A statistical model for activation of Factor C by binding to LPS aggregates Abstract Published data on Factor C activity at various LPS and Lipid A concentrations (Nakamura et al. in Eur J Biochem 176:89, 1988; Kobayashi et al. in J Biol Chem 37:25987, 2014) were rearranged to show that Factor C exhibited its maximum activity at a specific concentration of LPS. A statistical model was proposed for examining whether a single LPS molecule binding activates Factor C (monomeric activation) or dimerization of Factor C is necessary for the activation (dimeric activation). In the monomeric activation model the plots of the relative activity of Factor C against the molar ratio of LPS to Factor C were different from those in the published data. The plots in the dimeric activation model lie on a bell-shaped curve, whatever the Factor C concentration, matching the published data and indicating the appropriateness of that model. We suggest that Factor C is activated by multiple molecular interactions of Factor C with LPS aggregates on which it dimerises and that this explains why larger aggregates are less effective at activating Factor C than smaller ones.

Effects of electrically-induced constant tension on giant unilamellar vesicles using irreversible electroporation Abstract Stretching in membranes of cells and vesicles plays important roles in various physiological and physicochemical phenomena. Irreversible electroporation (IRE) is the irreversible permeabilization of the membrane through the application of a series of electrical field pulses of micro- to millisecond duration. IRE induces lateral tension due to stretching in the membranes of giant unilamellar vesicles (GUVs). However, the effects of electrically induced (i.e., IRE) constant tension in the membranes of GUVs have not been investigated yet in detail. To explore the effects of electrically induced tension on GUVs, firstly a microcontroller-based IRE technique is developed which produces electric field pulses (332 V/cm) with pulse width 200 µs. Then the electrodeformation, electrofusion and membrane rupture of GUVs are investigated at various constant tensions in which the membranes of GUVs are composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). Stochastic electropore formation is observed in the membranes at an electrically induced constant tension in which the probability of pore formation is increased with the increase of tension from 2.5 to 7.0 mN/m. The results of pore formation at different electrically-induced constant tensions are in agreement with those reported for mechanically-induced constant tension. The decrease in the energy barrier of the pre-pore state due to the increase of electrically-induced tension is the main factor increasing the probability of electropore formation. These investigations help to provide an understanding of the complex behavior of cells/vesicles in electric field pulses and can form the basis for practical applications in biomedical technology.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions Abstract It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0–100 g dm−3 Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically. The data were analyzed based on a two-state model assuming the native protein dimer separates into two monomers and then unfolds. The standard free energy of unfolding (ΔG°UN) increases with increasing concentration of each crowding agent in a manner suggesting that high concentrations of PEG 6000 and Ficoll 70 favour the native CgGST relative to the unfolded form. Ficoll 70 stabilizes the native CgGST better than PEG 6000 at low w/v concentration. A mixture of equal g/cm3 concentrations of both crowding agents, however, stabilizes the native form more effectively than either Ficoll 70 or PEG 6000 at equivalent w/v total concentration and is less sensitive to GdHCl. This is in strong agreement with the results of refolding studies, and suggests that a mixture of molecular crowders of widely different molecular weights might show enhanced excluded volume effects compared to a single crowder. Thus, mixed crowding agents more effectively protect the enzyme against denaturation and assist in renaturation better than a single crowder. This suggests a heterogeneous solution of crowders, as will be found within cells, enhances the beneficial effect of crowding on the folded protein stability.

A new six-electrode electrical impedance technique for probing deep organs in the human body Abstract Electrical impedance measurements of biological tissue have many potential applications and tetrapolar impedance measurement (TPIM) with four electrodes is traditionally used which eliminates high skin contact impedance. A linear array of four electrodes for TPIM on the horizontal plane of a cylindrical volume conductor of diameter D, where the length of the array is πD/2 with potential electrodes near the centre of the array, will give a high sensitivity near the surface which reduces rapidly with depth. A recently proposed six-electrode variation of TPIM uses an additional pair of potential electrodes on the opposite side of the volume conductor in the same horizontal plane around the circumference, with the expectation that the sensitivity of the deeper regions will thereby be enhanced. The present work carries out a finite element simulation (using COMSOL) and an experimental phantom study (saline phantom) to quantitatively evaluate the improvement obtained by this new method. The new configuration doubled the sensitivity at the central region, which was reasonably uniform over a wider zone, gradually increasing towards the potential electrodes on both sides. This would be useful for a range of biological studies of deep body organs such as lungs, stomach, and bladder. where the respective external body shapes may be approximated by an oval cylinder and where electrical impedance techniques have shown promise.

Thermodynamics of DNA: heat capacity changes on duplex unfolding Abstract The heat capacity change, ΔCp, accompanying the folding/unfolding of macromolecules reflects their changing state of hydration. Thermal denaturation of the DNA duplex is characterized by an increase in ΔCp but of much lower magnitude than observed for proteins. To understand this difference, the changes in solvent accessible surface area (ΔASA) have been determined for unfolding the B-form DNA duplex into disordered single strands. These showed that the polar component represents ~ 55% of the total increase in ASA, in contrast to globular proteins of similar molecular weight for which the polar component is only about 1/3rd of the total. As the exposure of polar surface results in a decrease of ΔCp, this explains the much reduced heat capacity increase observed for DNA and emphasizes the enhanced role of polar interactions in maintaining duplex structure. Appreciation of a non-zero ΔCp for DNA has important consequences for the calculation of duplex melting temperatures (Tm). A modified approach to Tm prediction is required and comparison is made of current methods with an alternative protocol.

On the motion of magnetotactic bacteria: theoretical predictions and experimental observations Abstract The movement of magnetotactic bacteria is done in a viscous media in the low Reynolds number regime. In the present research, the simple model for magnetotactic bacteria motion, proposed by Nogueira and Lins de Barros (Eur Biophys J 24:13–21, 1995), was used to numerically simulate their trajectory. The model was done considering a spherical bacterium with a single flagellum and a magnetic moment positioned in the sphere center and parallel to the flagella. The numerical solution shows that the trajectory is a cylindrical helix and that the body Euler angles have linear dependencies on time. Using that information, analytical expressions were obtained for the first time for the center-of-mass coordinates, showing that the trajectories are helixes oriented to the magnetic field direction. They also show that the magnetic moment does not align to the magnetic field, but it precesses around it, being fully oriented only for very high magnetic fields. The analytical solution obtained permits to relate for the first time the flagellar force to the axial velocity and helical radius. Trajectories of uncultivated magnetotactic bacteria were registered in video and the coordinates were obtained for several bacteria in different magnetic fields. The trajectories showed to be a complex mixture of two oscillating functions: one with frequency lower than 5 Hz and the other one with frequency higher than 10 Hz. The simple model of Nogueira and Lins de Barros shows to be incomplete, because is unable to explain the trajectories composed of two oscillating functions observed in uncultivated magnetotactic bacteria.

Investigation of spectral and kinetic properties of polymer films based on some analogs of bacteriorhodopsin Abstract We investigated the characteristics of modified forms of bacteriorhodopsin in which the native retinal chromophore is replaced by a chemical analog (“bacteriorhodopsin analogs”), embedded in a polymer film. We found they displayed differential absorption spectra and kinetic curves for the most long-lived intermediates of the BR photocycle. We also studied the influence of chemical reagents on the functioning of bacteriorhodopsin analogs in polymeric films. We found that the immobilization of BR analogs in polymer leads, as in the case of native BR, to a slowing down of their photocycles. Kinetic analysis showed that M-like state intermediates of all the BR analogs have a longer dark relaxation time than native BR. The retention and retardation of the photocycle in these films suggest that films based on BR analogs can be used as photochromic materials. Moreover, 4-keto BR seems to be more promising for this application as compared not only with native BR, but also with other analogs of BR studied in this work.

Obtaining precise and accurate results by ITC Abstract Acquisition of precise and accurate results by isothermal titration calorimetry (ITC) can be achieved through thoughtful experimental design and modeling and careful experimental operations. Large reported errors in ITC results in determinations of stoichiometries, equilibrium constants and enthalpy changes for ligand binding to proteins are the consequence of poor experiment design, failure to properly calibrate and test instruments and protocols, lack of controls, errors in solution preparation, and incorrect data analyses. Analysis of a recent report that claimed to have determined the “repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein–ligand binding reaction” by ITC is used to illustrate how to improve ITC operations and results. The analysis shows that the reported results are misleading because calorimeters were not calibrated, operating parameters were not optimized, errors were made in solution preparations, and data analysis was not optimized. As a consequence, the results do not provide a valid comparison of the capabilities of the calorimeters included in the study. A proposal that reaction of acetazolamide with carbonic anhydrase II be used as a comparison standard for testing ITCs and procedures is problematic because the binding constant is too large and for several other reasons discussed in the paper. Requirements for obtaining precise and accurate results by ITC are discussed and experimental results are presented to illustrate the precision and accuracy attainable with low volume ITCs. The problem of the blank correction is identified as the limiting factor in obtaining accurate results by ITC.

Study of the complex coacervation mechanism between ovalbumin and the strong polyanion PSSNa: influence of temperature and pH Abstract We studied the complex between ovalbumin and long flexible poly-(sodium 4-styrene sulfonate) as a function of pH and temperature. We used various techniques [turbidimetry, conductometry, dynamic light scattering, viscosimetry, and ultra-small-angle light scattering (USALS)] to fully characterize the coacervate complex. Different phases of complexation versus temperature were determined by turbidimetric analysis (pHc, pHϕ1, and pHϕ2). The optimal protein/polyelectrolyte interaction occurred at pHopt 4. An increase in temperature made the hydrophobic interactions more favorable in the case of the soluble complex and complex coacervation phases (pH > pHϕ2). We systematically determined the activation energy to follow the conformational changes of the complex at different temperatures. At pHopt, the size of the formed complex showed a remarkable decrease with temperature increase. USALS was used to determine simultaneously the radius of gyration (Rg) and fractal dimension Df of the coacervate.

Effects of bovine serum albumin (BSA) on the excited-state properties of meso -tetrakis(sulfonatophenyl) porphyrin (TPPS 4 ) Abstract To infer changes in the photophysical properties of porphyrins due to complexation with albumin, a combination of Z-scan and conventional spectroscopic techniques was employed. We measured the characteristics of excited states of meso-tetrakis(sulfonatophenyl) porphyrin bound to bovine serum albumin and observed that the binding reduces the intersystem crossing quantum yield and increases the internal conversion one. A reverse saturable absorption process was observed in the nanosecond timescale. These results are important for prediction of the efficiency of this complex in medical and optical applications, because associating porphyrins to proteins enables better accumulation in tumors and improves its stability in optical devices, but at the same time, decreases its triplet quantum yield.