Characterization of thermal stability of the Escherichia coli Fapy-DNA glycosylase

Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence. Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degrees C. Heat aggregation is preceded by tertiary conformational changes of Fpg. However, the secondary structure of the fraction that does not aggregate remains unchanged up to approximately 60 degrees C. The kinetics of heat aggregation occurs with an activation enthalpy of approximately 21 kcal/mol. The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above approximately 3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins. With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of approximately 3.6 kcal/mol at 25 degrees C. An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg. A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented.     

pH-dependent phase transition of chlorpromazine micellar solutions in the physiological range

The effects of pH and drug concentration on aggregation properties of chlorpromazine-HCl (CPZ) are examined. The critical micelle concentration (cmc) changes from 0.2 mM at pH 7.3 to 2 mM at pH 5.6 as estimated from the stearic acid spin label solubility measurements. For concentrations above the cmc CPZ micelles undergo a concentration-, temperature- and pH-dependent transition leading to phase separation. This phase transition is followed by a sudden increase of light scattering. The phase diagram pH vs. concentration is obtained by observation of the cloud point for concentrations ranging from 0.01 to 10 mM. The intramicellar environment is probed at pH ranging from 5.5 to 8.0 using a stearic acid spin label. The intramicellar compactness increases smoothly with increasing pH suggesting the weakening of polar heads repulsion due to charge decrease. The reported results indicate that pH effects are relevant and should be properly taken into account in the performance and interpretation of experiments with CPZ.     

Potentiating effect of chlorpromazine on glyceryl guaiacolate ether induced hemolysis of rat,dog and human blood in vitro

The hemolytic effect of glyceryl guiacolate ether (GGF) with and without chloromazine (CPZ) was studied in vitro on rat, dog and human blood. The lowest concentration of GGE which could produce hemolysis of rat red cells was 0.15 M. The time fpr 50% hemolysis (TH50) of blood depended upon the concentration of drug and dilution of blood. A higher concentration of GGE hemolyzed blood much faster than the lower. There was a progressive increase in the TH50 when 0.15 M GGF was tested on blood samples containing increasing numbers of red cells. CPZ in all cases had its own hemolytic effect at higher concentrations. In this regard rat blood was 10 times more sensitive than dog, and human. A striking potentiating effect of CPZ was observed on the hemolytic effect of GGE. The magnitude of potentiation in all cases was directly related to the concentrations of CPZ. Dog blood was found relatively more sensitive to the hemolytic effect of the combination of CPZ and GGE as compared to the rat and human, which acted alike.     

The effects of ethanol on the structural stability of acetylcholine receptor and the activity of various molecular forms of acetylcholinesterase

The actions of ethanol on the structural stability of acetylcholine receptor (AchR)-enriched membrane vesicles and the activity of various molecular forms of acetylcholinesterase (AchE) were investigated, using the receptor and the enzyme isolated from the electric organ of Torpedo californica. In the presence of ethanol up to 200 mM, the thermogram of AchR-enriched membranes exhibited no significant decrease in the temperature (td) of receptor transition at 57 degrees C, but a decrease in the enthalpy change (delta Hd) indicated a slight ethanol-induced structural perturbation. The presence of 12.5 nmol alpha-bungarotoxin also caused a decrease in delta Hd. A complete loss of the receptor transition was observed at a higher concentration 500 nmol of alpha-bungarotoxin and no recovery of the transition was found with the addition of 200 mM ethanol. The results suggested a noncompetitive interaction of ethanol with the receptor. In the presence of 200-1000 mM ethanol, the activity of two soluble forms of AchE, a higher (117 S) aggregate and a lower (10 S) aggregate was not significantly affected. Comparing the activity of these two aggregates over a wide concentration range of ethanol (200-2000 mM) revealed no obvious difference in the level of ethanol effect between them. However, after removal of ethanol, the higher aggregate form of AchE exhibited a greater recoverability of the activity, suggesting a possible slightly greater structure-functional stability for it. Studies of soluble AchE and membrane-bound AchE showed that the presence of 200 or 600 mM ethanol caused a greater level of inhibition in membrane-bound enzyme than in soluble enzyme, possible due to a disruption of protein-lipid interaction needed to maintain the conformation of membrane-bound AchE. Interestingly, at a much higher concentration of ethanol (2.0 M), membrane-bound AchE became more resistant to ethanol than did the soluble forms of AchE. In this case, the effective concentration of ethanol felt by the enzyme was expected to be less for membrane-bound AchE, owing to ethanol's solubility in lipids.     

Supramolecular self-assembly of d(TGG)4, synergistic effects of K+ and Mg2+.

Spectral evidence indicates that molar concentrations of K+ can induce aggregate formation in d(TGG)4. The 320-nm turbidity monitoring indicates that more than 1 M KCl is needed for the onset of aggregation to occur at 20 degrees C within the time span of 24 h. The kinetic profile is reminiscent of autocatalytic reactions that consist of a lag period followed by accelerative and levelling phases. Progressive shortening of lag periods and more rapid accelerative phases accompany further increases in [K+]. Interestingly, the presence of Mg2+ greatly facilitates the aggregate formation and results in the prominent appearance of an intense psi-type CD. For example, whereas 1 M K+ fails to induce aggregate formation of d(TGG)4 within 24 h, the addition of 1 mM Mg2+ to a 1 M K+ solution is sufficient to induce the onset of aggregation in approximately 12 h. Furthermore, adjustment of the buffer to 16 mM Mg2+/1 M KCl reduces the lag time to less than 10 min and aggregation is nearly complete in 2 h. The requirement of [K+] for aggregation is reduced to 2 mM in the presence of 16 mM Mg2+, a reduction of nearly three orders of magnitude when compared to solutions without Mg2+. The effects of K+ and Mg2+ ions are synergistic, because the presence of 16 mM Mg2+ alone does not induce aggregate formation in this oligomer. Thermal stabilities of the aggregates are strongly dependent on the concentrations of these two ions. Although aggregates formed in the presence of 2 M KCl alone melt around 55 degrees C, those formed with added 16 mM Mg2+ melt at approximately 90 degrees C, with some aggregates remaining unmelted even at 95 degrees C. The slow kinetics of aggregate formation led to the appearance of gross hystereses in the cooling profiles. The interplay of these two ions appears to be specific, because the replacement of K+ by Na+ or the replacement of Mg2+ by other divalent cations does not lead to the observed self-assembly phenomenon, although Sr2+ can substitute for K+. A possible mechanism for the formation of self-assembled structures is suggested.     

The denaturing action of lysophosphatidylcholine as studied by calorimetric and rheological techniques

The potency of lysophosphatidylcholine to perturb protein structure was investigated by differential scanning calorimetry and rheological measurements using myosin as a model protein. At physiological ionic strength (0.15 M NaCl) 5mM lysophosphatidylcholine produced a detectable reduction in the protein's enthalpy of denaturation, while concentrations less than or equal to 2 mM had no effect. At higher salt concentrations (0.6 M NaCl) lower concentrations of lysophosphatidylcholine were needed in order to reduce the enthalpy of denaturation. Also, the changes in myosin conformation, as judged from calorimetric measurements, became more extensive as the incubation temperature for myosin-lysophosphatidylcholine systems was increased from 10 degrees to 30 degrees C. Rheological techniques allowed detection of changes in the structure of filaments of myosin (in 0.15 M) upon addition of 0.2 mM lysophosphatidylcholine. The denaturing action of lysophosphatidylcholine is compared to the more familiar detergent sodium dodecyl sulphate.     

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.     

Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.     

Membrane binding induces lipid-specific changes in the denaturation profile of bovine prothrombin. A scanning calorimetry study.

Prothrombin denaturation was examined in the presence of Na2EDTA, 5mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-phosphatidylglycerol (DOPG). Heating denaturation of prothrombin produced thermograms showing two peaks, a minor one at approximately 59 degrees C previously reported to correspond to denaturation of the fragment 1 region (Ploplis, V. A., D. K. Strickland, and F. J. Castellino 1981. Biochemistry. 20:15-21), and a main one at approximately 57-58 degrees C, reportedly due to denaturation of the rest of the molecule (prethrombin 1). The main peak was insensitive to the presence of 5mM Ca2+ whereas the minor peak was shifted to higher temperature (Tm approximately 65 degrees C) by Ca2+. Sufficient concentrations of POPC/bovPS (75/25) large unilamellar vesicles to guarantee binding of 95% of prothrombin resulted in an enthalpy loss in the main endotherm and a comparable enthalpy gain in the minor endotherm accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). Peak deconvolution analysis on the prothrombin denaturation profile and comparison with isolated prothrombin fragment 1 denaturation endotherms suggested that the change caused by POPC/PS vesicles reflected a shift of a portion of the enthalpy of the prethrombin 1 domain to higher temperature (Tm approximately 77 degrees C). The enthalpy associated with this high-temperature endotherm increased in proportion to the surface concentration of PS. By contrast, POPC/DOPG (50/50) membranes shifted the prethrombin 1 peak by 4 degrees C to a lower temperature and the fragment 1 peak by 5 degrees C to a higher temperature. The data lead to a hypothesis that the fragment 1 and prethrombin 1 domains of prothrombin do not denature quite independently and that binding of prothrombin to acidic-lipid membranes disrupts the interaction between these domains. It is further hypothesized that PS containing membranes exert the additional specific effect of decoupling the denaturation of two subdomains of the prethrombin 1 domain of prothrombin.     

Differential binding of chlorpromazine to human blood cells: Application of the hygroscopic desorption method

Using a modified hygroscopic desorption method (HDM) the binding of chlorpromazine (CPZ) to human blood cells was investigated in the concentration range from 0.01 to 100 μmol/1. For erythrocytes and ghosts the ratio between cell bound and free drug concentration was constant up to 60 μmol/1 CPZ. Saturable binding, however, was observed for lymphocytes, granulocytes and less pronounced for platelets. In contrast to red cells, CPZ binding to white cells and platelets was strongly dependent on pH. For all blood cells a sharp decrease in binding occurred at drug concentrations higher than 60 μmol/1. This can hardly represent a true saturation of binding sites, since membrane damaging effects occur at these concentrations. Our results suggest that binding of CPZ to erythrocytes represents an interaction at the water-membrane interphase. For the different binding pattern of white cells, the cell organelles, the cytoplasma and the different composition of the membranes might be of importance.     

Hydrophilic carotenoids: surface properties and aggregation of an astaxanthin-lysine conjugate, a rigid, long-chain, highly unsaturated and highly water-soluble tetracationic bolaamphiphile

The surface and aggregation properties of a synthetic, highly water-soluble carotenoid, the tetracationic astaxanthin-lysine conjugate (Asly), have been examined through measurements of surface tension, optical absorption and dynamic light scattering. The following parameters were determined: critical aggregation concentration c(M), surface concentration Gamma, molecular area a(m), free energy of adsorption and aggregation (DeltaG(ad) degrees and DeltaG(M) degrees , respectively), and the aggregate size r(H). The compound forms true monomolecular solutions in water below c(M); aggregates emerge only at rather high concentrations (> or =2.18 mM).     

The permeation properties of small organic cations in gramicidin A channels.

The conductance properties of organic cations in single gramicidin A channels were studied using planar lipid bilayers. From measurements at 10 mM and at 27 mV the overall selectivity sequence was found to be NH4+ > K+ > hydrazinium > formamidinium > Na+ > methylammonium, which corresponds to Eisenman polyatomic cation sequence X'. Methylammonium and formamidinium exhibit self block, suggesting multiple occupancy and single filing. Formamidinium has an apparent dissociation constant (which is similar to those of alkali metal cations) for the first ion being 22 mM from the Eadie-Hofstee plot (G0 vs. G0/C), 12 mM from the rate constants of a three-step kinetic model. The rate-limiting step for formamidinium is translocation judging from supralinear I-V relations at low concentrations. 1 M formamidinium solutions yields exceptionally long single channel lifetimes, 20-fold longer than methylammonium, which yields lifetimes similar to those found with alkali metal cations. The average lifetime in formamidinium solution significantly decreases with increasing voltage up to 100 mV but is relatively voltage independent between 100 and 200 mV. At lower voltages (< or = 100 mV), the temperature and concentration dependences of the average lifetime of formamidinium were steep. At very low salt concentrations (0.01 M, 100 mV), there was no significant difference in average lifetime from that formed with 0.01 M methylammonium or hydrazinium. We conclude that formamidinium very effectively stabilizes the dimeric channel while inside the channel and speculate that it does so by affecting tryptophan-reorientation or tryptophan-lipid interactions at binding sites.     

Cationic amphiphiles induce fusion of acidic liposomes

Decylamine, dodecylamine and tetradecylamine induced aggregation and fusion of acidic liposomes at concentrations of about 1 mM, 75 μM and 75 μM, respectively. Aggregation was assayed as increase in turbidity. Fusion was assayed as intermixing of membranes and contents, and was observed in the electron-microscope to form large liposomes. Only at higher concentrations did these amphiphiles induce massive leakage of the liposomes' contents. Similar effects were caused by hexadecylpyridinium bromide (CP) and hexadecyltrimethylammonium bromide (CTAB). The trivalent cation 4-dodecyldiethylenetriamine and the more hydrophobic amphiphile, trioctylmethylammonium chloride, induced fusion at concentrations of about 10–20 μM. Octylamine and heptylamine induced size increase at mM concentrations. They induced membrane intermixing but little or no content intermixing. Thus, these amphiphiles seem to promote size increase either by transfer of lipid or mainly by ‘cracking and annealing’.     

Associative phenomena in galactomannan-deacetylated xanthan systems

The interaction between mesquite seed galactomannan (MSG; D-mannose to D-galactose ratio (M/G) approximately 1.1) and deacetylated xanthan (DX) in 5 mM NaCl leading to synergistic gel formation at 25 degrees C was investigated and compared with the far more studied system made of xanthan and locust bean gum (LBG; M/G approximately 3.5). Rheology and differential scanning calorimetry were used to measure temperatures of gel formation and transition enthalpy as a function of polymer composition, while circular dichroism was used to probe the conformation of DX in the LBG-DX system. MSG and DX associate at 25 degrees C with a well defined stoichiometry of 0.6:1.0 (w/w) at low ionic strength favouring the disordered coil state of DX. When LBG was used in place of MSG in water or 5 mM NaCl, two types of mechanisms of interpolymeric association are envisaged.     

Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 mM phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 mM surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 mM phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a "shoulder" appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.     

The effect of selected anions on dipalmitoylphosphatidylcholine phase transitions

The effect of three anions, Cl-, Br- and I-, on the phase transitions of dipalxnitoylphosphatidyicholine (DPPC) was measured. Main phase transition was modestly affected by these anions in the salt concentration range 0.2 M. For Cl- and Br- the temperature of main phase transition was lower (by about 0.5 degrees C), its half-width modestly larger and enthalpy practically unchanged, all three parameters were altered to a much larger deuce. Main phase transition temperature was 1.5 degrees C lower and the peak hall-width significantly smaller. These changes were not accompanied by any alteration in main phase transition enthalpy. Iodide shifted the pretransition temperature toward lower values and increased its half-width to such an extent that at concentrations above 100 mM it was practically undetectable. Besides cations, the presence of anions also has a distinct effect on lipid bilayer interface properties.     

Effects of polyols, saccharides, and glycoproteins on thermoprecipitation of phenylboronate-containing copolymers

The copolymer of 3-(acrylamido)phenylboronic acid and N-isopropylacrylamide (82:18, Mn = 47000 g/mol) was prepared by free radical polymerization. The copolymer showed typical thermoprecipitation behavior in aqueous solutions; its phase transition temperature (TP) was 26.5 +/- 0.2 degrees C in 0.1 M glycine-NaOH buffer containing 0.1 M NaCl, pH 9.2. Due to specific complex formation of the pendant boronates with sugars, TP was strongly affected by the type of sugar and its concentration at pH 9.2. Fructose, lactulose, and glucose caused the largest increase in TP (up to 4 degrees C) at 0.56 mM concentration, attributed to the high binding affinity of the sugars to borate and phenylboronate. Among the sugars typical of nonreducing ends of oligosaccharides, N-acetylneuraminic acid had the strongest effect on TP (ca. 2 degrees C at 0.56 mM concentration and pH 9.2), while the effects of other sugars are well expressed at the higher concentrations (16 and 80 mM) and decreased in the order xylose approximately galactose >or= N-acetyllactosamine >or= mannose approximately fucose > N-acetylglucosamine. The effect exerted on the phase transition by glycoproteins was the strongest with mucin from porcine stomach and decreased in the series mucin > horseradish peroxidase > human gamma-globulin at pH 9.2. As a first approximation, the weight percentage and/or the number of oligosaccharides in glycoproteins determined the character of their interaction with the pendant phenylboronates and, therefore, the effect on the copolymer phase transition.     

Photoaddition of chlorpromazine to DNA

Chlorpromazine, 2-chloro-N-(3-dimethylaminopropyl)phenothiazine (CPZ), is a frequently prescribed antipsychotic drug that causes cutaneous photosensitivity in man. CPZ is also phototoxic and photomutagenic in vitro. We have investigated the photoaddition of CPZ to DNA as a possible mechanism for these photobiologic effects. Prior to irradiation, CPZ binds non-covalently to double-stranded calf thymus DNA. At high nucleotide to CPZ ratios, the CPZ absorption maximum shifts from 305 nm to 340 nm with an isosbestic point at 323 nm and 90% of the CPZ fluorescence at 455 nm is quenched. The excitation and emission spectra for the unquenchable fluorescence are the same as those for unbound CPZ. The absorption and fluorescence spectra of unbound CPZ are restored at 0.1 mM magnesium acetate or 100 mM sodium acetate. Non-covalent binding of CPZ to heat-denatured DNA does not shift the CPZ absorption spectrum but quenches 65% of the CPZ fluorescence. Photolytic decomposition of CPZ was inhibited by binding to DNA. In the presence of high concentrations of double-stranded DNA or denatured DNA the photolysis rates were reduced by greater than 98% and 65%, respectively, compared to free CPZ. Formation of covalent photoadducts between CPZ and denatured DNA was 10-fold more efficient than photoadduct formation with double-stranded DNA. Approximately 10% of the CPZ which photodecomposed upon irradiation at 323 nm photoadded to denatured DNA. These results indicate that formation of a complex between CPZ and double-stranded DNA absorbing at 340 nm protects CPZ from photodecomposition and inhibits covalent photoadduct formation.     

Energetics of the interactions of human serum albumin with cationic surfactant

The heat capacity changes for interaction of human serum albumin (HSA) and a cationic surfactant—cetylpyridinium chloride (CPC), were studied at conditions close to physiological (50 mM HEPES or phosphate buffer, pH 7.4 and 160 mM NaCl) carrying out isothermal calorimetric titrations (ITC) at various temperatures (20-40 °C). ITC measurements indicated that the small endothermic changes associated with CPC demicellization were temperature independent at these conditions. Surprisingly, important enthalpy changes associated with binding of CPC to HSA were exothermic and temperature independent at lower concentrations (below 0.022 mM) of CPC and endothermic and temperature dependent at higher concentrations of CPC. The values of heat capacity changes were obtained for each studied concentration of CPC from the plot of enthalpy changes vs temperature. The obtained results demonstrate the temperature independence of heat capacity changes at entire range of studied CPC concentrations. Both enthalpograms and heat capacity curves indicate the two-step mechanism of HSA folding changes due to its interactions with CPC. The first step corresponds to transition from native state to partially unfolded state and the second to unfolding and to the loss of tertiary structure. The analysis of the results indicates that predominant cooperative unfolding occurs at CPC/HSA molar ratio region between 25 and 30. Such information could not be extracted from thermograms and describes the role of heat capacity as a major thermodynamic quantity giving insight on physical, mechanistic and even atomic-level into how HSA may unfold and interact with CPC. The effect of CPC binding on HSA intrinsic fluorescence, UV-Vis and CD spectra were also examined. Hence, the analysis of spectral data confirms the ITC results about the biphasic mechanism of HSA folding changes induced by CPC. The CD measurement also represents the conservation of considerable secondary structure of HSA due to interaction with CPC.     

Temperature effects on trimethylamine oxide accumulation and the relationship between plasma concentration and tissue levels in smelt (Osmerus mordax)

Rainbow smelt (Osmerus mordax) were maintained in a long term acclimation study to elucidate temperature effects on the accumulation of trimethylamine oxide (TMAO) and to determine if the activity of trimethylamine oxidase (TMAoxi) plays a role in modulating the seasonally variable levels of TMAO. In the same experiment, the TMAO content was determined for several tissues at varying plasma TMAO concentrations. TMAO accumulation begins at 5-7 degrees C, well above that which might be normally associated with an antifreeze response. The plasma concentration reached a plateau of 20 mM as temperatures reached 0 degrees C. Plasma TMAO concentration drops to pre-accumulation levels, less than 5 mM, when fish are held at elevated temperature (8-11 degrees C) and increases when fish are chilled below ambient seawater temperatures. However, despite temperatures near or below 0 degrees C, plasma TMAO decreases after the winter season. Changes in TMAoxi activity do not correlate with TMAO levels, suggesting that the activity of this enzyme does not play a key role in regulating TMAO concentrations in smelt. For the first time in any teleost fish, tissue TMAO contents in liver, kidney, brain, and intestine were found to strongly correlate with plasma TMAO concentrations. For these tissues, the intracellular and extracellular concentration of TMAO appears to be approximately equal. Conversely, the heart and white muscle accumulate TMAO, and in the case of white muscle, intracellular concentration is maintained at a constant level of approximately 35 mmol/kg, despite fluctuating plasma concentrations over a range from 0 to over 25 mM.