title: Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports.
abstract: 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 beta-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.
authors.affiliation: Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal.
authors.forename: V M
authors.initials: VM
authors.lastname: Balcão
authors.forename: C
authors.initials: C
authors.lastname: Mateo
authors.forename: R
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: F X
authors.initials: FX
authors.lastname: Malcata
authors.forename: J M
authors.initials: JM
authors.lastname: Guisán
mesh_terms: D001215:Asparaginase / Q000737:chemistry* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D020836:Protein Structure, Quaternary; D012685:Sepharose / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D012685:Sepharose; D001215:Asparaginase; D005976:Glutaral