A comparison of ceruloplasmin to biological polyanions in promoting the oxidation of Fe(2+) under physiologically relevant conditions.

ABSTRACT

BACKGROUND: Iron oxidation is thought to be predominantly handled enzymatically in the body, to minimize spontaneous combustion with oxygen and to facilitate cellular iron export by loading transferrin. This process may be impaired in disease, and requires more accurate analytical assays to interrogate enzymatic- and auto-oxidation within a physiologically relevant environment. METHOD: A new triplex ferroxidase activity assay has been developed that overcomes the previous assay limitations of measuring iron oxidation at a physiologically relevant pH and salinity. RESULTS: Revised enzymatic kinetics for ceruloplasmin (Vmax≈35µMFe(3+)/min/µM; Km≈15µM) are provided under physiological conditions, and inhibition by sodium azide (Ki for Ferric Gain 78.3µM, Ki for transferrin loading 8.1×10(4)µM) is quantified. We also used this assay to characterize the non-enzymatic oxidation of iron that proceeded linearly under physiological conditions. CONCLUSIONS AND GENERAL SIGNIFICANCE: These findings indicate that the requirement of an enzyme to oxidize iron may only be necessary under conditions of adverse pH or anionic strength, for example from hypoxia. In a normal physiological environment, Fe(3+) incorporation into transferrin would be sufficiently enabled by the biological polyanions that are prevalent within extracellular fluids.