Scatchard Approach Research Articles

Interactions of tetracationic porphyrins with DNA and their effects on DNA cleavage

The interaction of tetracationic porphyrins with DNA was studied using UV–Vis absorption, fluorescence spectroscopy and viscometry, and the particle sizes were determined. Аs cationic porphyrins, two isomer porphyrins, 3,3′,3″,3‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP3) and 4,4′,4″,4‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP4), were studied. They differ in the position of NCH3+ group in phenyl ring of the porphyrins and hence, in degree of freedom of rotation of the phenyl rings about the central macrocycle. It was found that intercalated complexes are formed at DNA/porphyrin molar ratios (R) of 2.2 and 3.9 for TMPyP3 и TMPyP4, respectively. Decreasing R up to 0.4 and 0.8 for TMPyP3 и TMPyP4, respectively, leads mainly to formation of outside complexes due to π-π stacking between the porphyrin chromophores interacting electrostatically with phosphate framework of DNA. Each type of the obtained complexes was characterized using Scatchard approach. It was ascertained that the affinity of TMPyP4 to DNA is stronger than TMPyP3, meanwhile the wedge effect of the latter is higher. The differences between the porphyrin isomers become more evident at irradiation of their complexes with DNA. It was established that irradiation of the intercalated complexes results in DNA fragmentation. In the case of TMPyP4, DNA fragments of different size are formed. The irradiation of the outside DNA/porphyrin complexes leads to cleavage of DNA (TMPyP3 and TMPyP4) and partial destruction of the complex due to photolysis of the porphyrin (TMPyP3).

Molecularly imprinted polystyrene-divinylbenzene adsorbents for removal of bisphenol A

The effect of acrylonitrile co-polymerization and molecular imprinting on bisphenol A (BPA) affinity by the polystyrene-divinylbenzene adsorbents was studied. Adsorption equilibria were measured using batch uptake experiments and equilibrium data were analyzed by means of Langmuir isotherm and Scatchard approach. The calculated parameters were utilized for modeling of adsorption–microfiltration hybrid system. The highest BPA capacity, 27 mg/g, is achieved for the material synthesized with 10% BPA template concentration in relation to monomer. Acrylonitrile does not improve BPA affinity or adsorption capacities of sorbents. BPA sorption rate can be described assuming pore diffusion control and using a tortuosity factor of 3. The estimated equilibrium and mass transport parameters describe reasonably well the removal of BPA in the adsorption–microfiltration hybrid system. Model calculations show that 97% reduction in BPA concentration is attained with sufficiently high amount of the adsorbent.

The Major Proteins of Bovine Seminal Plasma Interact with Caseins and Whey Proteins of Milk Extender

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.

Studies on the interaction of olive cake and its hydrophylic extracts with polyvalent metal ions (Cu(II), Eu(III)) in aqueous solutions

The paper presents and discusses the chemical affinity of two different forms of natural organic matter (e.g. in solid (SOM) and soluble form (DOM)) for Cu 2+ and Eu 3+ ions. In the this study SOM is represented by olive cake and DOM by its hydrophilic extracts. The investigations were performed in aqueous 0.1 M NaClO 4 solutions at pH 6, 23 °C and under atmospheric conditions by means of potentiometry using a copper ion selective electrode and competitions reactions between the two metal ions. The experimental data were evaluated according to an “operational” approach, which is based on the charge neutralization model and the Scatchard approach. Evaluation of the data resulted in the determination of conditional formation constants (β *), which are a measure for the chemical affinity of natural organic matter for a metal ion. For the olive cake metal ion binding the values of log β * for Cu(II) and Eu(III) were evaluated to be 5.1 ± 0.3 and 5.4 ± 0.5, respectively, whereas for the hydrophilic extracts the corresponding values for the Cu(II) and Eu(III) complexes were evaluated to be 5.3 ± 0.3 and 6.3 ± 0.5, respectively. The β * values show that natural organic matter in the two different forms presents almost similar affinity for the Cu 2+ ion, whereas the affinity of DOM for the Eu 3+ ion is significantly higher than the corresponding affinity of SOM. This is ascribed to the higher flexibility of DOM, which allows better coordination of its active sites around the Eu 3+ ion.

Isothermal titration calorimetric study of calcium association to lipid bilayers: influence of the vesicle preparation and composition

The association of Ca2+ ions with phospholipid bilayers was investigated using isothermal titration calorimetry. The study reveals that the binding enthalpy of these cations to bilayers formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) depends strongly on the method of preparation of the unilamellar vesicles. Extruded vesicles lead to an exothermic association, whereas sonicated ones lead to an endothermic association. In the later case, the calorimetric signal is sensitive to the length of the sonication period. It is proposed that a reorganization of the lipid bilayers under stress, obtained with sonicated small unilamellar vesicles, contributes to the calorimetric signal upon the titration with Ca2+. The analysis of the titrations indicates that, as expected, the nature of the association of Ca2+ with negatively charged phospholipid bilayers is essentially of electrostatic nature. Using a Scatchard approach, it is found that bilayers become saturated in Ca2+ approximately when the electroneutrality of the bilayer interface is reached. Moreover, the affinity constant was reduced by the increase of the ionic strength of the aqueous buffer. It was found that the intrinsic binding constant of Ca2+ to membranes containing 30 and 50 mol% of POPG was about 11 mM-1, in a MES buffer containing 10 mM NaCl, at pH 5.6.

A simple method based on IC-ICP-MS to determine conditional stability constants

A method was developed for the determination of conditional stability constants (K′f) and it is demonstrated here using a simple system, i.e., the complexation of Zn or Co by ethylenediaminetetraacetic acid (EDTA) in a 1∶1 ratio. The method involves separation of the free metal and its EDTA complex by ion-exchange chromatography (IC) with on-line detection by inductively coupled plasma mass spectrometry (ICP-MS). An anion-exchange guard column (Dionex AG-7, 4 × 50 mm), which also exhibits some cation-exchange properties, provided adequate separation using a mobile phase of 0.1 M NH4NO3 at pH 2.0 and 2.5 for Zn and Co, respectively. Since the peak area response of ICP-MS was independent of the metal species, K′f could readily be obtained by injecting a series of solutions with different metal and EDTA concentrations and then plotting log(peak area of complex/peak area of free metal) versus log(total free EDTA concentration). The resulting straight line had a slope equal to the chelation number and an intercept equal to log(K′f). Values of 3.10 ± 0.42 and 4.40 ± 0.24 were obtained for log K′f of Zn–EDTA2− (pH 2.0) and Co–EDTA2− (pH 2.5), respectively, in good agreement with expected values. The approach is more robust than the Scatchard approach, which yielded erroneous results as a result of drift in the ICP-MS response. In contrast, this drift, which was not significant during a chromatogram, was effectively compensated through ratioing the peak areas.

A Study of Heavy Metal Complexation in Grape Juice

AbstractDifferential pulse anodic stripping voltammetry, DPASV, has been used to monitor the initial stages of grape juice fermentation, focusing on Zn interactions with natural occurring ligands. Langmuir and Scatchard linearization methods have been employed. A 1 : 1 ratio has been found by either method; from Langmuir data analysis only one ligand population was found, while Scatchard approach gave rise to the detection of two ligand types. Both data analysis procedures led to the same total ligand concentration. When catechin was used as model ligand, a 1 : 1 ratio was found for Zn and also for Cu.

Amphetamine pretreatment induces a change in both D2-Receptor density and apparent affinity: a [11C]raclopride positron emission tomography study in cats

BackgroundMeasuring changes in dopamine (DA) levels in humans using radioligand-displacement studies and positron emission tomography (PET) has provided important empirical findings in disease and normal neurophysiology. These studies are based on the assumption that DA exerts a competitive inhibition on radioligand binding. To test this, we used PET and a Scatchard approach to investigate whether the decrease in [11C]raclopride binding following amphetamine results from competitive or noncompetitive interactions with DA. MethodsScatchard analyses of [11C]raclopride/PET data were used to quantify changes in apparent D2-receptor density (Bmax) and radioligand apparent affinity (K′D) at baseline and after amphetamine pretreatment (2 mg/kg; intravenous) in cats. ResultsAmphetamine induced a 46% decrease in [11C]raclopride binding in the striatum of five cats. Scatchard analyses revealed that this decrease in binding was due to a 28% decrease in Bmax and a concomitant 35% increase in K′D. ConclusionsCompetition with DA is an insufficient explanation for the decrease in [11C]raclopride binding observed after amphetamine. Noncompetitive interactions, likely representing D2-receptor internalization, also play an important role in this phenomenon. This finding may have important implications for the interpretation of amphetamine–raclopride PET studies in schizophrenia because dysregulation of the agonist-induced internalization of D2 receptors was recently suggested in this disorder.

The attraction between like-charged macroions—the crucial roles of macroion geometry andcharge distribution

A mechanism of attraction between like-charged macroions due to charge fluctuation hasbeen revisited for macroions of different geometries. For higher charge asymmetry,macroion geometry plays an important role in charge fluctuation, while for comparativelylower charge asymmetry the fluctuation is almost independent of the geometry. At very lowtemperatures and in a high Coulomb coupling regime, stable ionized states due toauto-ionization are able to exist, which leads to long range Coulomb attraction.Overcharging of a single macroion and its counterion distribution minimizing the totalenergy has been studied briefly to explain the attraction properties between twolike-charged macroions. The investigations have been carried out using a technique basedon total energy minimization which has been verified successfully for some special caseswith molecular dynamics simulation results previously published. A theoretical model,derived by modifying the Scatchard approach, is proposed to explain the overchargingcurves obtained from the simulation. It has been found that the model is equally efficientfor both spherical and non-spherical macroion geometries. Wigner crystal theory has alsobeen employed for spherical cases, and excellent agreement has been found between the twotheoretical approaches.

(Un)certainty of overall binding constants of Al with dissolved organic matter determined by the Scatchard approach

One of the best approaches to date to obtain overall binding constants ( K o) for Al and dissolved organic matter (DOM) from acidic soil solutions is to collect ‘free’ Al data with diffusive gradients in thin films (DGT) and to infer the K o values by fitting a continuous distribution model based on Scatchard plots. Although there is clear established literature demonstrating the usefulness of the Scatchard approach, relatively little attention has been given to a realistic assessment of the uncertainties associated with the final fitted K o values. In this study we present an uncertainty analysis of the fitted K o values using a synthetic dataset with different levels of random noise and a real data set using DGT data from an acidic soil solution. The parameters in the continuous distribution model and their corresponding upper and lower 95% uncertainty bounds were determined using the Shuffled Complex Evolution Metropolis (SCEM) algorithm. Although reasonable fits of the distribution model to the experimental data were obtained in all cases, an appreciable uncertainty in the resulting K o values was found due to three main reasons. Firstly, obtaining ‘free’ Al data even with the DGT method is relatively difficult, leading to uncertainty in the data. Secondly, before Scatchard plots can be constructed, the maximum binding capacity (MBC) must be estimated. Any uncertainty in this MBC propagates into uncertainty associated with the final plots. Thirdly, as the final fitted K o values are largely based on extrapolation, a small uncertainty in the fit of the binding data results in an appreciable uncertainty in the obtained K o. Therefore, while trends in K o for Al and DOM could easily be discerned and compared, the uncertainty in the K o values hinders the application in quantitative speciation calculation. More comprehensive speciation models that avoid the use of K o seem to fit better for this purpose.

Copper complexation by dissolved organic matter and uncertainty assessment of their stability constants.

The interaction of Cu with dissolved organic matter (DOM, extracted from an organic forest floor) was investigated and the resulting data was evaluated in terms of their uncertainty. The speciation of Cu over 'free' Cu (as analysed by diffusive gradients in thin films (DGT)), dissolved Cu-DOM complexes and precipitated Cu-DOM was determined as a function of pH (3.5, 4.0 and 4.5) and Cu/C ratio. The dissolved organically bound fraction was highest at pH 4.5, but this fraction decreased with increasing Cu/C ratio, which was observed for all pH levels. In the range of Cu/C = 7 x 10(-5) -2.3 x 10(-2) (mol/mol) the precipitated fraction was very small. The speciation of both Al and Fe was not affected by increasing Cu concentrations. From a continuous distribution model using the Scatchard approach, we calculated the optimal fit and corresponding upper and lower 95% uncertainty bounds of the overall stability constants (K(o)) with the shuffled complex evolution Metropolis (SCEM) algorithm. Although the optimal equation fitted the data very well, the uncertainty of the, according to literature, most reliable approach to establish stability constants, was still large. Accordingly, the usually reported intrinsic stability constants exhibited large uncertainty ranging from log K(i) = 6.0-7.1 (optimal 6.7) for pH 3.5, log K(i) = 6.5-7.1 (optimal 6.8) for pH 4.0, and log K(i) = 6.4-7.2 (optimal 6.8) for pH 4.5 and showed only little effect of pH.

A Spectroscopic Study on PtCl4(2−) Binding to Rabbit SkeletalMuscle G‐Actin

It was found that the binding of PtCl42− to G-actin and the consequent conformational changes are different with those for hard acids. It is a two-step process depending on molar ratio PtCl42−/actin (R). In the first step, R less than 25, the PtCl42− ions are bound to sulfur-containing groups preferentially. These high-affinity sites determined by Scatchard approach are characterized by n1 = 30 with average binding constant K1=1.0×107M-1. The conformational changes are significant as characterized by N-(1-pyrenyl) maleimide(NPM) labeled fluorescence, intrinsic fluorescence and CD spectra. EPR spectroscopy of maleimide spin labeled(MSL) actin demonstrated that even PtCl42−binding is limited to a very small fraction of high-affinity sites(R<1), it can bring about a pronounced change of conformation. In the range of R=25-40, high-affinity sites accessible are saturated. In the second step(R>40) , deep-buried binding sites turn out to be accessible as a result of the accumulated conformational changes. These new binding sites are estimated to be n2=26 with average binding constant K2=2.1×106M-1. Although in this step the quenching of intrinsic fluorescence goes on and the NPM-labled thiols moves to more hydrophilic environment, no change in α-helix content was found. These results suggested that with increasing in PtCl4(2−) binding, the G-actin turns to an open and loose structure in a discontinuous mode.

Isothermal binary and ternary diffusion in the system (C2H5)4 NBr–KBr–H2O

Isothermal free diffusion in the binary systems KBr–H2O, (C2H5)4NBr–H2O and in the ternary system (C2H5)4NBr–KBr–H2O has been studied at various compositions with a precision single-lens Gouy interferometer. For the binary systems, the observed diffusion coefficients have been compared with those of similar compounds and, in the case of (C2H5)4NBr, have been compared with theoretical values predicted from activity coefficient behaviour for which a parameter-fitting least-squares approach has been used to determine the activity coefficient variation with concentration. Ternary coefficients have been analysed qualitatively in terms of ionic attractions in the solution and have been compared with the binary coefficients at similar concentrations. The Onsager Reciprocal Relation has been verified for one composition of the ternary system, but the calculations differ from previous tests in that the more versatile Scatchard approach to multicomponent solutions has been used rather than Harned's rule.