The reaction between the Fe(II) chelating agent, bathophenanthrolinedisulfonate, and the iron-sulfur cluster in the Fe protein of nitrogenase from Clostridium pasteurianum has been studied. This reaction is greatly accelerated by the presence of MgATP. Analysis of the relationship between reaction rate and concentration of MgATP supports a model in which both of two binding sites for MgATP on the Fe protein must be occupied before the protein undergoes a conformational change, allowing the iron-sulfur site to react rapidly with chelator. This model is also consistent with presently available data on equilibrium binding of MgATP to the Fe protein. MgADP inhibits the effect of MgATP on the chelator reaction in a manner which suggests that MgADP binds strongly to one of the MgATP sites and more weakly to the other. Loss of enzymic activity due to exposure to O2 or 0 degrees C is accompanied by a decrease in the ATP-specific chelator reaction. Hence, this reaction was used to estimate the concentration of active iron-sulfur centers for the purpose of computing the extinction coefficient of the Fe protein, giving the value delta epsilon 430nm(ox-red) = 6600 M-1 cm-1.

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A new purification procedure that permits large-scale purification of dopamine P-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1 % Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis.The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH,-terminal amino acids do not support the suggestion that dopamine P-hydroxylase and chromogranin A contain identical peptide chains. Dopamine P-hydroxylase has been purified from bovine adrenal medulla [l-51 and from human pheochromocytoma [6]. The structure and catalytic mechanism of this copper-containing monooxygenase are still incompletely understood [7], but the published purification schemes cannot easily provide sufficient purified protein for the more extensive physical and chemical studies that are needed. Furthermore, conflicting reports on the amino acid composition [3,8, 9,101 and NH,-terminal amino acid [8,10] indicate that some of the published purification methods do not yield pure proteins.In the present publication we report a simple purification procedure that gives high yields of essentially homogeneous and highly active dopamine fl-hydroxylase. The method is suitable for large scale purification. We also report various chemical properties of the purified protein and compare these with previously published data. MATERIALS AND METHODS MaterialsC'oncanavalin A covalently linked to agarose (ConA-Sepharose) was obtained from Pharmacia.Diethylaminoethyl cellulose (DE52) was from Whatman. Polyethylene glycol (Carbowax 6000) was from Fluka. Methyl-or-D-mannopyranoside was from KochLight. Hydrochloric acid for hydrolysis was ultrapure grade from Pierce. Dansyl amino acid reference collection and polyamide layer sheets (manufactured by The Cheng Chin Trading Co.) were obtained from British Drug Houses. Dansyl chloride was from Pierce.[N-methyl-'4C]dansyl chloride (specific activity 20.3 Ci/ mol) was fromThe Radiochemical Centre (Amersham). Ampholine (Carrier Ampholytes) pH 3 -6 was from LKB-Produkter AB. Glucose oxidase reagent kit (Blood Sugar, GOD-Perid Method) was from Boehringer (Mannheim). Galactose oxidase reagent kit (Galax) was from AB Kabi (Stockholm). ...

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The enzyme‐bound copper of dopamine β‐monooxygenase reacted rapidly with the chelator bathocuproine disulfonate; the reaction in the presence of ascorbate was completed in 2 min at 25°C with 1 mM chelator. This reaction and also the reaction with EDTA could be used to prepare the apoenzyme, which in both cases was completely reactivated in less than 10 s. The reactivation data gave apparent Michaelis constants for copper of 0.03–0.2 μM. Trace amounts of copper in buffers and assay mixtures gave significant reactivation without added copper, unless they had been treated with a chelating resin. Titrations using the different chelation rates of free and enzyme‐bound copper indicated that four copper atoms are bound per enzyme molecule of four subunits. The native enzyme was more stable against thermal inactivation than the apoenzyme, but this stability was only partially restored by addition of copper to the apoenzyme.