G. A. Badun

In Situ Spatial Organization of Potato Virus A Coat Protein Subunits as Assessed by Tritium Bombardment

ABSTRACT Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing α-helices and β-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two β-strands, (ii) a C-terminal region including two α-helices, as well as three β-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four α-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Increase in the specific radioactivity of tritium-labeled compounds obtained by tritium thermal activation method

Abstract A method of tritium introduction into different types of organic molecules that is based on the interaction of atomic tritium with solid organic target is described. Tritium atoms are formed on the hot W-wire, which is heated by the electric current. Such an approach is called “tritium thermal activation method”. Here we summarize the results of labeling globular proteins (lysozyme, human and bovine serum albumins); derivatives of pantothenic acid and amino acids; ionic surfactants (sodium dodecylsulfate and alkyltrimethylammonium bromides) and nonionic high-molecular weight surfactants – pluronics. For the first time it is observed that if the target-compound is fixed and its radicals are stable the specific radioactivity of the labeled product can be drastically increased (up to 400 times) when the target temperature is ca. 295 K compared with the results obtained at 77 K. The influence of labeling parameters as tritium gas pressure, exposure time and W-wire temperature was tested for each target temperature that results in the optimum labeling conditions with high specific radioactivity and chemical yield of the resulting compound.

Collagen tissue treated with chitosan solutions in carbonic acid for improved biological prosthetic heart valves

Calcification of bovine pericardium dramatically shortens typical lifetimes of biological prosthetic heart valves and thus precludes their choice for younger patients. The aim of the present work is to demonstrate that the calcification is to be mitigated by means of treatment of bovine pericardium in solutions of chitosan in carbonic acid, i.e. water saturated with carbon dioxide at high pressure. This acidic aqueous fluid unusually combines antimicrobial properties with absolute biocompatibility as far as at normal pressure it decomposes spontaneously and completely into H2O and CO2. Yet, at high pressures it can protonate and dissolve chitosan materials with different degrees of acetylation (in the range of 16-33%, at least) without any further pretreatment. Even exposure of the bovine pericardium in pure carbonic acid solution without chitosan already favours certain reduction in calcification, somewhat improved mechanical properties, complete biocompatibility and evident antimicrobial activity of the treated collagen tissue. The reason may be due to high extraction ability of this peculiar compressed fluidic mixture. Moreover, exposure of the bovine pericardium in solutions of chitosan in carbonic acid introduces even better mechanical properties and highly pronounced antimicrobial activity of the modified collagen tissue against adherence and biofilm formation of relevant Gram-positive and Gram-negative strains. Yet, the most important achievement is the detected dramatic reduction in calcification for such modified collagen tissues in spite of the fact that the amount of the thus introduced chitosan is rather small (typically ca. 1wt.%), which has been reliably detected using original tritium labelling method. We believe that these improved properties are achieved due to particularly deep and uniform impregnation of the collagen matrix with chitosan from its pressurised solutions in carbonic acid.

A new technique for tritium labeling of humic substances

Abstract Humic substances (HS) of different origins have been labeled with tritium by the thermal activation method. Specific radioactivity of labeled HS ( 3H-HS) was sufficiently high and varied from 0.14 to 0.6 TBq/g. Parent HS and 3H-HS were analyzed by size exclusion chromatography with radioactivity and UV detection. The results allowed concluding that (1) neither partial decomposition nor polymerization of HS occurred during labeling and (2) tritium labeled molecules have a regular distribution among HS fractions of different molecular weights. The performed correlation analysis revealed that there was no significant relationship between HS properties and specific radioactivity of the obtained 3H-HS. Thus universality of the developed technique for radioactive labeling of HS with tritium could be demonstrated.

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium

The structure of the C‐terminal domain of the influenza virus A matrix M1 protein, for which X‐ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally‐activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C‐terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C‐terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C‐terminal domain is an almost flat layer with a three‐α‐helical structure. To explain the high level of tritium label incorporation into the C‐terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI‐TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C‐terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.

A novel approach radiolabeling detonation nanodiamonds through the tritium thermal activation method

Abstract Tritium labeling was introduced into detonation nanodiamonds (ND) through the tritium thermal activation method. Two target preparation techniques were developed to increase the radioactivity and the specific radioactivity of the labeled product: the desiccation of the waterless solvent suspension and the lyophilization of the hydrosol. The specific radioactivity of the labeled product was shown to correlate with the hydrogen content in the starting material and to achieve 2.6 TBq/g.

Liquid scintillation spectrometry of tritium in studying lysozyme behavior in aqueous/organic liquid systems. The influence of the organic phase.

Liquid scintillation spectrometry of tritium in the application of the scintillation phase method was used for studying the adsorption of lysozyme at the liquid/liquid interface and its distribution in the bulk of the system. The goal of this research was to reveal the influence of the nature of the organic phase on the distribution and adsorption ability of the protein when it is placed in a system containing two immiscible liquids. Based on the radiochemical assay distribution coefficients and adsorption isotherms obtained for aqueous/octane, aqueous/p-xylene and aqueous/octanol systems, it was concluded that the interaction of the protein with the interface plays a dominant role in protein behavior in aqueous/organic liquid systems.

Colloidal properties of binary mixtures of a nonionic surfactant and monomeric or gemini cationic surfactants

The behavior of binary mixtures composed of a nonionic surfactant Triton X-100 (TX-100) and monomeric dodecyltrimethylammonium bromide (DTAB) or gemini N,N’-bis(N-dodecyl-N,N-dimethyl)-1,2-diammonium ethane dibromide (DDAB) cationic surfactants is studied upon micellization, wetting of Teflon and adsorption at the solution-air and solution-Teflon interfaces. The compositions of mixed micelles and adsorption layers, as well as the parameters of interaction between the surfactants (mixture components), were calculated using the Rubingh-Rosen model. For both mixtures, the interaction parameters are negative, and their absolute values increase in the following order: mixed micelles ≈ adsorption layers at the solution-air interface < adsorption layers at the solution-Teflon interface. The absolute values of the interaction parameters for TX-100-DDAB mixtures are larger than those for TX-100-DTAB mixtures. The adsorption of both mixtures on Teflon demonstrates synergistic effects. In case of TX-100-DDAB mixtures, the synergistic effects are also observed upon micellization, reduction of the surface tension, and wetting of Teflon.

Nonequilibrium processes in reactions of hot tritium atoms with cooled solid targets. Influence of the atomizer temperature on formation of labeled substances

A system consisting of a cold target and “hot” atoms generated by dissociation of tritium on a tungsten wire was studied with the aim to determine conditions for preparing tritium-labeled organic compounds with the maximal radiochemical yield. The influence of the atomizer temperature on the result of the reaction of tritium atoms with amino acids and tetraalkylammonium bromides was studied; homological series of the substrates were examined with the aim to evaluate the contributions of functional groups and hydrocarbon tail to the processes occurring in the target. The dependence of the yield of the labeled parent compound on the atomizer temperature varied in the range 1600–2000 K was determined. The rates of decarboxylation and deamination sharply grew with increasing temperature of the tungsten wire. The highest yield of labeled amino acids was attained at an atomizer temperature of 1800–1900 K, and at higher temperature their yield decreased. The difference between the activation energies of the elimination of the carboxy and amino groups and of the isotope exchange of hydrogen for tritium in the C-H bond appeared to be 93 and 59 kJ mol−1, respectively. For alkyltrimethylammonium bromides with the alkyl radicals C12H25, C14H29, and C16H33, the yield of the labeled parent compound reached 80–90% and was virtually independent of the atomizer temperature. The capability of tritium atoms to penetrate into the targets was evaluated. For the exponential model of the attenuation of the flow of tritium atoms inside the target, the attenuation factor for freeze-dried amino acids and alkyltrimethylammonium bromides as targets was 1.8 nm−1.

Effect of the structure of ethylene oxide-propylene oxide block copolymers on their interaction with biological membranes

Partition coefficients of ethylene oxide-propylene oxide block copolymers between the lipid phase and water have been estimated via equilibrium dialysis. It has been shown that for the triblock copolymer (Pluronic L61), the partition coefficient is 45 ± 9, while for the diblock copolymer (REP), this parameter is as high as 78 ± 17. The effect of the copolymers on the permeation of the charged organic ion carboxyfluorescein across the lecithin bilayer membrane changes in the same direction. Even though the triblock copolymer binding is weaker, it shows a stronger effect on the rate of transbilayer migration of lipids and on the permeation of the uncharged substance (doxorubicin). The incorporation of cholesterol into the membrane decreases its sensitivity to the action of copolymers; however, the character of changes induced by both copolymers remains invariable. The experimental data of this study indicate that the triblock structure of amphiphilic macromolecules is responsible for their higher ability to disturb lipid bilayer membranes.