L-asparaginase (L-ase for short), an exogenous tetrameric enzyme produced by E. coli, is at present clinically used in the treatment of acute leukemia and lymphosarcoma, via depletion of circulating (blood) pools of L-asparagine through endovenous administrations of soluble enzyme. Such therapy, although effective to a certain degree, has the disadvantages of a short circulation time in blood, as well as immunological side-effects ranging in severity from mild allergenic reactions to anaphylactic shock, since the enzyme is an alien protein regarding the human body.

The development of strategies that may permit structural and functional stabilization of L-ase during immobilization onto highly activated supports may increase the biomedical applicability of this enzyme. These derivatives could be utilized in extracorporeal devices to eliminate asparagine from the plasma with several clear advantages: a.- the structural stabilization would prevent release of enzyme-subunits to the blood, thus greatly decreasing the risks of allergenic reactions; and b.- the functional stabilization would increase the operative live of the extracorporeal device, hence simplifying use and decreasing operation prices.

In the present research work, the production of derivatives from L-ase, with full stabilization of its multimeric structure, has been attempted via covalent multi-subunit immobilization onto activated supports. Several variables were studied, viz. concentration and nature of active groups, incubation time and temperature. Boiling of the optimal enzyme derivative in the presence of sodium dodecyl sulfate and b-mercaptoethanol did not release protein into the supernatant (as revaled by SDS-PAGE), thus evidencing the attachment of all enzyme subunits to the support, with concomitant structural stabilization of the enzyme. This derivative presented 50% of the intrinsic activity of the immobilized enzyme. Such L-ase derivative was also functionally stabilized, with thermo-stabilization enhancements of ca. 2 orders of magnitude when compared with that soluble form of enzyme. The use of these new derivatives in extracorporeal bioreactors would possess inherent advantages, when compared with the current endovenous administration of the soluble form of the enzyme, in that all subunits are covalently bound to the support, so the risk of subunit release into the circulating blood stream is virtually nil.