Inhibition of copper-catalyzed cysteine oxidation by nanomolar concentrations of iron salts.
Problems caused by the presence of adventitious metals in buffers and reagents are well recognized in studies of metal-catalyzed oxidation reactions. In most cases, metal contamination leads to an increase in rate, and chelating agents are inhibitory. In the present study, however, the rate of copper-catalyzed oxidation of cysteine was found to be increased by buffer purification with Chelex resin or by addition of micromolar concentrations of the specific iron chelator desferrioxamine (DFO). These effects are attributable to inhibition of copper-catalyzed oxidation by adventitious iron. In purified buffer at pH 7.25, containing 0.4 microM copper, cysteine was oxidized at a rate of 32 microM/min. Addition of iron salts to this buffer caused a dose-related decrease in this rate, up to a maximum of 85%. A 50% decrease in rate was recorded at an iron concentration of only 11 nM. Other transition metals were without effect. Similar effects of purification or addition of DFO on the rate of cysteine oxidation were seen in Tris, glycylglycine, Mops, and Pipes buffers. Catalase decreased the rate of cysteine oxidation, but the sensitivity to iron was similar in the presence and absence of catalase. Copper-catalyzed oxidation of cysteamine and reduced glutathione was much less sensitive to inhibition by iron. Our results offer an explanation for the conflicting literature reports of the effects of chelating agents and catalase on cysteine oxidation, and emphasize the need for buffer purification or addition of DFO in studies concerned with the oxidation or cytotoxicity of this thiol. The exceptional sensitivity of copper-catalyzed cysteine oxidation to iron makes this an attractive system for monitoring the iron content of buffers, and may also have application for determining the free iron content of physiological fluids.
Munday
Rex
R
AgResearch, Ruakura Research Centre, Hamilton, New Zealand. rex.munday@agresearch.co.nz
Munday
Christine M
CM
Winterbourn
Christine C
CC
D002021:Buffers; D002374:Catalase / Q000737:chemistry; D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D058085:Iron Compounds / Q000737:chemistry / Q000494:pharmacology*; D010084:Oxidation-Reduction; D012492:Salts / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry
D002021:Buffers; D002614:Chelating Agents; D058085:Iron Compounds; D012492:Salts; D013438:Sulfhydryl Compounds; D003300:Copper; D002374:Catalase; D003545:Cysteine