Study of the thermal stability and enzymatic activity of an immobilised enzymatic system for the bilirubin oxidation

Abstract

In this work, we have studied the immobilisation of the haemoglobin/glucose oxidase coupled enzymatic system in poly(vinyl alcohol) membranes. These are to be used as a reagent phase either in the development of an optical sensor or as an efficient bilirubin (BR) removal reactor. Poly(vinyl alcohol) (PVA) was chosen as the support for this purpose, due to its good biocompatibility, hydrophilicity and non-thrombogenic effects. A hydrogel containing the enzymatic system, consisting of PVA crosslinked with glutaraldehyde, was prepared and characterised by DSC, enzyme activity measurements and release tests. Investigating protein conformational changes as a function of temperature and the enzymatic system activity we have found that, in spite of the destabilizing effect of the glutaraldehyde in the acidic medium, the PVA insolubilisation conditions seem do not perturb either the conformation of the [`]native state' nor the enzymatic system activity. Moreover, it was found that PVA/glutaraldehyde membranes offer a simple way to hold enzymatic system, with the possibility of controlling the conditions to obtain either the effective prevention of leaching of the entrapped proteins or the in situ delivery of the haemoglobin.