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1.
M. Ollmann A. Robitzki G. Schwarzmann H.-J. Galla 《European biophysics journal : EBJ》1988,16(2):109-112
We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.Abbreviations DPPC
1-2 dipalmitoyl-sn-glycero-3-phosphocholine
- DSPC
1-2 distearoyl-sn-glycero-3-phosphocholine
- PyPC
1-acyl-2-[10(-1-pyrene)decanoyl]-sn-glycero-3-phosphocholine
- PyGM1
N-12-(1-pyrene)dodecanoyl-lyso GM1
- PyGM2
N-12-(1-pyrene)dodecanoyl-lyso GM2
- PyGM3
N-12-(1-pyrene) dodecanoyl-lyso GM3
- IM
fluorescence intensity of the monomeric pyrene probe
- ID
fluorescence intensity of the excimer 相似文献
2.
Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. 总被引:19,自引:4,他引:15
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H S Rye S Yue D E Wemmer M A Quesada R P Haugland R A Mathies A N Glazer 《Nucleic acids research》1992,20(11):2803-2812
The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide. 相似文献
3.
The methylotrophic yeast Hansenula polymorpha CBS4732 leu2 detoxifies electrophilic xenobiotics by glutathione (GSH)-dependent accumulation in vacuoles, as shown by fluorescence microscopy. GSH-dependent and GSH-independent export of xenobiotic derivatives were also demonstrated by high-performance liquid chromatography (HPLC). Conjugates of GSH and N-acetylcysteine with monobromobimane and N-[1-pyrene]maleimide were observed among the HPLC fractions, along with unidentified derivatives. 相似文献
4.
Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications 总被引:13,自引:3,他引:10
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The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure–property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above ~0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA) · poly(dT) and poly(dG) · poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least ~11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+. These structure–property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR. 相似文献
5.
Chowdhury N Dutta S Dasgupta S Singh ND Baidya M Ghosh SK 《Photochemical & photobiological sciences》2012,11(7):1239-1250
A new series of (E)-pyrene oxime ester conjugates of carboxylic acids including amino acids were synthesized by coupling with an environment sensitive fluorophore 1-acetylpyrene. (E)-Pyrene oxime esters exhibited strong fluorescence properties and interestingly their fluorescence properties were found to be highly sensitive to the surrounding environment. Direct irradiation of the (E)-pyrene oxime esters by UV light (≥350 nm) resulted in both the photo-Beckmann rearrangement product and products resulting from N-O bond homolysis. Photoproduct formation and their distribution were found to be solvent dependent. Further, we also showed (E)-pyrene oxime esters intercalated into DNA efficiently and photo-cleaved DNA. Finally we also showed these oxime esters can permeate cells efficiently and may cause cytotoxicity upon irradiation of light. 相似文献
6.
W B Mattes 《Nucleic acids research》1990,18(13):3723-3730
Various kinds of DNA damage block the 3' to 5' exonuclease action of both E. coli exonuclease III and T4 DNA polymerase. This study shows that a variety of DNA damage likewise inhibits DNA digestion by lambda exonuclease, a 5' to 3' exonuclease. The processive degradation of DNA by the enzyme is blocked if the substrate DNA is treated with ultraviolet irradiation, anthramycin, distamycin, or benzo[a]-pyrene diol epoxide. Furthermore, as with the 3' to 5' exonucleases, the enzyme stops at discrete sites which are different for different DNA damaging agents. On the other hand, digestion of treated DNA by lambda exonuclease is only transiently inhibited at guanine residues alkylated with the acridine mustard ICR-170. The enzyme does not bypass benzo[a]-pyrene diol epoxide or anthramycin lesions even after extensive incubation. While both benzo[a]-pyrene diol epoxide and ICR-170 alkylate the guanine N-7 position, only benzo[a]-pyrene diol epoxide also reacts with the guanine N-2 position in the minor groove of DNA. Anthramycin and distamycin bind exclusively to sites in the minor groove of DNA. Thus lambda exonuclease may be particularly sensitive to obstructions in the minor groove of DNA; alternatively, the enzyme may be blocked by some local helix distortion caused by these adducts, but not by alkylation at guanine N-7 sites. 相似文献
7.
J Jacob G Grimmer A Schmoldt 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(11):1525-1534
The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed. 相似文献
8.
The distance between specific sites on bacterial luciferase was estimated by energy transfer. Luciferase was fluorescently labeled by reaction of an essential sulfhydryl group with N-(1-pyrene)maleimide and N-[p-(2-benzoxazolyl)phenyl]meleimide. Both of the modified enzymes bind 8-anilino-1-naphthalenesulfonate (Ans) with affinities similar to that exhibited by the native luciferase. Using each of the two fluorescent probes as a donor and the bound Ans as an acceptor, the energy transfer efficiencies were determined by the resulting enhancement of fluorescence of the acceptor. The corresponding distance was calculated to be in the range of 21 to 37 A. Energy-transfer studies were also carried out using fluorescence lifetime measurements of bound ANS, acting as a donor with bound FMN as an acceptor. The corresponding distance was calculated to be between 30 and 58 A. Using samples of luciferase:Ans complex and luciferase modified with N-(1-pyrene)maleimide, the rotational correlation time of the enzyme-dye conjugate as awhole was found to be 47 +/- 2 ns. The observed rotational correlation time is much longer than that calculated for luciferase assuming a spherical structure, thus indicating an elongated form for the luciferase-dye conjugate. 相似文献
9.
Incubation of benzo[alpha] pyrene 4,5-oxide with poly(G) in neutral aqueous ethanol resulted in the formation of covalent adducts and in the production of free 4-hydroxybenzo[alpha]pyrene. This phenol, which was identified by its UV spectral properties and by its chromatographic characteristics, was also formed but at a much slower rate when the epoxide was incubated with DNA or with GMP. Phenol formation was not detected when benzo[alpha]-pyrene 4,5-oxide was incubated for prolonged periods in the presence of poly(A), poly(C) or poly(U) or in the absence of nucleic acid. Formation of 4-hydroxybenzo[alpha] pyrene from the epoxide in the presence of poly(G) was not accompanied by detectable base modifications or by breakage of phosphodiester linkages. 相似文献
10.
M Abramovich A S Prakash R G Harvey I S Zegar P R Lebreton 《Chemico-biological interactions》1985,55(1-2):39-62
The reversible DNA physical binding of a series of non-reactive metabolites and metabolite model compounds derived from benzo[a]pyrene (BP) has been examined in UV absorption and in fluorescence emission and fluorescence lifetime studies. Members of this series have steric and pi electronic properties similar to the highly carcinogenic metabolite trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and the less potent metabolite 4,5-epoxy-4,5-dihydrobenzo(a)pyrene (4,5-BPE). The molecules examined are trans-7,8-dihydroxy-7,8-dihydrobenzo[a]-pyrene (7,8-di(OH)H2BP), 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (tetrol) 7,8,9,10-tetrahydrobenzo[a]pyrene (7,8,9,10-H4BP), pyrene, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (4,5-di(OH)H2BP) and 4,5-dihydrobenzo[a]pyrene (4,5-H2BP). In 15% methanol at 23 degrees C the intercalation binding constants of the molecules studied lie in the range 0.79-6.1 X 10(3) M-1. Of all the molecules examined the proximate carcinogen 7,8-di(OH)-H2BP is the best intercalating agent. The proximate carcinogen has a binding constant which in UV absorption studies is found to be 2.8-6.0 times greater than that of the other hydroxylated metabolites. Intercalation is the major mode of binding for 7,8-di(OH)H2BP and accounts for more than 95% of the total binding. Details concerning the specific role of physical bonding in BP carcinogenesis remain to be elucidated. However, the present studies demonstrate that the reversible binding constants for BP metabolites are of the same magnitude as reversible binding constants which arise from naturally occurring base-base hydrogen bonding and pi stacking interactions in DNA. Furthermore, previous autoradiographic studies indicate that in human skin fibroblasts incubated in BP, pooling of the unmetabolized hydrocarbons occurs at the nucleus. The high affinity of 7,8-di(OH)H2BP for DNA may play a role in similarly elevating in vivo nuclear concentrations of the non-reactive proximate carcinogen. 相似文献
11.
Cypermethrin, a synthetic pyrethroid insecticide is shown to exert carcinogenic effects in rodents; however, its underlying mechanism remains elusive. Here, we showed the effect of cypermethrin on protein expression involved in neoplastic transformation in mouse skin. Comparative protein expression profiles between untreated control and cypermethrin-treated mouse skin were explored using 2-DE. A total of 27 spots that were statistically significant (p<0.05) and differentially expressed in response to cypermethrin exposure were identified by MALDI-TOF/TOF and LC-MS/MS. Among them, six up-regulated proteins (carbonic anhydrase 3 (Ca 3), Hsp-27, S100A6, galectin-7, S100A9, S100A11) and one down-regulated protein (superoxide dismutase [Cu-Zn] (Sod 1)) are associated with cancer-related key processes. These selected dysregulated proteins were further validated in 2-DE gels of mouse skin treated with known tumorigens (benzo-[a]-pyrene, 12-O-tetradecanoyl-phorbol-13-acetate and mezerein), respectively. Comparative studies showed that Ca 3, S100A6, S100A9, S100A11 and Sod 1 are specific for stages of development and progression of tumors whereas Hsp-27 and galectin-7 are specific for tumor promotion stage by cypermethrin in mouse skin. Furthermore, these chosen proteins confirmed by Western blotting and immunofluorescence staining were consistent with changes in 2-DE check. This proteomic investigation for the first time provides key proteins that will contribute in understanding the mechanism behind cypermethrin-induced neoplastic transformation. 相似文献
12.
The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction. 相似文献
13.
A comparative study of the photoluminescence properties of three kinds of tetra-ring aromatic hydrocarbon (1-sodium pyrenesulphonate, benz[alpha]anthracene and chrysene) solution in the absence of any protecting medium is described. It was found that a room temperature phosphorescence signal with different intensities can be induced for these solutions, using only TlNO3 or KI as a heavy atom perturber (HAP) and Na2SO3 as a deoxygenator. An appropriate amount of organic solvent added to the systems of pyrene, benz[alpha]anthracene and chrysene is necessary for increasing the solubility and phosphorescence intensity, and the preferable solvent is acetonitrile. For the pyrene, pyrenesulphonate and chrysene systems, a delayed excimer fluorescence accompanied with the room temperature phosphorescence (RTP) emission can be observed, but that for benz[alpha]anthracene cannot. The ratio of delayed excimer fluorescence and phosphorescence signals for pyrene, pyrenesulphonate and chrysene systems can be controlled by adjusting the concentration of luminophor, kinds and amount of both organic solvents and HAP. Under the optimal conditions, the RTP signals are proportional to the concentration of the four aromatic hydrocarbons, which means that the RTP properties of the four tetra-ring aromatic hydrocarbons can be used for quantitative analysis. 相似文献
14.
The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils 总被引:5,自引:0,他引:5
The effects of several bioremediation stimulants, including potentialmetabolism pathway inducers, inorganic/organic nutrients, and surfactants onthe metabolism of phenanthrene and pyrene, as well as the populationdynamics of PAH degrading microorganisms was examined in five soils withdiffering background PAH concentrations, exposure histories and physicalproperties. Most of the supplements either had no significant effect ordecreased the mineralization of [14C]-phenanthrene and[14C]-pyrene in soil slurry microcosms. The effect of aparticular supplement, however, was often not uniform within or acrosssoils. Decreased mineralization of [14C]-phenanthrene and[14C]-pyrene was usually due to either preferential use of thesupplement as carbon source and/or stimulation of non-PAH degradingmicroorganisms. Many of the supplements increased populations ofheterotrophic microorganisms, as measured by plate counts, but did notincrease populations of phenanthrene degrading microorganisms, as measuredby the [14C]-PAH mineralization MPN analysis or cellularincorporation of [14C]-PAH. These results suggest that the PAHdegrading community at each site may be unique in their response tomaterials added in an attempt to stimulate PAH degradation. Thecharacteristics of the site, including exposure history, soil type, andtemporal variation may all influence their response. 相似文献
15.
Jolanta Obniska Hanna Byrtus Krzysztof Kamiński Maciej Pawłowski Małgorzata Szczesio Janina Karolak-Wojciechowska 《Bioorganic & medicinal chemistry》2010,18(16):6134-6142
The synthesis and anticonvulsant properties of new N-Mannich bases of [7,8-f]benzo-2-aza-spiro[4.5]decane-1,3-diones (5a–h) and [7,8-f]benzo-1,3-diaza-spiro[4.5]decane-2,4-diones (7a–h) were described. Initial anticonvulsant screening was performed using intraperitoneal (ip) maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizures tests. The neurotoxicity was determined applying the rotarod test. The majority of compounds were effective in the MES or/and scPTZ screen. The quantitative studies showed that several molecules were more potent than phenytoin, used as reference drug. Selected derivatives were screened in the 6-Hz test and also assessed for potential activity against nerve agents using the Pilocarpine Induced Status Prevention model. To explain the possible mechanism of anticonvulsant action, for chosen active derivatives, their influence on voltage-dependent Na+ channel were tested in vitro. 相似文献
16.
Effects of the state of the succinimido-ring on the fluorescence and structural properties of pyrene maleimide-labeled alpha alpha-tropomyosin. 总被引:1,自引:0,他引:1
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Rabbit skeletal alpha alpha-tropomyosin was labeled at Cys-190 with pyrene maleimide to form (S-[N-(1-pyrene)succinimido])2-tropomyosin (pyreneI-Tm). The product with cleaved succinimido-rings, pyreneII-Tm was also prepared by incubation of pyreneI-Tm at pH greater than 7.5. The pH dependence of the rate of cleavage indicated that hydrolysis rather than aminolysis was the more likely reaction. PyreneI-Tm exhibited a loss in helix content and end-to-end polymerization compared with unlabeled Tm, which increased upon formation of pyreneII-Tm. The cleavage resulted in increased interchain excited state excimer fluorescence originating from pyrene-pyrene interaction between the chains. Thus, increased pyrene-pyrene interaction at Cys 190 leads to an increase in unfolding, the effects of which appear to be transmitted to the ends of tropomyosin. The fluorescence properties of the two types of pyrene-succinimide adducts of dithiothreitol were very similar to the corresponding adducts of pyrene-Tm indicating excimer formation through ground state pyrene-pyrene interaction. 相似文献
17.
The mutagenicity of nitrated benzo[a]pyrene (BP) and the related compounds, 1- and 3-nitrobenzo[a]pyrene (NBP), 1- and 3-nitro-6-cyanobenzo[a]pyrene (N-6-CBP), 1- and 3-nitro-6-azabenzo[a]-pyrene (N-6-ABP), 1- and 3-nitro-6-azabenzo[a]-pyrene-N-oxide (N-6-ABPO) and 1,6- and 3,6-dinitrobenzo[a]-pyrene (DNBP), was investigated. The mutagenic activities of 3-N-6-CBP and 3-N-6-ABP were 117 and 76 times, respectively, that of 3-NBP. In addition, 3,6-DNBP was more mutagenic than 1,6-DNBP. It is suggested that the mutagenic activation differs with the position of NO2 substitution in the chemical structure. A nitro derivative with NO2 substitution at the 3 position of the aromatic ring of BP was more mutagenic than that with the substitution at the 1 or 6 position. The reducibility of DNBPs was then determined by detecting 1- or 3-amino-6-nitrobenzo[a]pyrene (A-6-NBP), a metabolite of DNBP; 3,6- and 1,6-DNBP were reduced to 3- and 1-A-6-NBP at frequencies of 958 +/- 26 and 79 +/- 8, respectively, pmole per mg of protein, when the compound was incubated anaerobically with rat liver S9 mix at 37 degrees C for 15 min. NO2 substituted at the 3 position of the aromatic ring of BP was readily reduced by a microsome enzyme to form an amino derivative. The result suggests that these compounds have a structure-activity relationship between mutagenicity and NO2 substitution of BP. 相似文献
18.
It is possible to assay for trans-7,8-dihydroxy 7,8-dihydrobenzo[a]-pyrene (BP-7,8-dihydrodiol) in complex metabolite mixtures produced during microsomal metabolism of benzo[a]pyrene (BP) because only the BP-7,8-dihydrodiol metabolite will produce significant chemiluminescence (CL) in the NaOCl-H2O2 singlet oxygen-generating system. The limiting CL sensitivity is 30 pmol in a 1-ml CL reaction mixture. CL assays for BP-7,8-dihydrodiol in microsomal reaction solutions gave concentrations identical with those determined by calibrated high-performance liquid chromatography. 相似文献
19.
Ana Gradillas Carmen del Campo José Vicente Sinisterra Emilio F. Llama 《Biotechnology letters》1996,18(1):85-90
Summary The reaction yield of the esterification of (R,S) ibuprofen with n-propanol, catalyzed by physically adsorbed Candida antarctica lipase B on anionic resin, is improved by the addition of benzo-[18]-crown-6 or meso-tetraphenylporphyrin but reduced by the presence of metal-porphyrins. The interaction of benzo-[18]-crown-6 or meso-tetraphenyl porphyrin with the lid of pure lipase B on C. antarctica, would produce the activation of the lipase increasing the reaction rate but not the enantioselectivity. 相似文献
20.
Rabbit skeletal alphaalphatropomyosin was specificially labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-alphaalphaTm) or pH 7.5 (PyrII-alphaalphaTm). PyrII-alphaalphaTm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-alphaalphaTm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the--SH groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-alphaalphaTm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential instability in the molecule near cysteine 190. This work suggests that N-(1-pyrene)maleimide could be used to probe both--SH proximity and local conformation in any protein if the presence of two or more proximal--SH groups is suspected. 相似文献