Purification and immunochemical characterization of dopamine β hydroxylase from human pheochromocytoma

Dopamine β hydroxylase was purified from a human pheochromocytoma. Following sucrose density gradient centrifugation the enzyme was isolated in three different molecular forms. The bulk of the enzymatic activity was associated with fraction II (approximate mol wt 286,000), which possessed the highest specific activity. The specific activity of dopamine β hydroxylase in fraction I (approximate mol wt 164,000) and in fraction III (approximate mol wt 524,000) was 4 times lower than in fraction II. A specific antiserum to human dopamine β hydroxylase isolated from fraction II was produced in rabbits (antiserum H). Electrophoretic and immunological evidence supports the contention that dopamine β hydroxylase in fraction II was isolated in pure form. The lower homospecific activity of dopamine β hydroxylase in fraction I as compared with fraction II and the immunological titration data indicated that fraction I contained more enzyme protein than was apparent from the enzyme activity. The immunological titration studies showed that antiserum H, as well as the antiserum to bovine adrenal dopamine β hydroxylase (antiserum B), reduced the activity of the homologous enzyme more effectively than the activity of the heterologous enzyme. The poor interspecies crossreactivity suggests that for measurements of human serum dopamine β hydroxylase levels by radioimmunoassay a homologous system is required.