Efficient Immobilization of Phospholipase A₂

Abstract

Immobilized enzymes have been widely used for biomedical and industrial applications. Since phospholipase A₂ (PLA₂; E.C.3.1.1.4) catalyzes the hydrolysis of a wide range of phospholipid aggregates, immobilized PLA₂ should also have potential biomedical applications. For instance, a novel therapy for hypercholesterolemia has been recently developed that utilizes immobilized PLA₂ contained in an extracorporeal shunt, based on the finding that human serum low density lipoprotein modified by immobilized PLA₂ was removed from the circulation by liver more rapidly than unmodified low density lipoprotein (1). Also, immobilized PLA₂ has been used to study the mechanism of interfacial activation of PLA₂ (2,3). To date, the immobilization of PLA₂ has been carried out primarily with cobra venom enzymes using two different coupling methods; the coupling through the ɛ-amino group of lysines and the coupling through the carboxyl groups of asparatates and glutamates (3). These immobilization methods, however, often lead to a significant loss of enzymatic activity of PLA₂, thereby limiting the application of immobilized PLA₂. For instance, when the ɛ-amino groups are coupled to a solid support, the coupling yield is high but the activity of immobilized enzyme toward aggregated phospholipid substrates decreases drastically, which has been ascribed to the modification of the lysyl residues that play critical roles in the interaction of PLA₂s with anionic interfaces. On the other hand, the coupling of protein carboxylic groups to the solid support is hampered by a lower coupling yield and modification of catalytically important residues. On the other hand, the coupling of protein carboxylic groups to the solid support is hampered by a lower coupling yield and modification of catalytically important residues.