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Title: Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level

Abstract

Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{alpha}-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) inmore » which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.« less

Authors:
;  [1];  [2]
  1. Dept of Biochemistry and Molecular Biology, Oregon Health and Science University (OHSU), Portland, OR 97239-3098 (United States)
  2. Dept of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University (OHSU), Beaverton, OR 97006-8921 (United States)
Publication Date:
OSTI Identifier:
21054623
Resource Type:
Journal Article
Resource Relation:
Journal Name: AIP Conference Proceedings; Journal Volume: 882; Journal Issue: 1; Conference: XAFS13: 13. international conference on X-ray absorption fine structure, Stanford, CA (United States), 9-14 Jul 2006; Other Information: DOI: 10.1063/1.2644515; (c) 2007 American Institute of Physics; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; ABSORPTION SPECTROSCOPY; COORDINATION NUMBER; COPPER; CYSTEINE; DISEASES; FLUORESCENCE; GENES; LIGANDS; LIVER; LIVER CELLS; METABOLISM; MICE; MOLECULES; PHOSPHINES; PROTEINS; VALENCE; X-RAY FLUORESCENCE ANALYSIS; X-RAY SPECTRA; X-RAY SPECTROSCOPY

Citation Formats

Ralle, Martina, Lutsenko, Svetlana, and Blackburn, Ninian J. Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level. United States: N. p., 2007. Web. doi:10.1063/1.2644515.
Ralle, Martina, Lutsenko, Svetlana, & Blackburn, Ninian J. Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level. United States. doi:10.1063/1.2644515.
Ralle, Martina, Lutsenko, Svetlana, and Blackburn, Ninian J. Fri . "Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level". United States. doi:10.1063/1.2644515.
@article{osti_21054623,
title = {Using XAS and SXRF to Study Copper in Wilson Disease at the Molecular and Tissue Level},
author = {Ralle, Martina and Lutsenko, Svetlana and Blackburn, Ninian J.},
abstractNote = {Wilson disease (WD) is a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. In WD, the billiary copper excretion is impaired and copper accumulates in tissues, particularly in the liver and the brain. The affected gene, ATP7B, encodes the copper transporting ATPase, Wilson disease protein (WNDP). WNDP has six copper binding sites in the N-terminal portion of the molecule. Each site includes the conserved amino acid sequence MXCXXC, and binds 1 Cu(I) through its 2 cysteine residues. We performed X-ray absorption studies at the Cu K{alpha}-edge on the recombinant N-terminal domain of WNDP (N-WNDP). Copper was bound to N-WNDP either in vivo or in vitro in the presence of different reducing agents. We found that in N-WNDP copper is predominantly coordinated in a linear fashion by two cysteines, with the appearance of a Cu-Cu interaction when all metal binding sites are filled. Increasing amounts of reducing agents containing sulfide or phosphine groups led to binding of the exogenous ligands to copper thereby increasing the coordination number of copper from two to three. To better understand the role of copper in WD, we utilized livers of the 6-weeks-old Atp7b-/- mice (an animal model for WD) in which the copper concentration was 10-20-fold higher compared to that of the control mice. The distribution of copper in hepatocytes was evaluated by synchrotron based X-ray fluorescence microprobe (SXRF). We demonstrate that we can prepare liver slices that retain copper and can detect copper with subcellular resolution. On the same sections {mu}-XANES (spot size: 5 micron) was used to determine the oxidation state of copper.},
doi = {10.1063/1.2644515},
journal = {AIP Conference Proceedings},
number = 1,
volume = 882,
place = {United States},
year = {Fri Feb 02 00:00:00 EST 2007},
month = {Fri Feb 02 00:00:00 EST 2007}
}