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.     

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.     

EFFECTS OF PHOTODYNAMIC PROCESSES AND ULTRAVIOLET LIGHT ON DUCK AND HEN EGG-WHITE LYSOZYMES

Abstract— The photochemical yields for inactivation and amino acid destruction in hen and duck egg-white lysozyme are presented. Duck lysozyme II is devoid of histidine but it has two more tyrosine residues than does hen lysozyme. The data indicate that sensitized oxidation of the single histidine residue of hen lysozyme is of no significance for the inactivation of this lysozyme. The ultraviolet destruction of tryptophan and cystine residues appears to be equally related with the loss in enzymatic activity of hen lysozyme. In the case of duck lysozyme, however, the ultraviolet inactivation appears to be predominantly governed by the destruction of cystine residues.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

Laser flash photolysis and inactivation of carboxypeptidase A

Abstract— Laser photolysis of CPA at 265 nm photoionizes 3 to 4 Trp residues per molecule inactivated, leading to e^-_aq and the disulfide bridge electron adduct. The electron adduct is formed by an internal process and is not involved in the activity loss. Based on this work and published photochemical and pulse radiolysis studies on CPA it is proposed that photolysis of a key Trp residue, possibly Trp 73 adjacent to zinc ligand Glu 72 , mediates release of the zinc ion and consequent loss of peptidase activity.     

PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN

Abstract— It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo-dynamic inactivation of hen egg white lysozyme by eosin, using D₂O to enhance the solvent-induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air-saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H₂O to D₂O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re-examination of earlier work on eosin-sensitized photo-oxidation of I", it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.     

THE TRIPLET EXTINCTION COEFFICIENTS OF SOME BACTERIAL CAROTENOIDS

The extinction coefficients of the triplet states of the bacterial carotenoids, neurosporenc (Λ_max 489 nm). sphcroidene (Λ_max 510 nm). spheroidenone (Λ_max 550 nm) and spirilloxanthin (Λ_max 550 nm) in cyclohexane have been determined to be 27.4. 30.9. 6.06 and 9.20 × 10⁴ dm³ mol^?1 cm¹, respectively. These values were obtained by an energy transfer technique using a range of carotenoid concentrations. For the three that had been studied earlier, the extinctions now obtained are suhstantially higher than prcviously reported.     

FLASH PHOTOLYSIS OF RIBONUCLEASE A

Abstract— Flash photolysis spectra show that ultraviolet irradiation of RNase (Λ > 250 nm) at pH 11.5 generates the hydrated electron and a long-lived transient with absorption maxima at 390 nm and 410 nm, attributed to the phenoxyl type radical from tyrosyl residues. Comparison of the initial yields with flash photolysis spectra obtained from aqueous tyrosine and mixtures of the chromophoric amino acids indicates that 3–4 tyrosyl residues are photoionized in the primary act. This process is almost completely quenched at pH 1–9, even though the p -alanylphenoxyl radical is obtained with tyrosine over this pH range and the accompanying electron is observed at pH 7. The negative result is not altered by denaturation of RNase with 8 M urea or heating to 70°C, suggesting that a primary chain interaction is responsible for the suppression of tyrosyl residue photolysis. This mechanism is supported by flash photolysis spectra of small peptides, showing that the initial radical yield from tyrosylglycylglycine is strongly quenched compared to tyrosine when the phenolic group is protonated. Comparion of this work with published results on fluorescence and inactivation quantum yields indicates that photochemical electron ejection from RNase in alkaline solutions takes place in the dissociable residues and does not contribute to loss of enzymic activity.     

PHOTOCHEMICAL REDUCTION OF 5-BROMOURACIL BY CYSTEINE DERIVATIVES AND COUPLING OF 5-BROMOURACIL TO CYSTINE DERIVATIVES

Irradiation of pH 7, aqueous solutions of 5-bromouracil (BU) in the presence of cysteine peptide-like derivatives at 308 nm using a XeCl excimer laser yielded initial formation of only uracil (U) and the corresponding cystine derivative. Continued irradiation yielded an S-uracilylcysteinyl adduct as well as additional U and cystine derivative. Similar irradiation of a solution of BU and a cystine derivative yielded initial formation of U and the S-uracilylcysteinyl adduct. Formation of these products as well as secondary products of uracil photochemistry was observed upon irradiation of the respective solutions with 254 nm light. With 308 nm laser excitation, U-Cys adduct formation and reduction of BU to U are proposed to occur via initial electron transfer from the disulfide of the cystine derivative to triplet BU. The quantum yield of BU destruction with 308 nm excitation in the presence of cystine derivative is 1.1 X 10(-3). Reaction of triplet BU with the cysteine derivative does not yield U-Cys adduct but U and cystine derivative. A possible byproduct of reduction of triplet BU to U by a cysteinyl residue in a protein BU-DNA complex is a sulphenyl bromide which might yield a protein-DNA crosslink via nucleophilic substitution on sulfur by a nucleophilic site in DNA.     

MECHANISM OF ENZYME INACTIVATION BY ULTRAVIOLET LIGHT AND THE PHOTOCHEMISTRY OF AMINO ACIDS (AT 2537 Å)*

Abstract— >The inactivation of the enzymes chymotrypsin, lysozyme, ribonuclease, and trypsin by ultraviolet light can be accounted for quantitatively by summing the products of (1) the probability that light is absorbed by a given amino acid residue, the molecular extinc tion coefficient, and (2) the probability that absorbed light induces a chemical change in the residue, the quantum yield for the residue. The principal residues involved are cystyl and tryptophanyl. Peptide bond rupture is not important. Energy transfer among chromophores within molecules of enzymes need not be invoked in order to account for photochemical inactivation.
Quantum yields for the destruction of a number of amino acids at 2537 A have been measured.     

Photoinduced electron ejection from hydroxo complexes of thallium(I) and tin(II) in alkaline aqueous solution

In alkaline solution (1 M NaOH) irradiation (λ_ir=266 nm) of both TlOH and Sn(OH)⁻₃ leads to the formation of hydrated electron and oxidized complex in the primary photochemical step. In both cases nascent hydrated electrons react with the ground-state hydroxometalates to form the corresponding reduced compounds. The main reaction of these latter species is recombination (synproportionation) with the oxidized complexes, significantly diminishing the efficiency of the overall light-induced oxidation of TlOH and Sn(OH)⁻₃.     

THE ACTION OF U.V. LIGHT OF VARIOUS WAVELENGTHS ON PAPAIN

Abstract— Papain, prepared according to Kimmel and Smith is activated by irradiation with u.v. light of 254, 280 and 313 nm (φ_A= 0·022 ± 10 per cent). This activation is caused by the reductive splitting of a mixed disulfide in position 25. This disulfide is split with higher probability than any of the three structural disulfide links. This selectivity is likely caused by specific reduction of the mixed disulfide by thiol groups produced elsewhere in the molecule, although specific reduction by direct photochemical processes cannot be completely excluded on the basis of the data available. The quantum yields for destruction of structural cystine residues at the three wavelengths are comparable with the yields of cystine destruction in other proteins. The data also confirm that quanta absorbed by aromatic amino acid residues contribute to cystine destruction. In contrast to other enzymes with structural disulfide bonds, however, no correlation was found between the destruction of disulfide links and loss in activity. The results suggest that the mechanism of papain inactivation is not only dependent on the wavelength, but also on the dose.     

Biophysical aspects of lysozyme adduct with monocrotophos

The present study on in vitro formation and characterization of lysozyme adduct with monocrotophos (MP) evaluates the potential of lysozyme to be used as a sensitive biomarker to monitor exposure levels to the commonly used organophosphorus pesticide monocrotophos. Crystallization of lysozyme protein adduct with monocrotophos was also undertaken to understand the adduct formation mechanism at a molecular level. The binding of organophosphorus pesticides to lysozyme is one of the key steps in their mutagenicity. The formation and structural characterization of lysozyme adduct with monocrotophos was done using MALDI-TOFMS, fluorescence, UV/Vis spectroscopy, circular dichroism, and X-ray diffraction studies. We report the crystal structure of lysozyme adduct with monocrotophos at 1.9 Å. It crystallized in the P4₃ space group with two monomers in one asymmetric unit having one molecule of monocrotophos bound to each protein chain. The results proved that the fluorescence quenching of lysozyme by monocrotophos is due to binding of monocrotophos with a tryptophan residue of lysozyme. Monocrotophos interacts most strongly with the Trp-108 and Asp-52 of lysozyme. The interactions of the monocrotophos molecule with the lysozyme suggest the formation of a stable adduct. In addition, the alteration of lysozyme secondary structure in the presence of monocrotophos was confirmed by circular dichroism and fluorescence inhibition of lysozyme by increasing monocrotophos and UV/Vis spectrophotometry. The formation of lysozyme adduct with monocrotophos was confirmed by MALDI-TOFMS. Figure

Crystal Structure of lysozyme adduct with monocrotophos (MP) [ PDB ID 4TUN) and Ligplots shows the monocrotophos bonding distances and interactions with amino acid residues in lysozyme     

Studies on the Chemical Modification of the Essential Groups of <Emphasis Type="Italic">N</Emphasis>-Acetyl-β-<Emphasis Type="SmallCaps">d</Emphasis>-Glucosaminidase from Viscera of Green Crab (<Emphasis Type="Italic">Scylla Serrata</Emphasis>)

The chemical modification of N-acetyl-β-d-glucosaminidase (EC3.2.1.30) from viscera of green crab (Scylla serrata) has been first studied. The modification of indole groups of tryptophan of the enzyme by N-bromosuccinimide can lead to complete inactivation, accompanying the absorption decreasing at 275 nm and the fluorescence intensity quenching at 338 nm, indicating that tryptophan is essential residue to the enzyme. The modification of histidine residue, the carboxyl groups, and lysine residue inactivates the enzyme completely or incompletely. The results show that imidazole groups of histidine residue or sulfhydryl residues, the carboxyl groups of acidic amino acid, amino groups of lysine residue, and indole groups of tryptophan were essential for the catalytic activity of enzyme, while the results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme’s function.     

Photoionization of Indole, N-Methylindole and Tryptophan In Aqueous Solution upon Excitation at 193 nm

Abstract— The 193 nm photoionization of aqueous indole, A'-meth-ylindole and tryptophan–as a function of pH and under several saturating gas conditions–has been studied by laser photolysis using optical and conductometric detection methods. Monophotonic ionization leads to production of the cation radicals and hydrated electrons, the quantum yield of electron ejection is 0.3–0.4. The cation radicals have pK_a values of 4.5, <5 and 4.5 for indole, N -methylindole and tryptophan, respectively. Above these pH values, the cation radical deprotonate rapidly, having lifetimes of 1.0, ≅6 and 1.1 μs, respectively. Under N₂O, neutral indolyl radical production is accompanied by formation of an OH adduct radical (<1 μs). The conductivity results in Ar- and N₂O-saturated solution support the deprotonation mechanism and indicate that in the acidic pH range, the cation radical decays by release of protons with kinetics on the millisecond time scale.     

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.     

Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A₂ (PLA₂) activity. Therefore, the regulation of PLA₂ catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA₂ due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA₂ inhibition. A more detailed analysis of the plasma-treated PLA₂ identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.     

SPECTRAL AND PHOTOCHEMICAL PROPERTIES OF CURCUMIN

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione, is a natural yellow-orange dye derived from the rhizome of Curcuma longa, an East Indian plant. In order to understand the photobiology of curcumin better we have studied the spectral and photochemical properties of both curcumin and 4-(4-hydroxy-3-methoxy-phenyl)-3-buten-2-one (hC, half curcumin) in different solvents. In toluene, the absorption spectrum of curcumin contains some structure, which disappears in more polar solvents, e.g. ethanol, acetonitrile. Curcumin fluorescence is a broad band in acetonitrile (λ_max= 524 nm), ethanol (λ_max= 549 nm) or micellar solution (λ_max= 557 nm) but has some structure in toluene (λ_max= 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (φ= 0.011) but higher in acetonitrile (φ= 0.104). Curcumin produced singlet oxygen upon irradiation (φ > 400 nm) in toluene or acetonitrile (Φ= 0.11 for 50 μM curcumin); in acetonitrile curcumin also quenched ¹O₂ (k_q, = 7 × 10⁶ M^?1 s^?1). Singlet oxygen production was about 10 times lower in alcohols and was hardly detectable when curcumin was solubilized in a D₂O micellar solution of Triton X-100. In SDS micelles containing curcumin no singlet oxygen phosphorescence could be observed. Curcumin photogenerates superoxide in toluene and ethanol, which was detected using the electron paramagnetic resonance/spin-trapping technique with 5,5-dimethyl-pyrroline-.N-oxide as a trapping agent. Unidentified carbon-centered radicals were also detected. These findings indicate that the spectral and photochemical properties of curcumin are strongly influenced by solvent. In biological systems, singlet oxygen, superoxide and products of photodegradation may all participate in curcumin phototoxicity depending on the environment of the dye.     

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 ^–.     

芘四磺酸钠水溶液激光闪光光解机理

采用激光闪光光解-瞬态吸收光谱技术研究了355nm激光作用下芘四磺酸钠(PyTS)水溶液的光化学反应机理及其产生水合电子的动力学行为.研究首次发现PyTS水溶液激发单线态(PyTS1*)在260nm、激发三线态(PyTS3*)在300nm及阴离子自由基(PyTS-?)在330nm处的特征吸收峰;分析了生成的水合电子(e-aq)的主要反应途径包括自猝灭反应及与PyTS的反应,得到水合电子与PyTS反应的准一级速率常数为2.7′105s-1;并计算得到在此实验条件下,PyTS水溶液经双光子吸收产生的水合电子量子产率为3.2′10-2.