Use of TSK-SW columns for the high-performance liquid chromatographic analysis of proteins, isolated from sympathetic nerves and fractionated by fractogel TSK-HW chromatography. Purification of L-DOPA decarboxylase.

The soluble proteins isolated from sympathetic nerves were separated on Fractogel TSK-HW columns. With a mobile phase of 0.1 M phosphate + 0.1 M K2SO4, pH 6.8, the main fractions I-VI were obtained. These fractions were analysed by high-performance (HPLC) on TSK-SW columns. Fractogel fractions I-III showed peaks of molecular weights, Mr670,000, as estimated by HPLC. With sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) these fractions show no bands stainable with Coomassie Blue. The protein of fraction IV was L-DOPA decarboxylase (AADC E.C. 4.1.1.28) with Mr 150,000 existing of subunits with Mr 55,000, 45,000, 27,000 and purified according to Christenson et al. (Arch. Biochem. Biophys., 141 (1970) 356). The dopamine-beta-hydroxylase (E.C. 1.14.2.1) subunits with Mr 75,000 proteins were detected in Fractogel fraction V. Fraction VI was Mr 27,000 protein. Proteins with molecular weights Mr less than 5,000 were also detected. With Phenothiazine-Affigel the proteins of fraction V (mr 75,000) showed no affinity to the phenothiazine column equilibrated with application buffer containing Ca2+ X 50-70% fraction IV (Mr 150,000), eluted with Tris-EGTA buffer, and 100% fraction VI (Mr 27,000) showed affinity to the Phenothiazine-Affigel column.