Preparation methods and biocatalytic properties of covalent conjugates of rhodospirillum rubrum l-asparaginase
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Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow, Russia
V. N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia
Publication date: 2022-05-27
Public Health Toxicol 2022;2(Supplement Supplement 2):A4
Bacterial L-asparaginases have been successfully used for more than 50 years in the treatment of various types of leukemia, in particular for acute lymphoblastic leukemia (ALL). However, their use is associated with a number of side effects such as allergic reactions, blood clotting, and liver and nervous system disorders. Various methods of covalent modification of enzymes are used to improve the pharmaceutical characteristics of L-asparaginase preparations. For example, PEGylated E. coli L-asparaginase has a longer circulation time in the bloodstream and less immunogenicity. In our laboratory, the effect of different copolymers of chitosan, polyethylene glycol (PEG) and polyethyleneimine (PEI) on the properties of L-asparaginases from various bacterial sources is being studied. Special attention was paid to L-asparaginase of Rhodospirillum rubrum (RrA), which has a small amino acid sequence and high antitumor activity compared with commercial preparations.
We studied previously obtained covalent conjugates of L-asparaginase. The conjugates were obtained by modification of amino- and carboxyl groups of the protein. The first method was used to obtain RrA conjugates with chitosan-PEG (30 PEG chains) and chitosan-glycol (72 kDa). Carboxyl groups were modified using PEI (2 kDa), PEI-PEG (branched, 5 kDa), and polyamines (spermine, spermidine). Purification was performed on Amicon centrifugal filters. RrA preparations were characterized by infrared (IR) and CD spectrometry. The IR spectra of the native enzyme and the conjugates contain characteristic protein peaks of amide I (1600–1700 cm-1) and amide II (1500–1600 cm-1). The spectra of RrA-chitosan-PEG and RrA-chitosan-glycol conjugates contain a peak at 1080 cm-1 corresponding to vibrations of C-O-C bonds in the PEG chains and C-O-C, C-N and C-C bonds in the pyranose cycle of chitosan. The presence of these peaks confirms the modification of the protein by the mentioned copolymers. For the RrA-PEI-PEG conjugate one can also note a peak at 1100 cm-1 related to vibrations of the PEG chains and C-N bonds of PEI. The peak at 1465 cm-1 is responsible for vibrations of the N-H bonds as well as the scissor CH2 bonds. In the circular dichroism spectra of both the native enzyme and its modified forms, there is a distinct peak at 207–210 nm corresponding to alpha helices. To compare the specific catalytic activity of the conjugates, enzyme concentrations were determined from the ellipticity calibration dependence at wavelengths of 207 and 220 nm. The hydrolysis activity of 20 mM L-asparagine native RrA and its conjugates was determined by CD spectroscopy. The greatest increase in activity compared with the native enzyme (46 IU/mg) was observed for the RrA conjugate with chitosan-PEG (64 IU/mg). Cytotoxic activity was determined for the native enzyme and conjugates on cells of chronic myeloid leukemia (K-562) and T-cell acute lymphoblastic leukemia (Jurkat). RrA-chitosan-PEG and RrA-chitosan-glycol at a concentration of 10 IU/mL approximately equally reduced the survival of K-562 cells to 10–12% of the control, while the native enzyme only to 56%. On Jurkat cells, RrA-chitosan-glycol was more toxic at concentrations of 1–10 IU/mL than RrA-chitosan-PEG and native enzyme. Thus, the cell type and copolymer structure influence the toxicity of the L-asparaginase RrA conjugate. In addition, the cytotoxicity of the drug depends not only on its catalytic activity but also on its zeta potential.
The work was performed using equipment (FTIR spectrometer Bruker Tensor 27 and Jasco J-815 CD Spectrometer) of the program for the development of Moscow State University.
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