Inhibitor and substrate binding induced stability of HIV-1 protease against sequential dissociation and unfolding revealed by high pressure spectroscopy and kinetics

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure. © 2015 Ingr et al.INSERM; Grant Agency of the Czech Republic [P208-12-G016

A study of blends of amphiphilic polymers in solution

The viscometric coefficient, k(AB), characterizing the interaction of chemically dissimilar polymer segments was estimated from changes of the intrinsic viscosity of poly(vinyl alcohol) (PVA) measured in "pseudo-solvents," i.e., dilute solutions of poly(ethylene glycol) (PEG) in water and dimethyl sulfoxide (DMSO). The results provide evidence of the compatibility of these polymers in both solvents. The effects of hydroxyl end groups and the character of solvents has been found. The aggregation of PVA molecules in water is not significantly suppressed by PEG

A study of the interaction between chitosan and poly(ethylene glycol) by viscosity method

The viscometric coefficient, k(AB), characterizing the interaction of chemically dissimilar polymer segments was estimated from changes of the intrinsic viscosity of chitosan in "pseudosolvents,'' i.e., dilute solutions of poly( ethylene glycol) ( PEG) in a solution of 0.1 M acetic acid/0.2M sodium acetate ( 1: 1). The blends were studied at 25 degrees, 35 degrees, and 45 degrees C. The miscibility between chitosan and PEG was found to increase with the temperature of the measured systems and to decrease with increasing molar mass of PEG. In the cases where favorable compatibility was exhibited between chitosan and PEG, H-bonding interaction between the two polymers should be strong enough to overcome the intra- and intermolecular H-bonding among chitosan chains

Role of the shape of various bacteria in their separation by Microthermal Field-Flow Fractionation

Experimentální a teoretická studie vlivu tvaru separovaných částic na jejuich retenci v MikroTFFF.The steady-state movement of the spherical and non-spherical particles, such as prolate or oblate rotational ellipsoids, cylinders, or parallelepipeds. suspended in a liquid and exposed to a unidirectional temperature gradient, is analyzed theoretically. The differences in the ratios of the rotational to translational diffusion coefficients of the non-spherical to spherical particles, the heterogeneity of thermal conductivity of the particle body, and the heterogeneity in surface chemical nature make possible to separate the particles according to differences in shape. Preliminary experimental separations of Gram-positive and Gram-negative, nearly spherical and rod-shaped bacteria performed by Microthermal Field-Flow Fractionation confirmed that the fractionation of the cells according to differences in shape is possible. (C) 2010 Elsevier B.V. All rights reserved

Intrinsic viscosity and conformational parameters of chitosan chains

The dependence of the intrinsic viscosity [η] on the weight average degree of polymerization Pw of chitosans in aqueous acid media is discussed in terms of the Yamakawa-Fujii theory for the worm-like cylinder model. The conformational characteristics (Kuhn statistical segment length and Flory characteristic ratio) are estimated. They increase linearly with th reciprocal of the square root of salt concentration. The "intrinsic" values for uncharged chains are comparable to those for cellulose and lower man those for chitin. © 2011 RASYAN. All rights reserved

Effect of supermolecular particles in chitosan solutions on the correlation of intrinsic viscosity and degree of polymerization

Large differences in some reported relationships between the intrinsic viscosity [h] and the weight-average degree of polymerization PW of chitosans are discussed in terms of a simple model based on the assumption that the PW values estimated by light scattering can be significantly increased by large supermolecular particles which have not been removed from solutions and which do not affect significantly [h]. The effect depends on their content and size. Various shapes of the plots of [h] vs Pw can be explained. The hypothesis is presented that the supermolecular particles are residues of crystalline structures of chitosans. © 2011 RASĀYAN. All rights reserved

Separation of bacteria in temperature gradient: Micro-thermal focusing field-flow fractionation

Metodou mikro-termální field-flow frakcionace bylo dosaženo separace bakterií Staphylococcus epidermidis a Rhodococcus erythropolis. Je to vůbec první pokus separace živých biologických buněk termální difúzí (Ludwig-Soret) v kombinaci se vztlakovými silami, které způsobí fokusační mechanismus separace. Experimenty byly provedeny za přesně zvolených experimentálních podmínek, aby se zabránilo denaturaci bakterií.The separation of Staphylococcus epidermidis and Rhodococcus erythropolis bacteria was achieved with the use of Micro-Thermal Focusing Field-Flow Fractionation. This is the first performance of separation exploiting the Ludwig-Soret effect (thermal diffusion) of living biological cells, combined with lift forces and resulting in the focusing mechanism of separation. The experiments were carried out under carefully chosen experimental conditions preventing the denaturation of the bacteria

On the precision of particle size analysis by micro-thermal field-flow fractionation

Micro-Thermal Field-Flow Fractionation of polymer colloidal particles was performed in two different laboratories. Short term repeatability of the experimental retentions obtained in a single laboratory has been found to be very high. The short term precision (expressed as percent standard deviation) of the determination of average particle diameter can reach the values greater than 1%, relative. Average repeatability of the retentions in both laboratories was better than 3%, relative when using identical experimental protocol. No other method of particle size analysis can provide the results of a comparable precision. Average repeatability of the width of the raw fractograms, which contains the information on particle size distribution, is of the order of 5%, relative. However, this value cannot be considered as the ultimate limit because the experiments were not carried at the low flow rate of the carrier liquid permitted to reach much higher resolution. The effect of the stability of the most important operational variables, such as the temperature drop between the cold and hot walls, the temperature of the cold wall, and the flow rate of the carrier liquid, on the precision of the analytical results is discussed

Note on the relation betweeen the parameters of the Mark-Houwink-Kuhn-Sakurada equation

The relation between the parameters K and a of the equation [eta] = Km(a). which has been empirically established by several authors,, is discussed. Equations describing this relation are derived based on the two-parameter theory of the intrinsic viscosity for flexible, chain polymers (where a 0.8). The correspondence of calculated and empirical results is good

Micro-thermal field-flow fractionation of bacteria

Metodou mikro-termální field-flow frakcionace bylo dosaženo separace bakterií Staphylococcus epidermidis. Je to vůbec první pokus separace a charakterizace biomakromolekul a biologických částic termální difúzí (Ludwig-Soret). Experimenty byly provedeny za přesně zvolených experimentálních podmínek, aby se zabránilo denaturaci bakterií. Vztlakové síly, které se objevují při vysokých rychlostech mobilní fáze, způsobily fokusační mechanismus separace, která byla kratší než 10 minut.The retention of Staphylococcus epidermidis bacteria cells, achieved with the use of micro-thermal field-flow fractionation and described in this paper, represents the first experimental proof that the separation and characterization of the bio-macromolecules and biological particles is possible by exploiting Ludwig-Soret effect of thermal diffusion. The experiments were carried out under gentle experimental conditions preventing the denaturation of the bacteria. Lift forces, appearing at high linear velocities of the carrier liquid, generated the focusing mechanism of the retention which resulted in high-speed and high-performance separation performed in less than 10 minutes