Spectroscopic and equilibrium dialysis studies of the binding of acridine and 10-methylacridine to DNA in the pH range 4–10

For a better understanding of the interactions between DNA and various acridine dyes, the binding of acridine (Acr) and 10-methylacridine (MeAcr) to native and heat-denatured calf-thymus DNA was studied in the pH range between 4 and 10 by the equilibrium dialysis and spectroscopic methods. The binding between DNA and the dyes was predominantly electrostatic. The amount of bound Acr varied with pH, mixing ratio (P/D), and the DNA conformation, and reached a maximum at pH = 5.2. The amount of bound MeAcr was constant in the pH range 5–9. The apparent binding constants of these dyes were obtained at some pH, and they were found to vary with P/D for native DNA-dye complexes. The pure spectra of bound Acr and MeAcr could be unmasked. The bound Spectra were bathochromic and hypochromic relative to the spectra of free days. Acridine bound to native DNA was shown to undergo structural changes from an acridiniumlike to a neutral acridinelike form as the pH of solutions was varied. The pK value for the transition between the bound forms was evaluated to be 7.3. The extrinsic Cotton effects of the bound dyes were observed in the DNA-Acr and-MeAcr complexes and varied with pH and the conformation of DNA.     

Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.     

Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining.

A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes

The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.     

Decolorization of azo and triphenylmethane dyes by Pycnoporus sanguineus producing laccase as the sole phenoloxidase

Partial decolorization of two azo dyes (orange G and amaranth) and complete decolorization of two triphenylmethane dyes (bromophenol blue and malachite green) was achieved by cultures in submerged liquid culture producing laccase as the sole phenoloxidase. Enzyme production could be correlated with dye decolorization, with sorption of dye to mycelia accounting for less than 3% of dye removal.     

Feulgen type staining of mammalian tissues with Dahlia-SO2.

The use of the basic dye, Dahlia, which belongs to triphenylmethane group but without a primary amino group in its molecule has been described as useful in the staining of aldehyde groups of acid hydrolysed DNA in tissue sections following the conventional Feulgen procedure. Dahlia-SO2 prepared with sodium hydrosulphite is highly suitable when used at pH 4-0 to 5-0. The absorption characteristics of the stained nuclei indicate on the peak of maximum absorption at 560 nm, whereas, that of the aqueous dye solution is at 590 nm.     

染料的生物降解研究

生物降解法是染料污染治理的重要方法。针对目前使用量较大的偶氮染料、二苯基甲烷染料和蒽醌染料这3大类染料,重点介绍了厌氧和好氧条件下的偶氮还原及其机理、三苯基甲烷染料降解菌和蒽醌染料降解菌的研究进展。     

Antimicrobial dyes and mechanosensitive channels

The search for new and effective antimicrobial agents has never been as important; however, since the discovery of antibiotics, exploring the antimicrobial activity of dyes has been forgotten. Antimicrobial dyes are an untapped resource and have the ability to potentially combat the spread of drug-resistant bacteria either alone or as antimicrobial adjuvants. The mechanosensitive ion channel of large conductance (MscL) is highly conserved and ubiquitous in bacterial species. There is evidence to suggest that at least one triphenylmethane dye acts through the highly conserved MscL channel and combining the two approaches of exploring the mechanism of action of other triphenylmethane dyes or antimicrobial dyes in general and the novel MscL target provides a new opportunity for further exploration.     

Structural insight into bioremediation of triphenylmethane dyes by Citrobacter sp. triphenylmethane reductase

Triphenylmethane dyes are aromatic xenobiotic compounds that are widely considered to be one of the main culprits of environmental pollution. Triphenylmethane reductase (TMR) from Citrobacter sp. strain KCTC 18061P was initially isolated and biochemically characterized as an enzyme that catalyzes the reduction of triphenylmethane dyes. Information from the primary amino acid sequence suggests that TMR is a dinucleotide-binding motif-containing enzyme; however, no other functional clues can be derived from sequence analysis. We present the crystal structure of TMR in complex with NADP+ at 2.0-angstroms resolution. Despite limited sequence similarity, the enzyme shows remarkable structural similarity to short-chain dehydrogenase/reductase (SDR) family proteins. Functional assignments revealed that TMR has features of both classic and extended SDR family members and does not contain a conserved active site. Thus, it constitutes a novel class of SDR family proteins. On the basis of simulated molecular docking using the substrate malachite green and the TMR/NADP+ crystal structure, together with site-directed mutagenesis, we have elucidated a potential molecular mechanism for triphenylmethane dye reduction.     

Interactions of heteroaromatic compounds with nucleic acids. A - T-specific non-intercalating DNA ligands.

In the present paper we report the results of a study on the base specificity and affinity of eight dyes potentially able to interact with DNA. These compounds include four triphenylmethane dyes used in histochemistry, auramine, "Hoechst 33258" and two acridines substituted with t-butyl groups. They were selected with regard to their inability to intercalate between the base pairs of helical polynucleotides due to structural limitations. Hydrodynamic studies performed with the DNA complexes of crystal violet and Hoechst 33258 confirmed our assumptions that compounds of this type bind to the outside of DNA. The main results from DNA binding studies indicate that the triphenylmethane dyes except p-fuchsin are bound with high preference to two adjacent A - T pairs while Hoechst 33258 seems to need three A - T pairs as the binding site. Model studies with synthetic polynucleotides revealed that not only a sequence of A - T pairs, but also their structural arrangement in a helix, is crucial for the high affinities observed for most of the ligands when interacting with natural DNA. Methyl green and Hoechst 33258 can be used for increasing the resolution power of cesium chloride density gradients for DNAs with different (A + T) content.     

Constituents of Plant Leaf Wax Contained in Rice Callus Tissues

Two enzyme preparations having both nuclease and 3′-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography.

The enzyme activities for RNA, native DNA, heat-denatured DNA and 3′-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymes were shown to be endonucleases (EC 3.1.30.2) which liberated 5′-mononucleotides and oligonucleotides from both RNA and DNA with the following relative rate of hydrolysis: RNA > native DNA = heat-denatured DNA.     

Extractive biodecolorization of triphenylmethane dyes in cloud point system by Aeromonas hydrophila DN322p

The biological treatment of triphenylmethane dyes is an important issue. Most microbes have limited practical application because they cannot completely detoxicate these dyes. In this study, the extractive biodecolorization of triphenylmethane dyes by Aeromonas hydrophila DN322p was carried out by introducing the cloud point system. The cloud point system is composed of a mixture of nonionic surfactants (20 g/L) Brij 30 and Tergitol TMN-3 in equal proportions. After the decolorization of crystal violet, a higher wet cell weight was obtained in the cloud point system than that of the control system. Based on the results of thin-layer chromatography, the residual crystal violet and its decolorized product, leuco crystal violet, preferred to partition into the coacervate phase. Therefore, the detoxification of the dilute phase was achieved, which indicated that the dilute phase could be discharged without causing dye pollution. The extractive biodecolorization of three other triphenylmethane dyes was also examined in this system. The decolorization of malachite green and brilliant green was similar to that of crystal violet. Only ethyl violet achieved a poor decolorization rate because DN322p decolorized it via adsorption but did not convert it into its leuco form. This study provides potential application of biological treatment in triphenylmethane dye wastewater.     

Cultural Separation of Bacteria on the Basis of Triphenyl Methane Co-Efficients

The general parallelism between the Gram reaction and normal selective bacteriostasis by the triphenylmethane dyes is well established, as is also the existence of a small number of organisms in each group which do not follow the rule. Reverse extrinsic bacteriostasis has been demonstrated, but only within a very limited field. The discovery of substances possessing reverse selective power, comparable in extent to normal selective power, would be of value. In the absence of such substances, the slight quantitative differences in the behavior toward dyes, of organisms belonging to the same Gram group, may be turned to account. The authors have determined with great accuracy the crystal violet coefficients of five Gram-positive and five Gram-negative organisms. These observations show that all known aerobic organisms could probably, on the basis of their triphenylmethane coefficients, be placed on a curve, which would on the whole, parallel the Gram reaction. The possibilities of separating Gram-negative organisms from Gram-positives by means of the dyes are well understood. The authors cite experiments to show that similar separations may be made within the Gram groups by making use of the quantitative differences in triphenylmethane coefficients.     

Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

毛木耳对孔雀石绿染料降解条件的优化

随着我国印染工业的发展,废水对生态环境的危害日趋严重,亟需开发一种脱色明显且成本低廉的降解方法。本研究发现毛木耳Auricularia cornea菌株AC5对不同结构的染料均具有一定的降解作用,尤其是三苯甲烷类染料。利用26℃、160r/min振荡培养7d的粗酶液对染料（75.0mg/L）进行12h降解,结果显示三苯甲烷染料孔雀石绿、结晶紫,蒽醌染料活性蓝19和偶氮染料活性蓝222的降解效率分别为83.27%、71.77%、67.81%和63.92%。染料降解实验和酶活力测定结果表明,毛木耳对孔雀石绿的降解率达到最高时漆酶活性最高,为321.0U/mL,木质素过氧化物酶和锰过氧化物酶活性较低。因此,推测在降解过程中漆酶起到主要作用。研究表明利用毛木耳菌丝发酵液降解染料废水成本低且操作方便,为染料废水的降解研究提供了前期基础。     

The interaction of methylene blue,azure B,and thionine with DNA: Formation of complexes with polynucleotides and mononucleotides as model systems

The interactions of methylene blue, azure B, and thionine with calf thymus DNA, [poly (dG-dC)]₂, [poly(dA-dT)]₂, and the constituent mononucleotides 2′-deoxyguanosine-5′-monophosphate(dGMP), 2′-deoxyadenosine-5′-monophosphate(dAMP), 2′-deoxycytidine-5′-monophosphate(dCMP), and thymidine-5′-monophosphate(dTMP) have been studied by steady-state absorption spectroscopy and with equilibrium dialysis. Scatchard plots for binding of the dyes to the nucleic acid polymers were convex downward at low binding ratios, characteristic of intercalation, and binding constants for this mode were calculated under conditions of varying ionic strength. For each of the dyes, binding constants with [poly(dG-dC)]₂ and [poly(dA-dT)]₂ were of the same order of magnitude, so that previously reported (G-C) preferentially is not very marked. At high binding ratios, the Scatchard plots did not return to the abscissa but curved upward, indicative of a weaker cooperative binding mode, occurring under conditions where the dye is in excess, which is suggested to be external stacking of the dye molecules promoted by the polyanion. The dependence of the absorption spectra on added salt demonstrated a shift in the strong binding mode for the three dyes with [poly(dA-dT)]₂ with increasing ionic strength, while with [poly(dG-dC)]₂ this does not occur. The dyes were found to bind to purine but not pyrimidine mononucleotides with dGMP and dAMP, 1:1 complexes were formed initially and also 1:2 dye/nucleotide complexes with increasing nucleotide concentrations. Under low salt conditions, binding to dAMP was slightly stronger than to dGMP for the three dyes studied, while at high ionic strength, when the binding constants are significantly lower, all binding constants become very similar. Binding to mononucleotides is suggested to be primarily stabilised by π-π stacking interactions between the planar dyes and the nucleobases: for thionine and azure B there also appears to be H-bonds between the exocyclic amines and the sugar–phosphates conferring extra stability. Neither increasing the number of phosphate groups on the nucleotides nor changing from deoxyribose to ribose sugars had any significant effect on the binding constants. © 1995 John Wiley & Sons, Inc.