The efforts of this laboratory have focused on understanding the basic mechanisms by which trace elements are absorbed, transported, assimilated, stored and excreted. This knowledge makes possible the detection and prevention of pathologies associated with both deficiencies and toxicities related to these essential metals. Currently, our three principal research goals are to understand: 1) The mechanisms by which intracellular and intramolecular electron transport regulate and control Fe(lll) and Cu(ll) metabolism; 2) How Fe(lll) and Cu(ll) in the presence of reducing agents cause pathological damage to cells and tissue by """"""""oxidative stress""""""""; 3) The role of low molecular weight ligands in directing and regulating trace element binding to site-specific loci on proteins. Heme proteins including hemoglobin, myoglobin and cytochrome beta5 reduce complexes of Fe(lll) and Cu(ll). Transfer of heme-iron electrons to Fe(lll) varies as a function of the redox potential and stability constant of the Fe(lll)-complex, as well as the omega-bonding of the complex with heme. Hemoglobin, uniquely, manifests site-specific binding of Fe(lll) prior to reduction. Electron transfer to Cu(ll) requires site-specific binding to favorably modify both the half-cell potential of the Cu(ll) and the entropy of ligands surrounding the metal. We will characterize the nature of these binding sites using the techniques of high resolution NMR, """"""""suicide labeling,"""""""" stopped-flow kinetics with completing medals, and electrochemical potential measurements. Additionally, the role of certain amino acid groups in the site- specific binding of metal ions will be defined using other heme proteins such as leghemoglobin and various myoglobin derivatives with specifically modified histidine residues. The pathological damage associated with thalassemic red cells can be mimicked in normal red cells. Fe(lll) and Cu(ll) complexes in the presence of ascorbic acid generate hydroxyl free radicals (OH). The ability of competing metal complexes to inhibit site-specific damage by displacement of redox metals and the addition of compounds to scavenge OH will be tested. Strategies for preventing cellular damage in reperfused ischemic heart tissue and the toxic effects of paraquat in E. coli will be developed. Low molecular weight chelators are necessary both to prevent hydrolysis of trace metals at physiologic pH and to effectively present the metals to specific sites on proteins. We will develop models for chelator/medal/protein interaction and test these models experimentally. These studies will be directed toward understanding the mechanisms by which biological activities of Fe(lll)-adriamycin are affected by chemical changes both in the chelator and the metal. There is a remarkable coherence among the three research goals in that site-specific binding, oxidation-reduction and chelation play significant roles in cellular metabolism of trace elements.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK012386-26
Application #
3224865
Study Section
Nutrition Study Section (NTN)
Project Start
1977-05-01
Project End
1994-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
26
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Masuoka, J; Saltman, P (1994) Zinc(II) and copper(II) binding to serum albumin. A comparative study of dog, bovine, and human albumin. J Biol Chem 269:25557-61
Potuznik, S; Gelvan, D; Burda, P et al. (1993) Thiols, gold-thiols, zinc-thiols and the redox state of hemoglobin. Biochim Biophys Acta 1164:289-98
Masuoka, J; Hegenauer, J; Van Dyke, B R et al. (1993) Intrinsic stoichiometric equilibrium constants for the binding of zinc(II) and copper(II) to the high affinity site of serum albumin. J Biol Chem 268:21533-7
Gelvan, D; Moreno, V; Gassmann, W et al. (1992) Metal-ion-directed site-specificity of hydroxyl radical detection. Biochim Biophys Acta 1116:183-91