues are active fiuorophores in the latter, while the emission of all but two residues in the former is lost by energy transfer to retinal. The visible chromophore of BR is progressively bleached with increasing pH. Up to pH 12 this bleaching is reversed on reneutralization; but above this the change is irreversible with the appearance of a new absorption band characteristic of free retinal. Emission yields for both proteins decrease with increasingly alkaline pH in a manner typical of energy transfer to weakly-fluorescent tyrosinate. The limiting yields, reached at a pH corresponding to that producing irreversible bleaching of the visible chromophore, agree with an integral value of one remaining active Trp fluorophore in BR and between one and two in BO and show that the bulk of Trp are within the 11 Å Förster energy-transfer distance of Tyr accessible to OH⁻. Current models of the native protein structure of BR arrange the polypeptide chain primarily in a bundle of seven helical segments with axes perpendicular to the lipid bilayer plane and with buried polar residues, including Trp and Tyr, located at intrahelical surfaces. An interpretation of the observed accessibility of buried Tyr to OH⁻ is that an aqueous region exists within the protein structure. Moreover, this putative aqueous region must be close to the retinylidene chromophore and thus may be associated with the light-driven ion transport system. The results are also compatible with energy transfer to internal Tyr residues which are connected via a chain of phenolate hydrogen bonds to a surface Tyr.     

RADICAL CATION PRODUCTION IN THE PHOTOLYSIS OF 5-M-ETHOXY-1-M-ETHYLINDOLE

Abstract— The flash photolysis of 5-m-ethoxy-1-m-ethylindole in aqueous media was studied for the purpose of assigning the absorption spectrum of the radical cation. Transients produced in this study were analogous to those formed in the photolysis of 5-m-ethoxyindole. The major transient observed with an absorption maximum of 460 nm was O₂-s_-ensitive and had a lifetime of 20 μs in nitrogen saturated solutions. One radical species is produced with absorption maxima at 445 and 530 nm. Ionic strength effects on the reaction of this species with I⁻ confirms that it is the radical cation of 5-m-ethoxy-l-methylindole. The effect of H⁺ and Br⁻ on the fluorescence, radical cation and triplet yields is discussed in relation to the mechanism of transient formation.     

DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract— The triplet-triplet absorption spectra in aqueous solution of the acid (³LfH₂⁺), the neutral (³LfH) and the basic (³Lf^-) forms of lumifiavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The p K _a values of the corresponding protolytic equilibria of the lumifiavin triplet were found to be 4.4₅±0.1 and 9.8±0.15.     

丙酮三重态与含芳香氨基酸肽的物理化学过程

用激光闪光光解瞬态吸收光谱研究了水溶液中含芳香氨基酸残基肽的光敏化反应过程.结果表明,在丙酮存在的含色氨酸残基肽(Trp-Gly,n-f-Met-Trp,Trp-Phe)体系的光解,丙酮三重态与Trp分别通过三重态-三重态(T-T)激发能转移和电子转移生成Trp激发三重态和N中心自由基(Trp/N^·);丙酮三重态仅与含酪氨酸残基肽(Phe-Tyr)通过电子转移生成Tyr酚氧自由基(Tyr/O^·).在色氨酰酪氨酸(Trp-Tyr)与丙酮的光解体系中,观察到分子内的电子转移,即由Trp/N^·-Tyr→Trp-Tyr/O^·自由基的生成过程     

ULTRAVIOLET RESONANCE RAMAN STUDY ON PURPLE AND BLUE MEMBRANES OF Halobacterium halobium

Abstract— Two hundred and forty and 213 nm excited resonance Raman spectra of purple membrane (PM) and blue membrane (BM) of Halobacterium halobiurn were studied. Generally intense Trp scattering and a strong relative intensity of the W3 band at 1553 cm^-1 in the 240 nm spectrum of PM indicate red-shifted B_b absorptions of some Trp sidechains. A high intensity ratio of Trp doublet at 1360 and 1340 cm^-1 suggests interactions with highly hydrophobic Trp environments. These Trp are not strongly H-bonded and their N₁ sites are located in positions easily reached by solvent water molecules. Tyrosines are also in very hydrophobic environments and H-bonded. The mainchain consists of normal and distorted α-helices whose amide NH are hardly deuterated in D₂O suspension, and some NH exchangeable irregular segments on the membrane surface. Upon acidification, the ratio of Trp doublet with 240 nm excitation decreases concomitant with increase in retinal absorbance at 600 nm, and the W3 relative intensity and overall Trp scattering also decrease. These observations strongly indicate that the counterpart of Trp interactions in PM is the retinal and that the interactions partly diminish upon acidification. The Tyr environment also changes with the color. Although the 240 nm amide I intensity is greater in acid BM than in PM, the change is not related to the color change because the amide I intensity of deionized BM is practically the same as that of PM. The amide I intensity increase in acid BM is ascribable to a structural change of the surface peptides due to acid induced aggregation.