N. V. Penkov

On singularities of molecular relaxation in water solutions

In this paper, we analyzed spectra of liquid water and water solutions in a frequency domain, characteristic of the collective dynamics of water molecules (from 0 to 200 cm−1). Particular attention is paid to the relaxation processes, one of which is observed in the terahertz region of the spectrum (∼5–50 cm−1). The physical essence of this process at the molecular level is still unclear. Based on data obtained this process is strongly suggested to interpret as a monomolecular relaxation of unbound water molecules.

Terahertz Spectroscopy Applied for Investigation of Water Structure.

The absorption spectra of liquid water and various aqueous solutions were analyzed in a terahertz frequency domain (from 6 to 200 cm(-1)) which characterize the collective dynamics of water molecules. Particular attention was paid to the relaxation process in the range of ∼6-80 cm(-1). The physical essence of this process on the molecular level is still unclear. We found that the amplitude of this relaxation process correlates with the degree of destruction of water structure. The obtained data allowed us to interpret this process as a monomolecular relaxation of free water molecules. On the basis of a consideration of the water polarization in the electric field, we proposed a method of calculation of the amount of free water molecules in solution.

Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions.

The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10-50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening.

Smooth muscle titin forms in vitro amyloid aggregates

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimers disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro. This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700–4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.

[The influence of spermine on Ca(2+)-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,Ω-hexadecanedioic acids].

The effect of spermine on Ca2+-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,ω-hexadecanedioic acid was studied. It has been shown that spermine inhibited the cyclosporin A-insensitive mitochondrial swelling induced by palmitic acid and Ca2+ and α,ω-hexadecanedioic acid and Ca2+. 100 μM spermine did not influence the mitochondrial respiration in state V2 and the respiration stimulated by palmitic acid, α,ω-hexadecanedioic acid and Ca2+. Preincubation of liposomes with 100 μM spermine resulted in inhibition of the palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced release of a fluorescent dye sulforhodamine B from liposomes. At the same time, spermine added to fatty acid-containing liposomes stimulated Ca2+-dependent release of sulforhodamine B from liposomes. Addition of spermine to liposomes resulted in a significant increase in the ζ -potential of liposomal membranes (from −39.8 to −18.6 mV). A possible mechanism of spermine influence on palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced permeability transition in mitochondria and liposomes is discussed.

Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes

The work examines the effect of inhibitors of cytosolic Ca2+-dependent and Ca2+-independent phospholipases A2 on bilayer lipid membranes. It was established that trifluoroperazine (TFP) and, to a lesser extent, arachidonyl trifluoromethyl ketone (AACOCF3) and palmitoyl trifluoromethyl ketone (PACOCF3) were able to permeabilize artificial lipid membranes (BLM and liposomes). It was shown that AACOCF3 lowered the temperature of phase transition of DMPC liposomes, inducing disordering of the hydrophobic region of lipid bilayer. TFP disordered membranes both in the hydrophobic region and in the region of hydrophilic heads, this being accompanied by changes in the membrane permeability: appearance of a channel-like BLM activity and leakage of sulforhodamine B from liposomes. In contrast to AACOCF3 and TFP, PACOCF3 increased membrane orderliness in the hydrophobic region (heightened the temperature of phase transition of DMPC liposomes) and in the region of lipid heads. The effectiveness of AACOCF3 and PACOCF3 as inductors of BLM and liposome permeabilization was considerably lower comparatively to TFP. As revealed by dynamic light scattering, incorporation of TFP, AACOCF3 and PACOCF3 into the membrane of liposomes resulted in the increase of the average size of particles in the suspension, presumably due to their aggregation or fusion. The paper discusses possible mechanisms of the influence of phospholipase A2 inhibitors on bilayer lipid membranes.

Calculation of the amount of free water molecules in aqueous solutions by means of spectral parameters from the terahertz frequency domain taking into account processes of screening

In this paper we derive a formula to calculate the amount of free water molecules in solution. Physical values in this formula may be obtained by analyzing the spectra of aqueous solutions in the terahertz frequency range. Formula is derived on the basis of considering water polarization process in electric field. It is shown that without processes of shielding the electric field in the water calculations lead to very high estimation of a share of free water molecule.

Calculation of the portion of free water molecules in water solutions by means of spectral analysis

In this paper we present the results of spectral studies of water solutions and water at different temperatures. The calculation method for estimation of the portion of free water molecules is offered. The method is based on the analysis of transmissions spectra features in the terahertz region. The calculation using the proposed method shows that the portion of free water molecules at room temperature ranges from 10 to 16% for a variety of water solutions. In case of pure water, the temperature increase from 20 to 50°C results in the increase of the free molecule fraction from 12 to 19%. Moreover, the increase in the number of free water molecules within a specified temperature range exhibits significant nonuniformity: the most intense growth was observed in the range of 30–40°C. This is particularly interesting because this temperature range includes the optimum temperatures for the existence of warm-blooded animals.

The Role of Intermolecular Disulfide Bonds in Stabilizing the Structure of Peroxiredoxins

The comparative characterization of thermal stability of human peroxiredoxins 1–6 (Prx1–Prx6) has been performed by physicochemical and biochemical methods and the role of disulfide bonds in stabilizing their structure has been shown. Prx1 and Prx2 among the tested peroxiredoxins exhibit the highest peroxidase activity and thermal stability. Prx1 and Prx2 are more than 2 times more active on average with H2O2 and tert-butyl hydroperoxide as substrates compared to other peroxiredoxins and retain at least 50% activity after 30 min heating at a temperature of 64°C, which is more than 10°C higher compared to Prx3–Prx6. The reduction of the disulfide bonds between Prx1 and Prx2 leads to a decrease of their thermal stability, comparable to the thermal stability of Prx3–Prx6, which confirms the important role of the intermolecular S–S bonds in stabilizing the structure of these proteins.

A Study of the Effect of a Protein on the Structure of Water in Solution Using Terahertz Time-Domain Spectroscopy:

Terahertz time-domain spectroscopy (THz-TDS) was used to determine the spectra (range = 1.2–120 cm−1) of aqueous solutions of bovine serum albumin (BSA) at pH range 2.5–10. Under each of the selected pH, BSA molecules exist in a different conformation, compared to other pH values. The spectra were used to calculate the functions of the dielectric permittivity of BSA solutions. Dielectric functions of the aqueous phase of BSA solutions were calculated based on the Bruggeman model, without the contribution of BSA itself. Fitting of the dielectric functions was performed using a model which includes three water spectral bands: two relaxation bands with relaxation times of about 8.28 and 0.3 ps and a vibrational band with a maximum of about 180 cm−1. The parameters of these bands were determined through fitting and physical interpretation at the molecular level can be provided for each of them. A comparison between the values of model parameters of solutions with BSA and without BSA allowed to conclude that the main effect of BSA is the formation of strongly bound hydration shells in the immediate proximity to the protein molecule. At the same time, the structure of more distant layers of the hydration shells is destroyed, with an increased formation of free water molecules. Some differences are observed in the effect of different BSA conformations on the aqueous phase of solution. The proposed approach can be generalized and applied for studying of a wide class of biological macromolecules in aqueous solutions.