Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.     

Physical binding of pyrene and phenanthrene to native and denatured DNA: Measurements by spectral and coupled-column liquid chromatography methods

The physical (noncovalent) binding of pyrene and phenanthrene to calf-thymus DNA in aqueous NaCl solutions was measured by a spectral method (analysis of absorption spectra by Benesi-Hildebrand plots) and a coupled-column liquid chromatography method (equilibration of DNA solutions with solid hydrocarbon in a generator column and analysis of dissolved hydrocarbon by liquid chromatography). The measurements yielded values of an affinity constant K′ = nK, where n is the apparent number of binding sites per nucleotide and K is the apparent binding constant. The affinity of native DNA for pyrene decreases monotonically with increasing NaCl concentration, whereas the affinity of heat-denatured DNA exhibits a maximum at 0.10M NaCl.K′ for the binding of phenanthrene to native DNA is an order of magnitude lower than K′ for pyrene. The molar enthalpy for the binding of pyrene to native DNA in 10 mM NaCl is (?34.0 ± 1.0) kJ mol^?1. The spectral method data indicate that 50 is an upper limit for the average number of base pairs between intercalation sites for pyrene along the DNA helix and that only a fraction of these sites are bound at the highest binding ratios.     

Removal of surfactant solubilized polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium in a rotating biological contactor reactor

White rot fungi can oxidize surfactant solubilized polycyclic aromatic hydrocarbons (PAH). The objective of this study was to evaluate the performance of immobilized white rot fungus, Phanerochaete chrysosporium, to remove surfactant Tween 80 solubilized PAH i.e. phenanthrene, pyrene and benzo(alpha)pyrene in a rotating biological contactor (RBC) reactor. Results indicated that the immobilized P. chrysosporium in the RBC reactor system in continuous operation could effectively remove the three tested PAH at specific hydraulic loading rates and concentrations tested for each individual PAH. Batch operation of RBC reactor showed that the immobilized P. chrysosporium was stable and effective for the eight successive batch treatments of PAH in solution medium i.e. PAH removal was greater than 90% after 60 h, although only low levels of ligninolytic enzyme activity were detected. The removal of phenanthrene and pyrene in solution medium has been found to be a first order reaction in batch operation. A mass balance calculation indicated that biological oxidation was the main factor for removal of benzo(alpha)pyrene i.e. 95.7% in the RBC reactor. However, for phenanthrene and pyrene, both biological oxidation (i.e. 49 and 56%, respectively) and RBC disc foam adsorption (i.e. 44 and 34%, respectively) made a significant contribution to the removal of PAH.     

Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1)

Micellization of sodium deoxycholate (NaDC) and sodium ursodeoxycholate (NaUDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle, where sodium cholate (NaC) was used as a reference. The fluorescence probe technique of pyrene was employed to determine accurately the CMC values for the bile salts, which indicated that a certain concentration range of CMC and a stepwise aggregation for micellization were reasonable. The temperature dependences of micellization for NaDC and NaUDC were studied at 288.2, 298.2, 308.2, and 318.2 K by aqueous solubility change with solution pH. Aggregations of the bile salt anions were analyzed using the stepwise association model and found to grow in size with increasing concentration, which confirmed that the mass action model worked quite well. The average aggregation number was found to be 2.5 (NaUDC) and 10.5 (NaDC) at the concentration of 20 mM and at 308.2 K. The aggregation number determined by static light scattering also agreed well with those by the solubility method in the order of size: NaUDC相似文献   

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Covalent binding of benzo[a]pyrene to dna in fish liver

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ($\text{Parophrys vetulus}$) force fed tritiated benzo[a]pyrene. No statistically signïficant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 10⁶ nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

Enhancement of stability of 7 beta,8 alpha-dihyroxy-9 alpha epoxybenzo(a)pyrene by complex formation with serum albumin

The biologically active and chemically unstable metabolite of benzo(a)pyrene, 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxybenzo(a)pyrene (BPDE), binds to human serum albumin in aqueous solutions with an association constant of 2.6 X 10(5)M-1. At pH 7.2, 24 degrees C and in 5mM sodium cacodylate buffer solution, this binding increases the lifetime of the diol epoxide by a factor of nearly 3. It is suggested that the formation of such physical complexes with proteins having hydrophobic interiors or with lipids may provide a mechanism by which highly reactive metabolites are transported from the site of metabolic synthesis to biological target molecules (e.g., DNA), in a reactive aqueous environment.     

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

Polycyclic aromatic hydrocarbon oxidation by the white-rot fungus Bjerkandera sp. strain BOS55 in the presence of nonionic surfactants

The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures.     

Sodium hydroxide based non-detergent decellularizing solution for rat lung

Lung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.     

Mechanism and rate of permeation of cells by polycyclic aromatic hydrocarbons

The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.     

Pyrene as a Sensitive Probe for DNA Conformational Changes Due to Protonation

Abstract

Planar pyrene molecules can acquire optical activity upon binding to the disymmetric environment of duplex DNA. Positive induced Cotton effects are observed for pyrene in basic and neutral DNA solutions but revert to strongly negative CD bands in moderately acidic solutions. pH titrations indicate that this change over is strongly cooperative until acid denaturation sets in. The negative induced CD for pyrene results from its binding to a protonated duple.x state which shows a striking 40°C decrease in the melting temperature from its neutral counterpart with 0.18 M NaCI. Further studies with synthetic polynucleotidesreveal that pyrene strongly prefers the guanine containing sequences in general and dG-dC (and/or dC-dG) sequence(s) in particular for this protonated duplex state, in distinct contrast to the dA-dT (and/or dT-dA) sequence specificity in the neutral solutions. The observed pyrene CD sign reversal upon protonation is thus the manifestation of DNA conformational change and the accompanied alteration in base sequence specificity.     

Solubilization of cholesterol and polycyclic aromatic compounds into sodium bile salt micelles (part 2)

The aqueous solubility of cholesterol was determined over the temperature range from 288.2 to 318.2 K with intervals of 5 K by the enzymatic method. The solubility was (3.7+/-0.3)x10(-8) mol dm(-3) (average +/- S.D.) at 308.2 K. The maximum additive concentrations of cholesterol into the aqueous micellar solutions of sodium deoxycholate (NaDC), sodium ursodeoxycholate (NaUDC), and sodium cholate (NaC) were spectrophotometrically determined at different temperatures. The cholesterol solubility increased in the order of NaUDC相似文献   

Quantification of sodium dodecyl sulfate in microliter biochemical samples by gas chromatography

A novel gas chromatography (GC) method has been developed to accurately quantitate sodium dodecyl sulfate (SDS) in aqueous biochemical samples. This method is based on the quantitative conversion of SDS to 1-dodecanol in the GC injection port at elevated temperature, and the thermal degradation product 1-dodecanol was analyzed to determine SDS concentration. It was found that the addition of guanidinium chloride (GnHCl) to SDS samples (via direct dilution with GnHCl/MeOH solution) is necessary to ensure accurate quantitation. The presence of GnHCl enables quantitative conversion of SDS to 1-dodecanol, improves sensitivity, and virtually eliminates interference from proteins and other chemicals commonly present in biochemical samples. The method features direct analysis of diluted SDS samples, is free from interference, and is capable of quantifying less than 1 ng SDS in biochemical samples. It is also suitable for samples with limited volume, with as little as 1 microl sample being sufficient for quantitation.     

Enhanced solubility of proteins and peptides in nonpolar solvents through hydrophobic ion pairing

A new technique for enhancing the solubility of peptides and proteins in organic solvents is described. Complexation of polypeptides with stoichiometric amounts of an anionic detergent, such as sodium dodecyl sulfate (SDS), produces diminished aqueous solubility, but enhanced solubility in organic solvents. Consequently, the partitioning of a polypeptide into a nonpolar solvent can be increased by two to four orders of magnitude. In the case of an insulin–SDS complex, the solubility in 1-octanol is more than tenfold greater than in water. In 1-octanol, the native structure of insulin remains intact, as determined by CD spectroscopy, and the thermal denaturation temperature (T_m) is increased by approximately 50°C relative to unfolding in water. Finally, peptides and proteins can be extracted back into an aqueous phase provided the chloride concentration is sufficient to displace bound detergent molecules. © 1993 John Wiley & Sons, Inc.     

Microstructures formed in aqueous solutions of a hydrophobically modified nonionic cellulose derivative and sodium dodecyl sulfate: a fluorescence probe investigation

The association behavior of hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC) and its interaction with the anionic surfactant sodium dodecyl sulfate (SDS) has been studied in the dilute concentration regime. Steady-state fluorescence probe techniques have been utilized to obtain microstructural information of the system properties and combined with macroscopic bulk information from equilibrium dialysis experiments in order to determine binding isotherms of SDS to HM-EHEC. HM-EHEC was found to self-associate and form polymeric micelles in semi-dilute aqueous solutions. c^* for the self-association process was determined to be approximately 0.4%. The microviscosity of the polymeric micelles is much higher, and the micropolarity slightly higher, than that of ordinary SDS micelles. The onset of interaction between HM-EHEC and SDS was evidenced by a simultaneous strong increase in microviscosity and decrease in micropolarity upon successive addition of SDS. There is a minor, noncooperative SDS binding to the HM-EHEC starting from low concentrations of SDS (<5 mM) followed by a highly cooperative binding region at SDS concentrations ≥5 mM. The polymer–surfactant aggregates are rigid and hydrophobic with a maximum in microviscosity in the noncooperative binding region at a very low degree of SDS-adsorption.     

Functional robustness of a polycyclic aromatic hydrocarbon metabolic network examined in a nidA aromatic ring-hydroxylating oxygenase mutant of Mycobacterium vanbaalenii PYR-1

In this study, we obtained over 4,000 transposon mutants of Mycobacterium vanbaalenii PYR-1 and analyzed one of the mutants, 8F7, which appeared to lose its ability to degrade pyrene while still being able to degrade fluoranthene. This mutant was identified to be defective in nidA, encoding an aromatic ring-hydroxylating oxygenase (RHO), known to be involved in the initial oxidation step of pyrene degradation. When cultured with pyrene as a sole source of polycyclic aromatic hydrocarbon (PAH), high-pressure liquid chromatography analysis revealed that the nidA mutant showed a significant decrease in the rate of pyrene degradation compared to the wild-type PYR-1, although pyrene was still being degraded. However, when incubated with PAH mixtures including pyrene, phenanthrene, and fluoranthene, the pyrene degradation rate of the mutant was higher than that of the mutant previously incubated with pyrene as a sole source of PAH. There was no significant difference between wild-type PYR-1 and the mutant in the rates of phenanthrene and fluoranthene degradation. From the whole-cell proteome analysis of mutant 8F7 induced by pyrene, we identified expression of a number of RHO enzymes which are suspected to be responsible for pyrene degradation in the nidA mutant, which had no expression of NidA. Taken together, results in this study provide direct evidence for the in vivo functional role of nidA in pyrene degradation at the level of the ring-cleavage-process (RCP) functional module but also for the robustness of the PAH metabolic network (MN) to such a genetic perturbation.