Abstract

Several investigators have reported multiple molecular forms of dopamine-β-hydroxylase purified from human pheochromocytoma and human plasma. We purified the soluble dopamine-β-hydroxylase from the chromaffin vesicles of two human pheochromocytomas, using carbohydrate affinity chromatography on Concanavalin A-Sepharose. The purified products were electrophoretically homogeneous. No evidence of multimeric forms of the enzyme were found by analytical polyacrylamide gel electrophoresis, preparative polyacrylamide gel electrophoresis with activity elution, analytical gel filtration chromatography, or zonal sedimentation on sucrose density gradients. By combining the hydrodynamic parameters of s_20,w and Stokes radii, the tumor enzyme was characterized as having a molecular mass of 300,000 to 317,000 daltons, as compared to the bovine adrenomedullary enzyme at 282,000 daltons. Its frictional ratio was 1.68 to 1.69, leading to anomalously high molecular weights when estimated by gel filtration alone. The tetrameric enzyme was composed of monomeric subunits of 78,000 to 80,500 daltons each, joined by disulfide linkages to form dimeric subunits of 155,000 to 160,000 daltons, two of which join by noncovalent interaction to form each tetramer.