In order to survive in an oxygen environment, aerobic organisms have developed numerous mechanisms to protect against oxygen radicals and singlet oxygen. One such mechanism, which appears to have attained particular significance during primate evolution, is the direct scavenging of oxygen radicals, singlet oxygen, oxo-haem oxidants and hydroperoxyl radicals by uric acid. In the present paper we demonstrate that another important ‘antioxidant’ property of uric acid is the ability to form stable co-ordination complexes with iron ions. Formation of urate-Fe3+ complexes dramatically inhibits Fe3+-catalysed ascorbate oxidation, as well as lipid peroxidation in liposomes and rat liver microsomal fraction. In contrast with antioxidant scavenger reactions, the inhibition of ascorbate oxidation and lipid peroxidation provided by urate9s ability to bind iron ions does not involve urate oxidation. Association constants (Ka) for urate-iron ion complexes were determined by fluorescence-quenching techniques. The Ka for a 1:1 urate-Fe3+ complex was found to be 2.4 × 10(5), whereas the Ka for a 1:1 urate-Fe2+ complex was determined to be 1.9 × 10(4). Our experiments also revealed that urate can form a 2:1 complex with Fe3+ with an association constant for the second urate molecule (K9a) of approx. 4.5 × 10(5). From these data we estimate an overall stability constant (Ks approximately equal to Ka × K9a) for urate-Fe3+ complexes of approx. 1.1 × 10(11). Polarographic measurements revealed that (upon binding) urate decreases the reduction potential for the Fe2+/Fe3+ half-reaction from −0.77 V to −0.67 V. Thus urate slightly diminishes the oxidizing potential of Fe3+. The present results provide a mechanistic explanation for our previous report that urate protects ascorbate from oxidation in human blood. The almost saturating concentration of urate normally found in human plasma (up to 0.6 mM) represents 5-10 times the plasma ascorbate concentration, and is orders of magnitude higher than the ‘free’ iron ion concentration. These considerations point to the physiological significance of our findings.
