17 alpha (haloacetamidoalkyl) estradiols alkylate the human estrogen receptor at cysteine residues 417 and 530

Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17 alpha-(haloacetamidoalkyl) estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine --> alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong evidence that only C417 and C530 were sites for electrophile covalent attachment. Since C530 was also alkylated by tamoxifen aziridine, a nonsteroidal affinity-labeling agent, we propose a selective mode of superimposition of tamoxifen-class antiestrogens with estradiol, which could account for the relative positioning of the two types of ligands in the receptor hormone-binding pocket. According to the structure of the hormone-binding pocket of nuclear receptors, as inferred from crystallographic studies and general sequence alignment of hormone-binding domains, C417 and C530 appear to be (1) located at the extreme border or in structural elements involved in delineation of the hormone-binding pocket, (2) spatially in close proximity to each other, and (3) in positions highly homologous to those of glucocorticoid receptor sites alkylated by affinity- and photoaffinity-labeling agents, respectively.     

Separation of nickel from cobalt using electrodialysis in the presence of EDTA

Optimum conditions are determined for the removal of nickel from cobalt solutions by electrodialysis exploiting the greater stability of the EDTA complex with nickel. The Ni–(EDTA)^2– complex and hydrated Co²⁺ ions are transferred from the feed solution to the electrodialysis anolyte and catholyte chambers, respectively. A three compartment cell is required to prevent the transfer of hydrated Ni²⁺ from the anolyte chamber as the EDTA present is destroyed at the anode. Complete removal of nickel from cobalt can be achieved but there is a compromise between cobalt purity and the percentage of cobalt transferred to the catholyte chamber for recovery.     

Anodic oxidation of the dye materials methylene blue,acid blue 25, reactive blue 2 and reactive blue 15 and the characterisation of novel intermediate compounds in the anodic oxidation of methylene blue

Anodic oxidation of the dye molecules, methylene blue, acid blue 25, reactive blue 2 and reactive blue 15 in chloride solution leads to colour destruction but UV and TOC data show that the oxidation reactions do not lead to complete destruction of the organic molecules. Analysis of the anodic oxidation products of [3,7‐bis (dimethylamino) phenothiazinium] chloride (methylene blue) in a chloride solution provides evidence for formation of seven neutral and two charged intermediates. The main intermediate is identified by its X‐ray diffraction crystal structure and accurate mass spectrometry as the novel leuco dye 4,6‐dichloro‐7‐dimethylamino‐3H‐phenothiazin‐3‐one, C₁₄H₁₀Cl₂N₂OS (I) formed by replacement of one of the dimethylamino groups of methylene blue with oxygen accompanied by regiospecific chlorination of the carbocyclic systems. The mass spectra of other intermediates formed are interpreted in terms of the structure of I. © 2002 Society of Chemical Industry