Extractions of pyrroloquinoline quinone from crude biological samples

The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaCl did not improve the solvent extraction. PQQ, which had been extracted into n-butanol, could be re-extracted into an aqueous phase by addition of either n-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of 1 micrograms PQQ, which had been added to 1 g human liver, brain and 1 ml plasma and had undergone the n-butanol and the Sep-Pak extractions, was 50, 75 and 105%, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 micrograms/g or ml, respectively, if present.     

Solvent effects on fluorescence properties of protochlorophyll and its derivatives with various porphyrin side chains

Fluorescence spectra and fluorescence lifetimes of protochlorophyll (Pchl) were measured in organic solvents having different physical and chemical properties and were analyzed taking into account the nonspecific (dependent on bulk solvent parameters), and specific (e.g. H bonds, Mg coordination) solvent–solute interactions. The energy of the fluorescence emission band decreased, while the Stokes shift increased for increasing solvent orientation polarizability, which is a function of both the dielectric constant (ε) and the refractive index (n). The extent of the dependence of the Stokes shift on solvent orientation polarizability was higher in protic (i.e. those able to form hydrogen-binding) than in aprotic solvents. High value of the Stokes shift was also observed in pyridine and methanol, i.e. in solvents hexacoordinating the central Mg atom. The fluorescence decay of Pchl was monoexponential in all of the investigated solvents. The fluorescence lifetime decreased for increasing solvent orientation polarizability from 5.5 ± 0.1 ns in 1,4-dioxane to 3.3 ± 0.1 ns in methanol. Longer lifetime values were observed in the case of aprotic solvents than in protic solvents. The hexacoordination of Mg had no effect on the fluorescence lifetime. The present data are discussed with respect to results found for protochlorophyllide (Pchlide) (My?liwa-Kurdziel et al. in Photochem Photobiol 79:62–67, 2004), and they indicate that the presence of phytol chain in the porphyrin ring influences the spectral properties of the whole chromophore. This is the first complex analysis comparing the fluorescence emission and fluorescence lifetimes of purified Pchl and Pchlide.     

Sequence-dependence of the energetics of opening of at basepairs in DNA

Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the energetics of opening of AT/TA basepairs in the DNA dodecamer 5'-d(GCTATAAAAGGG)-3'/5'-d(CCCTTTTATAGC)-3'. The dodecamer contains the TATA box of the adenovirus major late promoter. The equilibrium constants for opening of each basepair are measured from the dependence of the exchange rates of imino protons on ammonia concentration. The enthalpy, entropy, and free energy changes in the opening reaction of each basepair are determined from the temperature dependence of the exchange rates. The results reveal that the opening enthalpy changes encompass a wide range of values, namely, from 17 to 29 kcal/mol. The largest values are observed for the AT basepairs in 7th and 8th positions. These values, and the exchange rates of the corresponding imino protons, suggest that these two basepairs open in a single concerted reaction. The enthalpy changes for opening of the central six basepairs are correlated to the opening entropy changes. This enthalpy-entropy compensation minimizes the variations in the opening free energies among these central basepairs. Deviations from the enthalpy-entropy compensation pattern are observed for basepairs located close to the ends of the duplex structure, suggesting a different mode of opening for these basepairs.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

The effects of organic solvent/water mixtures on the structure and catalytic activity of porcine pepsin

The effects of aqueous solutions of ethanol, acetonitrile and 1,4-dioxane in the concentration range 10–90% (v/v) on the activity of porcine pepsin were studied. The enzyme retained its activity in aqueous ethanol and aqueous acetonitrile with increasing organic solvent concentration up to 60%, and in aqueous 1,4-dioxane up to 30%, but thereafter a considerable decrease in activity was observed. The changes caused in the catalytic activity by the water-miscible organic solvents may be related to structural changes, which were followed by means of intrinsic fluorescence and circular dichroism spectroscopy measurements.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii

The effect of various organic solvents on the catalytic activity, stability and substrate specificity of alchohol dehydrogenase from Haloferax volcanii (HvADH2) was evaluated. The HvADH2 showed remarkable stability and catalysed the reaction in aqueous?Corganic medium containing dimethyl sulfoxide (DMSO) and methanol (MeOH). Tetrahydrofuran and acetonitrile were also investigated and adversely affected the stability of the enzyme. High concentration of salt, essential to maintain the enzymatic activity and structural integrity of the halophilic enzyme under standard conditions may be partially replaced by DMSO and MeOH. The presence of organic solvents did not induce gross changes in substrate specificity. DMSO offered a protective effect for the stability of the enzyme at nonoptimal pHs such as 6 and 10. Salt and solvent effects on the HvADH2 conformation and folding were examined through fluorescence spectroscopy. The fluorescence findings were consistent with the activity and stability results and corroborated the denaturing properties of some solvents. The intrinsic tolerance of this enzyme to organic solvent makes it highly attractive to industry.     

A combined spectroscopic and TDDFT investigation of the solute‐solvent interactions of two coumarin derivatives

The UV/Vis absorption and fluorescence characteristics of 3‐cyano‐7‐hydroxycoumarin [ CHC ] and 7‐amino‐4‐methyl‐3‐coumarinylacetic acid [ AMCA‐H ] were studied at room temperature in several neat solvents and binary solvent mixtures of 1,4‐dioxane/acetonitrile. The effects of solvent on the spectral properties are analyzed using single and multi‐parameter solvent polarity scales. Both general solute/solvent interactions and hydrogen bond interactions are operative in these systems. The solvation of CHC and AMCA‐H dyes in 1,4‐dioxane/acetonitrile solvent mixtures has been studied. The solutes CHC and AMCA‐H are preferentially solvated by acetonitrile and a synergistic effect is observed for both molecules in dioxane/acetonitrile solvent mixtures. In addition, using the solvatochromic method the ground‐ and the excited‐state dipole moments of both the dyes were calculated. The ground‐ and excited‐state dipole moments, absorption and emission maxima and HOMO–LUMO gap were also estimated theoretically using B3LYP/6–311+ G (d,p) level of theory in the gaseous phase, dioxane and acetonitrile solvents. Furthermore, changes in dipole moment values were also calculated using the variation of Stokes shift with the molecular–microscopic empirical solvent polarity parameter ( ). The observed excited‐state dipole moments are larger than their ground‐state counterparts, indicating a substantial redistribution of the electron densities in a more dipolar excited state for both coumarins investigated.     

SER-HIS-ASP catalytic triad in model non-aqueous solvent environment: a computational study

Emerging new properties and applications of enzymes in organic solvents and ionic liquids are unabating. By applying a combined Quantum Mechanics/Continuum Mechanics computation on a prototypical catalytic triad serine-histidine-aspartate (SER-HIS-ASP) interacting with ethanol or acetonitrile molecules, the major difference between protic and aprotic solvents in effecting transition-state stabilization has been analyzed. Moderately polar aprotic solvent acetonitrile is predicted to be unable to stabilize the transition state in replacing the role of the oxyanion-hole environment, whereas protic ethanol solvent molecules of similar polarity to acetonitrile are adequate in re-gaining the enzymatic activities.     

Effect of water activity on enzyme hydration and enzyme reaction rate in organic solvents

The effects of water on enzyme (protein) hydration and catalytic efficiency of enzyme molecules in organic solvents have been analyzed in terms of the thermodynamic activity of water, which has been estimated by the NRTL or UNIFAC equations. When the amount of water bound to the enzyme was plotted as a function of water activity, the water adsorption isotherms obtained from the water-solvent liquid mixtures were similar to the reported water-vapor adsorption isotherms of proteins. The water adsorption of proteins from the organic media was not significantly dependent on the properties of the solvents or the nature of the proteins. It is also shown that there is a linear relationship between the logarithm of the enzyme reaction rate and water activity. However, the dependence of the enzyme reaction rate on water activity was found to be different depending on the properties of the solvent. The relationship between water activity and other solvent parameters such as solvent hydrophobicity and the solubility of water in the solvent is also discussed.     

Photophysical properties of dibenzofluorescein and the presence of its tautomers or prototropic forms in organic solvents.

The UV/Vis absorption and fluorescence properties of dibenzofluorescein (DBFL) in organic solvents were measured and used to shed light on the possible presence of its tautomers or various prototropic forms. DBFL in aprotic solvents mainly exists in two tautomeric forms, viz. quinoid and lactone, but neither are efficiently fluorescent. In protic solvents, such as methanol and ethanol, both the monoanion and neutral quinoid are present and showed the highest fluorescence quantum yield. In contrast, DBFL is fully dissociated to the monoanion and dianion in deionized water.     

Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents.

Calorimetric heat effects and structural rearrangements assessed by means of Fourier transform infrared (FTIR) amide I spectra were followed by immersing dry human serum albumin and bovine pancreatic alpha-chymotrypsin in low water organic solvents and in pure water at 298 K. Enthalpy changes upon immersion of the proteins in different media are in a good linear correlation with the corresponding IR absorbance changes. Based on calorimetric and FTIR data the solvents were divided into two groups. The first group includes carbon tetrachloride, benzene, nitromethane, acetonitrile, 1,4-dioxane, n-butanol, n-propanol and pyridine where no significant heat evolution and structural changes were found during protein immersion. Due to kinetic reasons no significant protein-solvent interactions are expected in such systems. The second group of solvents includes dimethyl sulfoxide, methanol, ethanol, and water. Immersion of proteins in these media results in protein swelling and involves significant exothermic heat evolution and structural changes in the protein. Dividing of different media in the two groups is in a qualitative correlation with the solvent hydrophilicity defined as partial excess molar Gibbs free energy of water at infinite dilution in a given solvent. The first group includes the solvents with hydrophilicity exceeding 2.7 kJ/mol. More hydrophilic second group solvents have this energy values less than 2.3 kJ/mol. The hydrogen bond donating ability of the solvents also assists in protein swelling. Hydrogen bonding between protein and solvent is assumed to be a main factor controlling the swelling of dry solid proteins in the studied solvents.     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Effect of organic solvents on a chloroperoxidase biotransformation

The effect of organic solvents on the chlorination activity of chloroperoxidase (CPO) was identified for use in biotransformations with CPO. CPO was found to chlorinate monochlorodimedon (MCD) in the presence of organic solvents with log P values less than 0. The relative rates of chlorination with chloride ion in the presence of H₂O₂, buffer and 2.5–20% of either dimethyl sulfoxide, N,N-dimethyl formamide, methanol or acetonitrile, were in the range of 10–58% of that in buffer (pH 2.8) at the same reactant concentrations. The presence of such organic solvents was found to alter CPO catalysis by altering the protein conformation and the local environment at the active site. CPO did not display chlorination activity in the presence of organic solvents which had log P values greater than 0.     

Influence of solvents and leucine configuration at position 5 on tryptophan fluorescence in cyclic enkephalin analogues

The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. In this study we carried out fluorescence measurements of the tryptophan residue of cyclic enkephalin analogues of a general formula X-c[D-Dab(2)-Gly(3)-Trp(4)-Y(5)] where X = Cbz or H and Y = D- or L-Leu, in four solvents [water, methanol, acetonitrile, and dimethyl sulfoxide (DMSO)]. An analysis of the tryptophan fluorescence decays using a discrete-exponential model indicates that tryptophan fluorescence decay can be described by a double exponential function in all solvents studied. Lifetime distribution analysis yields a bimodal distribution in protic solvents (water and methanol), whereas an asymmetric, unimodal distribution in an aprotic solvent (DMSO) and uni- or bimodal distributions in acetonitrile solution, depending on leucine configuration. The data are interpreted in terms of the rotamer model, in which the modality and the relative proportions of the lifetime components are related to the population distribution of tryptophan chi(1) rotamers about the C(alpha)--C(beta) bond. The chirality of the Leu(5) residue and solvent properties affect the local environment of the tryptophan residue and therefore influence the distribution of side-chain rotamers. These results are consistent with the results of theoretical conformational calculations.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibitory effect of organic solvents on the mutagenicity of N-nitrosodialkylamines in Salmonella

The influence of organic solvents on the mutagenicity of 11 N-nitrosamines was examined in Salmonella typhimurium TA100 using the Ames's liquid incubation assay in the presence of rat-liver S9. The mutagenic activities of N-nitrosodimethylamine, N-nitrosodiethylamine, 6 oxidative derivatives of N-nitrosopropylamine and N-nitroso-2,6-dimethylmorpholine were considerably decreased by addition of dimethyl sulfoxide, dimethyl formamide, acetone, 95% ethanol or acetonitrile, which are recommended for use as solvents in the assay by Ames's group, to the incubation mixture. The mutagenic activities of N-nitrosodipropylamine and N-nitrosodibutylamine, which are barely soluble in water, were also suppressed by increasing concentrations of dimethyl sulfoxide. These organic solvents did not appear to exert their influence by desmutagenic and antimutagenic actions. In contrast, the recoveries of unmetabolized carcinogens from preincubation mixtures and from agar plates were significantly higher in the presence of organic solvents than in their absence. The results indicate that the inhibitory effect is a result of interference with the process of metabolic activation by liver S9.     

Interaction of quinacrine mustard with mononucleotides and polynucleotides

1. The interaction between quinacrine mustard and mononucleotides and polynucleotides was investigated by fluorimetry and absorbance spectrophotometry. 2. The fluorescence spectrum of quinacrine mustard is independent of the ionic strength and pH. The dependence of the quinacrine mustard fluorescence intensity on ionic strength, pH and anions is described. 3. The fluorescence intensity of quinacrine mustard was enhanced with the mononucleotide adenylic acid and polynucleotides such as poly(rA), poly(rU) and poly(rA,rU). 4. Quenching of the fluorescence intensity of quinacrine mustard occurred with the mononucleotide guanylic acid and with poly(rG) and poly(rC,rG). 5. The mononucleotide cytidylic acid or poly(rC) showed no effect on the fluorescence intensity of quinacrine mustard. 6. The interaction between the dye and native DNA species was also dependent on the presence of base-specific binding sites in the DNA. The higher the (G+C) content was in the native DNA tested the higher was the quenching effect on the fluorescence intensity of quinacrine mustard. 7. No interaction was found between the dye and methylated DNA. The binding between quinacrine mustard and apurinic DNA was confirmed to be in the phosphate groups of the purines.