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M. A. Malakhova, M. V. Pokrovskaya, S. S. Alexandrova, N. N. Sokolov, E. V. Kudryashova
Regulation
of catalytic activity of recombinant L-asparaginase from Rhodospirillum
rubrum by conjugation with a PEG-chitosan copolymer
Abstract
A new approach for improvement of biocatalytic properties of
L-asparaginase is developed. The approach is based on modification of the
enzyme by copolymers of polyethylene glycol (PEG) and chitosan
(chitoPEGylation). One of the promising preparations of L-asparaginases is an
enzyme from Rhodospirillum rubrum (RrA). This medicine is
immunologically distinct from used preparations from L-asparaginase E. coli,
which determines the prospects for its use as an alternative in the development
of hypersensitivity. Advantage of RrA is its high antitumor activity, as well
as low activity on L-glutamine, which significantly reduces the likelihood of
side effects from the use of such a drug. In the present work, the synthesis of
RrA conjugates with PEG-chitosan with various modification degrees was carried
out. The composition of the resulting conjugates was analyzed by IR
spectroscopy. The activity of RrA conjugates for L-asparagine and L-glutamine
in comparison with the native enzyme was determined. It was demonstrated that
chitoPEGylation improved catalytic activity of the enzyme. It was shown that
the affinity for the main substrate (L-asparagine) in the conjugate is higher
than for the native enzyme. It varies from 56 IU/mg for the native enzyme to
61–72 IU / mg for conjugates. The secondary structure of RrA conjugates with
PEG-chitosan was studied by CD and IR spectroscopy. It was found that the
structure of the enzyme undergoes only slight changes during the formation of
conjugates with PEG-chitosan: the content of α-helixes varies from 36% for native to 30–33% for
conjugates, the content of β-sheets
varies from 15% for the native enzyme to 18% for the conjugate. The results
obtained determine the outlook for the use of RrA conjugates with PEG-chitosan
for the development of highly effective new-generation L-asparaginase medicine
with improved biopharmaceutical properties.
Key words: L-asparaginase, PEG-chitosan, conjugates, catalytic
activity.
Copyright (C) Chemistry Dept., Moscow State University, 2002
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