Vol 71, No 2 (2016)

Purification of the 11S proteasome regulator from mouse brain was optimized; the subunit composition of the isolated protein was determined by Western blot. The dependency of 20S proteasome peptidase activity on the molar concentration of the 11S regulator was examined. The Michaelis constants of hydrolysis of the specific fluorescent substrates Suc–Leu–Leu–Val–Tyr–AMC, Ac–Arg–Leu–Arg–AMC, and Z–Leu–Leu–Glu–AMC by the 20S proteasome from BALB/c mouse brain and the 20S–11S complex were determined. It was shown that the 11S particle has almost no influence on binding of specific fluorescent substrates to the 20S proteasome, but strongly accelerates hydrolysis of all three substrates, while not affecting the rate of peptide substrate hydrolysis by the 26S proteasome.

Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires.

The ChitoPEGylation method, which is a novel approach to regulating the catalytic properties of enzymes that is based on the formation of a covalent conjugate of an enzyme with branched copolymers of chitosan, has been developed. The efficiency of this method has been demonstrated using a new recombinant preparation of L-asparaginase from Erwinia carotovora (EwA) as a model. The molecular architecture and composition of EwA conjugates with PEG–chitosans have been optimized. It has been shown that the decisive factors that affect the activity of the EwA conjugates are the molecular weight of and PEGylation degree of chitosan. It has been found that the EwA conjugation with PEG–chitosan increases, its cytostatic activity against human chronic myeloid leukemia K562 cells, Burkitt’s lymphoma Raji cells, and acute lymphoblastic leukemia Jurkat cells. These data provide new approaches to the synthesis of L-asparaginase preparations with improved biocatalytic properties.

A comparative study of the exhaled-breath-condensate (EBC) proteome that was obtained for four donor groups was carried out using ion cyclotron resonance mass spectrometry with electrospray ionization. The groups included subjects with diagnosed lung cancer, chronic obstructive pulmonary disease, community-acquired pneumonia, and healthy nonsmoking control subjects. More than 300 proteins were identified, while 19 of them were found in the EBC samples of the donors who were diagnosed with lung cancer in the early stages and are potentially significant in the development of a diagnostic lung-cancer biomarker panel. It was shown that the EBC protein profiles of different donor groups can be distinguished. It may be possible to highlight a specific protein group that is typical for certain conditions/diseases of the respiratory system. Thus, the EBC analysis could be a promising non-invasive method for early diagnosis of lung cancer.