Author(s):
Balcão, Vitor M. 
; Mateo, Cesar ; Fernández-Lafuente, R. ; Malcata, F. Xavier ; Guisán, José M.
Date: 2001
Persistent ID: http://hdl.handle.net/10400.14/6917
Origin: Veritati - Repositório Institucional da Universidade Católica Portuguesa
Description
A new protocol for the stabilization of the quaternary structure of multimeric enzymes
has been attempted using as model enzyme (tetrameric) L-asparaginase from Escherichia
coli. Such strategy is based upon multisubunit covalent immobilization of the
enzyme onto activated supports (agarose-glutaraldehyde). Supports activated with
different densities of reactive groups were used; the higher the density of groups, the
higher the stabilization attained. However, because of the complexity of that enzyme,
even the use of the highest densities of reactive groups was not enough to encompass
all four subunits in the immobilization process. Therefore, a further chemical
intersubunit cross-linking with aldehyde-dextran was pursued; these derivatives
displayed a fully stabilized multimeric structure. In fact, boiling the modified enzyme
derivative in the presence of sodium dodecyl sulfate and â-mercaptoethanol did not
lead to release of any enzyme subunit into the medium. Such a derivative, prepared
under optimal conditions, retained ca. 40% of the intrinsic activity of the free enzyme
and was also functionally stabilized, with thermostabilization enhancements of ca. 3
orders of magnitude when compared with its soluble counterpart. This type of
derivative may be appropriate for extracorporeal devices in the clinical treatment of
acute leukemia and might thus bring about inherent advantages in that all subunits
are covalently bound to the support, with a longer half-life and a virtually nil risk of
subunit release into the circulating blood stream.
