Том 73, № 4 (2018)

The results of molecular modeling allow us to associate the catalytic activity of aspartoacylase towards the hydrolysis of N-acetylaspartate with the dynamic properties of a dimeric molecule of the enzyme. The availability of the enzyme’s active site for the substrate is controlled by the conformational dynamics of the peptide loops that form a gate to the transport channel in one of the monomers. It is shown that this model explains the results of the experimental studies according to which the point mutation K213E does not affect the catalytic function of the enzyme.

Cells and tissues are composed from atoms of chemical elements, some of which have two kinds of stable isotopes, magnetic and nonmagnetic ones. Not long ago, magnetic isotope effects (MIEs) have been discovered in experiments with cells enriched with magnetic or nonmagnetic isotopes of magnesium. These MIEs can stem from higher efficiency of the enzymes of bioenergetics in the cells enriched with magnetic magnesium isotope. In the studies of MIEs in biological systems, it is needed to monitor the ATP concentrations as the major energy source in cells. The most sensitive and rapid method of the ATP measurements is based on the use of the firefly luciferase–luciferin system. Since luciferase is the ATP-dependent enzyme and activated by Mg-ions, it is necessary to elucidate whether this enzyme is sensitive to magnetic field of the magnesium isotope’s nuclear spin. Herein we present the results of studying the effects of different isotopes of magnesium, magnetic ²⁵Mg and nonmagnetic ²⁴Mg and ²⁶Mg, on bioluminescence spectra and enzymatic activity of firefly luciferase. It was shown, that neither kinetics of the bioluminescence signal nor the bioluminescence spectra manifest any statistically significant dependence on the type of magnesium isotope. So, no MIEs have been revealed in the luciferase-catalyzed oxidation of luciferin. It means that firefly luciferase bioluminescence can serve as the tool for search and studies of magnetic isotope effects in ATP-dependent enzyme reactions in biological systems, including the enzymatic synthesis and hydrolysis of ATP.

We present a method for the simultaneous detection of thyroglobulin and thyroid-stimulating hormone on microchips using gold nanoparticles for antibody labeling. Nitrocellulose and silicon were used as supports for the microchips. The detection principle was based on a sandwich immunoassay using a pair of antibodies that simultaneously bind to the analyte. Specific monoclonal antibodies that are most sensitive and specific in detecting the hormones and signal-to-background ratio are selected. The analytical signal was the intensity of the staining of the microchip zones on the membrane and the nanoparticle density on the silicon substrate. The detection limit for thyroglobulin and thyroid-stimulating hormone was 0.25 ng/mL and 0.016 μIU/mL for the membrane support and 0.1 ng/mL and 0.013 μIU/mL for silicon substrate, respectively. The method can be further used to develop nanoscale sensor devices for the express diagnostics of thyroid pathologies.

A new approach for the regulation of catalytic properties of the medically significant enzyme L-asparaginase is suggested based on the formation of conjugates with PEG-chitosan (chitoPEGylation). The efficiency of this approach is demonstrated using recombinant L-asparaginase from Rhodospirillum rubrum (RrA). This preparation is immunologically different from the one used in medical practice preparations of L-asparaginase from E. coli, which offers a promising alternative for applications in the case of hypersentsitivity development. The low level of activity of RrA towards L-glutamine, which decreases significantly the chance of side effects developing, is an advantage of RrA. The technique for the synthesis of the RrA conjugates with PEG-chitosan (chitoPEGylation) of a varying modification degree is developed. It is established that conjugation of RrA with PEG-chitosan increased the specific activity of the enzyme in comparison with the native one. The activity changes from 56 IU/mg (for the native enzyme) to 61–72 IU/mg (for the conjugates) depending on the degree of chitosan PEGylation. The secondary structure of the Rhodospirillum rubrum asparaginase conjugates with PEG-chitosan is examined using CD- and IR-spectroscopy. It is found that the enzyme structure changed only slightly as a result of conjugation with PEG-chitosan: the content of α-helices changed from 36% (for the native enzyme) to 30–33% (for the conjugates). The content of β-structures changed from 15% (for the native enzyme) to 18% (for the conjugate). The obtained data open new opportunities for the synthesis of L-asparaginase preparations with improved biocatalytic properties.

The effect of medium pH and ion strength on the thermal stability of formate dehydrogenases (EC 1.2.1.2, FDH) from model plant Arabidopsis thaliana (AthFDH) and soybean Glycine max (SoyFDH) are studied. The dependence of the residual activity on time is described by the kinetics of the reaction of the first order in all the experimental conditions. The dependence of the first order thermal inactivation constants on phosphate buffer concentration has the form of a bell-shaped curve. It was found that an increase in the pH value resulted in an increase and decrase of inactivation rate constans for SoyFDH and AthFDH, respectively. The activation parameters of the thermal inactivation process, ΔH^≠ and ΔS^≠ are calculated from the experimental data at different medium pH values and phosphate buffer concentrations.