Protein partitioning in aqueous two-phase systems composed of a pH-responsive copolymer and poly(ethylene glycol)

The effect of pH and salt concentration on the partitioning behavior of bovine serum albumin (BSA) and cytochrome c in an aqueous two-phase polymer system containing a novel pH-responsive copolymer that mimics the structure of proteins and poly(ethylene glycol) (PEG) was investigated. The two-phase system has low viscosity. Depending on pH and salt concentration, the cytochrome c was found to preferentially partition into the pH-responsive copolymer-rich (bottom) phase under all conditions of pH and salt concentrations considered in the study. This was caused by the attraction between the positively charged protein and negatively charged copolymer. BSA partitioning showed a more complex behavior and partitioned either to the PEG phase or copolymer phase depending on the pH and ionic strength. Extremely high partitioning levels (partition coefficient of 0.004) and very high separation ratios of the two proteins (up to 48) were recorded in the new systems. This was attributed to strong electrostatic interactions between the proteins and the charged copolymer.     

Photophysical and photodynamic properties of Pyronin Y in micellar media at different temperatures

In this study, photophysics and photodynamical properties of Pyronin Y (PyY) in different liquid media were investigated. Interactions of PyY, which is a positively charged pigment compound pertaining to the xanthene derivatives with surfactants possessing distinct charges, were determined by using the molecular absorption and fluorescence spectroscopy techniques. It was observed that band intensities of absorption and fluorescence spectra belonging to PyY increase in proportion to the water when compared to three micelle systems, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X‐100 (TX‐100). This suggests that interactions in micelle systems are different from those in deionized water, and solvation and surface interactions modify. It is determined that the strongest interaction occurs between PyY dye and SDS, anionic surfactant, and this interaction arises from the electrostatic forces. Calculated photophysical parameters indicated that the microenvironment of PyY in SDS micelle is different to that of other systems. In temperature studies, it was reported that increasing the temperature of the samples increased non‐radiative transitions. Steady‐state fluorescence anisotropy values were calculated by using fluorescence intensities of PyY compound in pre‐micellar, micellar and post‐micellar systems. Once the PyY fluorescence probe is added to the surfactant containing solutions below the critical micelle concentrations, the measured anisotropy values were found to be low because the probe remains in the deionized water phase. When the surfactant concentration of the medium becomes closer to the critical micelle concentrations, the steady‐state anisotropy value prominently increases. This is because of the restrictions on the rotational diffusion of the probe in micellar solution. It is observed that positively charged PyY shows a higher affinity to the negatively charged SDS compared with the positively charged CTAB and neutral TX‐100 surfactants. This can be explained by Coulombic interactions.     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis

Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Using this nondisruptive fluorescent tracer system, we characterized the effects of sodium chloride and ammonium sulfate concentrations on lysozyme-lysozyme interactions by steady-state and time-resolved fluorescence anisotropy measurements and the introduction of a novel interaction parameter, k_rot. The results suggested that the specific attractive interaction, which was reflected in the retardation of the protein rotational diffusion, was induced depending on the salt type and its concentration. The change in the attractive interactions also correlated with the crystallization/precipitation behavior of lysozyme. Moreover, we discuss the validity of our rotational diffusion analysis through comparison with the osmotic second virial coefficient, B₂₂, previously reported for lysozyme and those estimated from k_rot.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Salt dependence and thermodynamic interpretation of the thermal denaturation of small DNA restriction fragments.

The influence of cation concentration on the thermal denaturation of DNA restriction fragments from the E. coli lac regulatory region and from pVH51, ranging in size from 43- to 880- bp, is described. Upon increasing the ionic strength, the melting transitions broaden in a cooperative manner at salt concentrations characteristic for the specific fragment. For three fragments studied in detail, the salt concentration dependence at the midpoint varied between 0.03 and 0.19 M Na+. Along with the broadening, the melting transitions become more symmetrical. This result is discussed with respect to the irreversibility of melting transitions at low ionic strength. After a cooperative broadening, the shape of the melting curves remains constant up to salt concentrations of 0.5 M Na+. The dTM/dlog[Na+] values for three fragments fall between 15.7 and 16.7. An easily applicable approximation of the van't Hoff equation is used to evaluate the enthalpies of 13 transitions arising from the denaturation of 43 to 600 bp. The results of this analysis are compared to calculations of the expected enthalpies based on calorimetric measurements. The TMs of most transitions were directly related to the base composition, but several deviations from the predicted behavior were observed. The possible influences of fragment length and sequence on the thermal stability are discussed. The experimental and mathematical procedure to resolve a thermal denaturation transition with a width f 0.17 +/- 0.01 degrees and its distinction from another preceeding transition only approximately 0.15 degrees away in an 880-bp Hae III fragment from pVH51 is described. This transition is about half as wide as the smallest one reported to date.     

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.     

Structure and interactions of aggrecans: statistical thermodynamic approach

Weak polyelectrolytes tethered to cylindrical surfaces are investigated using a molecular theory. These polymers form a model system to describe the properties of aggrecan molecules, which is one of the main components of cartilage. We have studied the structural and thermodynamical properties of two interacting aggrecans with a molecular density functional theory that incorporates the acid-base equilibrium as well as the molecular properties: including conformations, size, shape, and charge distribution of all molecular species. The effect of acidity and salt concentration on the behavior is explored in detail. The repulsive interactions between two cylindrical-shaped aggrecans are strongly influenced by both the salt concentration and the pH. With increasing acidity, the polyelectrolytes of the aggrecan acquire charge and with decreasing salt concentration those charges become less screened. Consequently the interactions increase in size and range with increasing acidity and decreasing salt concentration. The size and range of the forces offers a possible explanation to the aggregation behavior of aggrecans and for their ability to resist compressive forces in cartilage. Likewise, the interdigitation of two aggrecan molecules is strongly affected by the salt concentration as well as the pH. With increasing pH, the number of charges increases, causing the repulsions between the polymers to increase, leading to a lower interdigitation of the two cylindrical polymer layers of the aggrecan molecules. The low interdigitation in charged polyelectrolytes layers provides an explanation for the good lubrication properties of polyelectrolyte layers in general and cartilage in particular.     

Brownian dynamics simulation of probe diffusion in DNA: effects of probe size, charge and DNA concentration

We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model.     

Kinetics of cellulose regeneration from cellulose--NaOH--water gels and comparison with cellulose--N-methylmorpholine-N-oxide--water solutions

The regeneration kinetics of cellulose from cellulose--NaOH--water gels immersed in a nonsolvent bath is studied in detail. Cellulose concentration, bath type, and temperature were varied, and diffusion coefficients were determined. The results were compared with data measured and taken from the literature on the regeneration kinetics of cellulose from cellulose--N-methylmorpholine-N-oxide (NMMO) monohydrate solutions. Different theories developed for the transport behavior of solutes in hydrogels or in porous media were tested on the systems studied. While the diffusion of NaOH from cellulose--NaOH--water gels into water has to be described with "porous media" approaches, the interpretation of NMMO diffusion is complicated because of the change of NMMO's state during regeneration (from solid crystalline to liquid) and the high concentration of NMMO in the sample. The activation energies were calculated from diffusion coefficient dependence on temperature for both systems and compared with the ones obtained from the rheological measurements. The activation energy of cellulose--NaOH--water systems does not depend on cellulose concentration or the way of measurement. This result shows that whatever the system is, pure NaOH--water solution, cellulose--NaOH--water solution, or cellulose--NaOH--water gel, it is NaOH hydrate with or without cellulose in solution, which is moving in the system. The swelling of cellulose in different nonsolvent liquids such as water or different alcohols during regeneration was investigated and interpreted using the Hildebrand parameter.     

Temperature dependence of macromolecular interactions in dilute and concentrated hemoglobin solutions

We studied the temperature-dependent effects of intramolecular interactions on the mutual diffusion coefficient of normal human oxygenated hemoglobin in salt solution. We used photon correlation spectroscopy to observe this temperature dependence of the mutual diffusion coefficient of two protein concentrations (1.25 and 17.0 g %) between 13 and 37°C. This coefficient was our probe for monitoring temperature-dependent structural changes of hydrated hemoglobin in solution. Comparison of our measured diffusion coefficient with that predicted by the Stokes-Einstein relationship in terms of solvent or solution viscosity showed a clear transition in the conformation of hemoglobin at approximately 22°C, independent of the hemoglobin concentration. We postulated that at this physiological temperature, a considerable quaternary rearrangement of the hemoglobin chains takes place. We believe this rearrangement changes the effective volume and the hydration sphere of the hemoglobin macromolecule.     

Possible basis for the apparent surface selectivity of the contact activation of human blood coagulation factor XII

The activation of factor XII by the proteases factor XIIa and kallikrein is known to be greatly enhanced by certain negatively charged surfaces. Studies that compared factor XII surface binding to factor XII activation found that binding alone was insufficient to account for surface enhancement of the activation rate. The temperature dependence of the reaction showed unusual behavior that may be related to the conformational change of factor XII following binding; the rate of factor XII activation had a relatively low temperature optimum (0-47 degrees C) that was sensitive to choice of surface and salt concentration. In temperature studies, below 47 degrees C, the decrease in the activation rate was not related to the thermal denaturation of enzyme or substrate, nor to the choice of activator enzyme (factor XIIa or kallikrein), nor to the species of factor XII (human or bovine) but to a behavior, designated a thermal transition, associated with the surface or the protein-surface interaction. The previously reported surface selectivity of contact activation is possible due to the temperature characteristics and other properties of the thermal transition; a surface that has a low-temperature thermal transition and that is highly sensitive to salt will be a "poor" contact surface under the usual choice of reaction conditions (approximately 150 mM ionic strength and 37 degrees C). However, solution conditions were identified that allowed the following negatively charged surfaces to function, in nearly equal potency, in the activation of factor XII: phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol 4-phosphate, heparin, and 5-kDa dextran sulfate, as well as the previously characterized sulfatide and 500-kDa dextran sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic interactions modulate the conformation of collagen I

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.     

On the temperature- and salt-dependent conformation change in human erythrocyte pyruvate kinase

The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.     

Effect of NaCl and CaCl2 on the lateral diffusion of zwitterionic and anionic lipids in bilayers

The influence of addition of NaCl or CaCl₂ (0.3 and 0.1 M, respectively) on the lateral diffusion coefficient (D_L) of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) was measured by the pulsed field gradient NMR technique. D_L of DOPC was unaffected, whereas the DOPG diffusion decreased with salt concentration. ²³Na NMR quadrupole splittings of DOPG between 20 and 60 °C and added NaCl between 0 and 15 wt% decreased only slightly with salt content, but increased with increasing temperature. Similar results were obtained for palmitoyloleoylphosphatidylglycerol, in which the palmitoyl chain order parameter increased slightly with salt. A model with free and “bound” ions was used to interpret the splitting data.With increasing salt content a decrease in the water permeability for DOPG was observed, but not for DOPC, as measured by water diffusion perpendicular to the oriented lipid bilayers.It was concluded that calcium and sodium ions interacted with the DOPG head-groups resulting in a decrease in the “free area” per lipid molecule due to a screening of the charged lipid head-groups. Thus, there was a closer packing of DOPG, leading to a decrease in D_L and water permeability. DOPC did not show any changes in the bilayer properties upon the addition of ions.     

Thermal expansion measurements of cryoprotective agents. Part II: measurements of DP6 and VS55, and comparison with DMSO

As part of an ongoing effort to characterize the mechanical behavior of biological tissues in the cryogenic temperature range, the current study focuses on thermal expansion measurements of cryoprotective agents. Utilizing the experimental apparatus described in the previous report (Part I), the current report (Part II) includes thermal expansion measurements of the cryoprotectant mixtures DP6 and VS55, and comparison with available data from the literature on DMSO. In the temperature range in which the cryoprotectant mixture behaves like low viscosity liquid, results of this study show that the thermal expansion coefficient of VS55 and DP6 is 22% and 40% lower than that of 3M DMSO, respectively, where 3M DMSO is only one component of each cryoprotectant mixture. This significant difference is attributed to the presence of 3M formamide in VS55.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.