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1.
The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC相似文献
2.
Luiz M Biasutti MA García NA 《Redox report : communications in free radical research》2004,9(4):199-205
Benzylhexadecyl dimethylammonium chloride (BHDC) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) water-in-oil micro-emulsions were employed to study the influence of medium heterogeneity on singlet molecular oxygen [O(2)((1)Delta(g))]-mediated degradation of 1-hydroxynaphthalene (1-OHN) and 2-hydroxynaphthalene (2-OHN) in their naphthoate form. The kinetic study in the micellar system, that was considered as a closer model for the environment of contaminants in natural polluted waters, consisted of a comparative work with the process in homogeneous solution, by varying surfactant structure and water content of the micro-emulsion. While it is known that 1-OHN and 2-OHN are rapidly and efficiently photo-oxidised in aqueous medium, time-resolved phosphorescence detection of O(2)((1)Delta(g)) and stationary photolysis experiments demonstrate that both the values for the overall and reactive rate constants for the quenching of O(2)((1)Delta(g)) and the photo-oxidation efficiencies are lowered in BHDC micelles, whereas the photo-oxidative process in AOT micro-emulsions was totally inhibited. Results are interpreted and discussed on the basis of different locations of the probe in the micellar environment. 相似文献
3.
Yppolito R Pappano N Debattista N Miskoski S Bertolotti SG García NA 《Redox report : communications in free radical research》2002,7(4):229-233
The ability of the widely employed therapeutic drugs 4-aminosalicylic acid and 5-aminosalicylic acid to act as singlet molecular oxygen (O(2)((1)delta(g))) scavengers was investigated at pH 7 and pH 12. The isomer 3-aminosalicylic acid was also included in the study for comparative purposes. All three compounds quench photochemically generated O(2)((1)delta(g)) with rate constants in the range of 10(7)-10(8) x M(-1)s(-1), depending on the experimental conditions. No chemical reaction (oxidation of the aminosalicylic acids) was detected at the neutral pH, whereas at pH 12 both chemical and physical interactions with O(2)((1)delta(g)) operated. The physical process implies the de-activation of the oxidant species without destruction of the aminosalicylic acid. The quotients between the overall and reactive rate constants for O(2)((1)delta(g)) quenching at pH 12 (k(r)/k(t) ratios), which account for the actual effectiveness of photodegradation, were relatively low (0.22, 0.04, and 0.06 for 3-, 4- and 5-aminosalicylic acids, respectively). This indicates that the drugs, particularly the 4- and 5-amino derivatives, de-activate the excited oxygen species, at both pH values studied, mainly in a physical fashion, preventing its photodegradation and providing an antioxidative protection for possible photo-oxidizable biological targets in the surroundings. 相似文献
4.
Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic studies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum yields (phi(F)) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell line. The production of singlet molecular oxygen, O(2)((1)Delta(g)), was determined using 9,10-dimethylanthracene yielding values of approximately 0.56 for both sensitizers. Under these conditions, the addition of beta-carotene (Car) suppresses the O(2)((1)Delta(g))-mediated photooxidation. In biological medium, no dark cytotoxicity was found for cells incubated with 0.1 microM of phthalocyanines 3 and 4 for 24 h. However, under similar conditions 0.5 microM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1muM of sensitizer, reaching values of approximately 0.05 nmol/10(6) cells after 3h of incubation at 37 degrees C. The cell survival after irradiation of the cultures with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15 min of irradiation). Also, these studies were performed treating the cells with 0.5 microM of ZnPc 3. In this case, an increase in the uptake (approximately 0.28 nmol/10(6) cells) was observed, which is accompanied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed. 相似文献
5.
Criado S Guardianelli C Tuninetti J Molina P García NA 《Redox report : communications in free radical research》2002,7(6):385-394
The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage. 相似文献
6.
Reinhard Schmidt 《Photochemical & photobiological sciences》2005,4(6):481-486
A charge transfer (CT) channel and a non-CT deactivation channel, both leading to formation of O(2)((1)Sigma (g)(+)), O(2)((1) Delta(g)) and O(2)((3)Sigma(g)(-)), compete in the quenching of triplet states by O(2). Recent studies by our group demonstrated that these channels are described by rather simple and general quantitative relations. In the present paper we use the detailed kinetic data on the quenching by O(2) of pi pi* triplet sensitizers of three homologous aromatic series in CCl(4) to derive a parameter, which describes the balance between CT and non-CT deactivation. This quantity, p(CT), is the relative contribution of CT mediated deactivation and is easily calculated for a sensitizer of known triplet energy from its quenching rate constant. The parameter p(CT) quantitatively describes the balance between both deactivation channels without requiring any knowledge of oxidation potentials. It is shown how the variation of p(CT) influences the efficiencies and the rate constants of O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)) and O(2)((3)Sigma(g)(-)) formation in the quenching process. 相似文献
7.
The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments. 相似文献
8.
Bensasson RV Brettreich M Frederiksen J Göttinger H Hirsch A Land EJ Leach S McGarvey DJ Schönberger H 《Free radical biology & medicine》2000,29(1):26-33
Using pulse radiolysis and laser flash photolysis, we have investigated the reactions of the deleterious species, e(-)(aq), HO&z.rad;, O(2)(*)(-) and O(2)((1)Delta(g)) with 10 water-soluble cyclopropyl-fused C(60) derivatives including a mono-adduct dendro[60]fullerene (d) and C(60) derivatives based on C(60)[C(COOH)(2)](n=2-6), some of which are known to be neuroprotective in vivo. The rate constants for reactions of e(-)(aq) and HO&z.rad; lie in the range 0.5-3.3 x 10(10) M(-1) s(-1). The d and bis-adduct monoanion radicals display sharp absorption peaks around 1000 nm (epsilon = 7 000-11 500 M(-1) cm(-1)); the anions of the tris-, tetra-, and penta-adduct derivatives have broader, weaker absorptions. The monohydroxylated radicals have their most intense absorption maxima around 390-440 nm (epsilon = 1000-3000 M(-1) cm(-1)). The anion and hydroxylated radical absorption spectra display a blue-shift as the number of addends increases. The radical anions react with oxygen (k approximately 10(7)-10(9) M(-1) s(-1)). The reaction of O(2)(*)(-) with the C(60) derivatives does not occur via an electron transfer. The rate constants for singlet oxygen reaction with the dendrofullerene and eee-derivative in D(2)O at pH 7.4 are k approximately 7 x 10(7) and approximately 2 x 10(7) M(-1) s(-1) respectively, in contrast to approximately 1.2 x 10(5) M(-1) s(-1) for the reaction with C(60) in C(6)D(6). The large acceleration of the rates for electron reduction and singlet oxygen reactions in water is due to a solvophobic process. 相似文献
9.
Emanuel A Dupouy Debora Lazzeri Edgardo N Durantini 《Photochemical & photobiological sciences》2004,3(11-12):992-998
The photodynamic activity of a cationic Zn(II) tetramethyltetrapyridinoporphyrazinium salt (ZnPc ) was compared with that of a non-charged Zn(II) tetrapyridinoporphyrazine (ZnPc 1), both in vitro using human red blood (HRB) cells and a typical Gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were analyzed in different media. Fluorescence quantum yields (phi(F)) of 0.35 for ZnPc 1 and 0.30 for ZnPc 2 were calculated in N,N-dimethylformamide (DMF). The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of Phi(Delta)= 0.56 for ZnPc 1 and 0.50 for ZnPc 2. In biological medium, the photodynamic effect was first evaluated in HRB cells. Both phthalocyanines produce similar photohemolysis of HRB cells, reaching values >90% of lysis after 5 min of irradiation with visible light. The photodynamic effect is accompanied by an increase in the membrane fluidity of HRB cells. However, these studies on E. coli cells showed that the cationic ZnPc 2 produces a higher photoinactivation of Gram-negative bacteria than ZnPc 1. Also, these results were established by stopped of growth curves for E. coli. Therefore the studies show that cationic ZnPc 2 is an efficient phototherapeutic agent with potential applications in tumor cell and Gram-negative bacteria inactivation by photodynamic therapy. 相似文献
10.
Estevam ML Nascimento OR Baptista MS Di Mascio P Prado FM Faljoni-Alario A Zucchi Mdo R Nantes IL 《The Journal of biological chemistry》2004,279(38):39214-39222
This work compares the effect of photogenerated singlet oxygen (O(2)((1)Delta(g))) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB(+)) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 microm) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mm SDS. Over a pH range in which cytochrome c is in the native form, O(2) ((1)Delta(g)) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g approximately 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB(+) dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met(65) and Met(80)) and tyrosine (Tyr(74)) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by beta-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2). 相似文献
11.
On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin
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The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open. 相似文献
12.
We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation. 相似文献
13.
Martinez GR Gasparutto D Ravanat JL Cadet J Medeiros MH Di Mascio P 《Free radical biology & medicine》2005,38(11):1491-1500
It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides. 相似文献
14.
Soriano GM Guo LW De Vitry C Kallas T Cramer WA 《The Journal of biological chemistry》2002,277(44):41865-41871
The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex. 相似文献
15.
The reaction rates (k(r)) of 5,7-diisopropyl-tocopheroxyl radical (Toc) with catechins (epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG)) and related compounds (methyl gallate (MG), 4-methylcatechol (MC), and 5-methoxyresorcinol (MR)) have been measured by stopped-flow spectrophotometer. The k(r) values increased in the order of MR < < MG < EC < MC approximately ECG < EGC < EGCG in ethanol and 2-propanol/H(2)O (5/1, v/v) solutions, indicating that the reactivity of the OH groups in catechins increased in the order of resorcinol A-ring < < gallate G-ring < catechol B-ring < pyrogallol B-ring. The catechins which have lower oxidation potentials show higher reactivities. The rate constants for catechins in micellar solution showed notable pH dependence with one or two peaks around pH 9-11, because of the dissociation of various phenolic hydroxyl protons in catechins. The structure-activity relationship in the free-radical-scavenging reaction by catechins has been clarified by the detailed analyses of the pH dependence of k(r) values. The reaction rates increased remarkably with increasing the anionic character of catechins, that is, the electron-donating capacity of catechins. The mono anion form at catechol B-and resorcinol A-rings and dianion form at pyrogallol B-and gallate G-rings show the highest activity for free-radical-scavenging. It was found that catechins (EC, ECG, EGC, and EGCG) have activity similar to or higher than that of vitamin C in vitamin E regeneration at pH 7-12 in micellar solution. 相似文献
16.
The interface between the c-subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Delta(phi)) at low transmembrane pH difference (Delta(pH)). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0, and the mechanism determining the relative contribution of Delta(pH) and Delta(phi) in driving ATP synthesis. 相似文献
17.
Milanesio ME Morán FS Yslas EI Alvarez MG Rivarola V Durantini EN 《Bioorganic & medicinal chemistry》2001,9(8):1943-1949
A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2',6'-dinitro-4'-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen, O(2)((1)Delta(g)), production. 相似文献
18.
Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1 总被引:4,自引:0,他引:4
van Thor JJ Borucki B Crielaard W Otto H Lamparter T Hughes J Hellingwerf KJ Heyn MP 《Biochemistry》2001,40(38):11460-11471
The P(r) to P(fr) transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Delta2 is accompanied by net acidification in unbuffered solution. The extent of this net photoreversible proton release was measured with a conventional pH electrode and increased from less than 0.1 proton released per P(fr) formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Delta2) H(+)/P(fr) at pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transient absorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constants of approximately 4 and approximately 20 ms that were also observed in parallel measurements of the photocycle (tau(3) and tau(4)). The number of transiently released protons per P(fr) formed is about one. This H(+) release phase is followed by a proton uptake phase of a smaller amplitude that has a time constant of approximately 270 ms (tau(5)) and is synchronous with the formation of P(fr). The acidification observed in the P(r) to P(fr) transition with pH electrodes is the net effect of these two sequential protonation changes. Flash-induced transient absorption measurements were carried out with Cph1 and Cph1Delta2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (tau(1)-tau(5): 5 and 300 micros and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B(1), B(3), and B(5)) showed significant pH dependence. Measurements of the P(r)/P(fr) photoequilibrium indicated that it is pH independent in the range of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested that the phycocyanobilin chromophore deprotonates at alkaline pH in both P(r) and P(fr) with an approximate pK(a) of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both P(r) and P(fr). The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pK(a) changes in the protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome. 相似文献
19.
Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity. 相似文献
20.
Molina-Garcia AD de Pieri LA Ludlow I Waites WM de la Torre JG Harding SE 《Applied and environmental microbiology》1996,62(8):3073
Volume 62, no. 5, p. 1702, column 2, equation 3: the equation should read as follows. g(sup1)((tau)) = [g(sup2)((tau)) - 1](sup1/2) = exp[-K(sup2)(D(inf1) cos(sup2)(alpha) + D(inf2) sin(sup2)(alpha))(tau)] (3) [This corrects the article on p. 1699 in vol. 62.]. 相似文献