Tetranuclear and dinuclear phenoxido bridged copper complexes based on unsymmetrical thiosemicarbazone ligands.

We report on the synthesis of new dinucleating phenol-based "end-off" compartmental ligands HLMeH and HLMe2 bearing two different binding sites, one bis(2-methylpyridyl)aminomethyl (BPA) and one thiosemicarbazone (TSC) site, and their corresponding copper(ii) complexes 1t and 2d. With the ligand HLMeH, a tetranuclear entity (1t) has been isolated in the solid state, whereas with HLMe2, which differs from HLMeH by a methyl substituent on the N-terminal amino group of the TSC arm, a dinuclear form (2d) is obtained. X-ray crystallography analysis shows that the nuclearity di vs. tetra is modulated by interactions between copper atoms and hydroxido bridges along with the sulphur atoms of TSC arms. From a magnetic point of view, 1t can be considered as an association of two dinuclear forms leading for both complexes to overall antiferromagnetic coupling. Analysis in acetonitrile solution of structure-property relationships has been carried out by comparing their UV/Vis, electrochemistry, ESI-MS, and NMR (variable temperature and DOSY = diffusion ordered spectroscopy) properties with trends from computational calculations (DFT). HRMAS-DOSY (High Resolution Magic Angle Spinning) NMR spectroscopy has been performed to evaluate the presence of different species in solution at room temperature.

Dopamine beta-hydroxylase inactivation generates a protein-bound quinone derivative.

Bovine dopamine beta-hydroxylase (DbH) was inactivated by hydrogen peroxide and ascorbate in the presence of dioxygen. Both inactivated forms of the enzyme were investigated. We could highlight the presence of a quinone derivative bound to the protein, assumed as being dopa-quinone, that is absent from active enzyme. Such results suggest that a tyrosinyl radical transiently forms during catalysis. Moreover we could show that addition of substrate tyramine to H2O2 incubates is responsible for a partial protection of DbH against inactivation.

New approaches to chromatographic purification of bovine dopamine-beta-hydroxylase.

The use of the traditional scheme for the isolation of bovine dopamine-beta-hydroxylase (bDBH) from bovine adrenal medulla resulted in active but not pure bDBH, containing about 50% of admixtures. Immobilized metal chelate affinity chromatography on agarose modified with iminodiacetic acid residues and charged with cobalt ions was applied in the final stage to obtain more than 90% pure and active bDBH. Final purification of bDBH using step elution with 0-0.5 M methyl-D-mannoside in buffer solution from concanavalin A-Sepharose was studied. The determination of bDBH in various samples was performed using size-exclusion chromatography.