Development of an immobilized brain glutamine synthetase liquid chromatographic stationary phase for on-line biochemical studies

Glutamine synthetase (GS) plays a key role in the regulation of glutamate availability to neurons. In the present study glutamine synthetase was immobilized on a silica-based immobilized artificial membrane liquid chromatographic stationary phase (IAM-SP) to create the GS-IAM. The stability of GS was improved by immobilization, but the enzyme’s affinity for the substrates l-glutamate and d-glutamate was significantly decreased. In contrast, immobilization significantly increased GS sensitivity to inhibition by methionine sulfoximine. The GS-IAM was packed into a chromatography column to create an immobilized enzyme reactor (GS-IMER). On-line experiments with the GS-IMER demonstrated that the immobilized enzyme was comparable to the non-immobilized enzyme with regards to retention of activity and selectivity toward substrates and inhibitors and was reusable for several weeks.