Synthesis and characterization of immobilized dopamine beta-hydroxylase inmembrane-bound and solubilized formats

Dopamine beta -hydroxylase (DBH) catalyzes the beta -hydroxylation of dopam ine to norepinephrine. The enzyme in chromaffin granules occurs in a solubl e form and a form confined to the surrounding membrane. DBH was noncovalent ly immobilized in the hydrophobic interface of an immobilized artificial me mbrane (IAM) liquid chromatographic stationary phase and the resulting DBH- IAM stationary phase was enzymatically active and was shown to mimic the me mbrane-bound form of the enzyme. DBH was also covalently immobilized onto a silica-based support containing, glutaraldehyde-P (Glut-P), The resulting DBH-Glut-P interphase was also enzymatically active, reproducible and shown to display characteristics of the solubilized enzyme. The results demonstr ate that the different immobilization methods utilized for the enzyme can b e used to quantitatively and qualitatively determine the enzyme kinetic con stants associated with enzyme/ substrate and enzyme/inhibitor interactions for the two distinct forms of the enzyme. These new entities can be used in basic biochemical studies as well as in high throughput screening of subst ances for DBH substrate/inhibitor properties. (C) 2001 Elsevier Science B.V . All rights reserved.