Simple assay procedure for tyrosine hydroxylase activity by high-performance liquid chromatography employing coulometric detection with minimal sample preparation

Abstract

A simple assay procedure for tyrosine hydroxylase activity in crude tissue samples was devised that requires minimal sample preparation and use of high-performance liquid chromatography with coulometric electrochemical detection. After incubation of enzyme samples, such as human brain homogenates or rat pheochromocytoma PC12h cells, with L-tyrosine and a tetrahydropterin cofactor, in the presence or absence of p-bromobenzyloxyamine, an inhibitor of aromatic L-amino acid decarboxylase, the reaction was terminated by addition of an equal volume of 0.1 M perchloric acid. For quantitation of L-DOPA produced, the sample was centrifuged, filtered and directly applied to the chromatographic apparatus connected to a coulometric electrochemical detector. This method makes redundant a time-consuming step in the previous methods, purification and concentration of L-DOPA or dopamine using alumina. The reaction conditions for the assay of tyrosine hydroxylase activity in brain homogenates and PC12h cells were re-examined by this method. Both tyrosine hydroxylase samples required a naturally occurring cofactor, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4], catalase and NSD-1055 for the full activity, and tyrosine hydroxylase in human brain homogenates required Fe2+ ions for its full activity. (6R)BH4 proved to be a more effective cofactor than a synthetic cofactor, (6RS)-methyl-5,6,7,8-tetrahydropterin, which is commonly used for this assay.