ABSTRACT
As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.
Subject(s)
Copper/metabolism , Fatty Acids/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Antioxidants/chemistry , Antioxidants/metabolism , Ascorbic Acid/chemistry , Ascorbic Acid/metabolism , Copper/chemistry , Cysteine/chemistry , Electron Spin Resonance Spectroscopy , Ethylmaleimide/chemistry , Ethylmaleimide/metabolism , Humans , In Vitro Techniques , Macromolecular Substances , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Oxidants/chemistry , Oxidants/metabolism , Oxidation-Reduction , Protein ConformationABSTRACT
A new fluorescent test developed at the Institute of Physicochemical Medicine (Ministry of Health of the Russian Federation) allows to determine not only blood concentration of albumin, but also to evaluate the state of its molecule. The test is feasible and enables express-analysis of the plasma and serum without fractionation and other preliminary procedures. This test reveals abnormalities in albumin molecule caused by toxic metabolites or conformational changes. Many diseases are accompanied by conformational changes in albumin, while its concentration often remains unchanged. Changes in albumin conformation can serve a diagnostic and prognostic criterion in some pathologies.
