title: 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.
  
  
  METHODS
  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
  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.
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Bruce X
authors.initials: BX
authors.lastname: Wong
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Scott
authors.initials: S
authors.lastname: Ayton
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Linh Q
authors.initials: LQ
authors.lastname: Lam
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Peng
authors.initials: P
authors.lastname: Lei
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Adlard
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia. Electronic address: ashleyib@unimelb.edu.au.
authors.forename: Ashley I
authors.initials: AI
authors.lastname: Bush
authors.affiliation: Oxidation Biology Unit, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia; School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, United Kingdom. Electronic address: J.A.Duce@leeds.ac.uk.
authors.forename: James A
authors.initials: JA
authors.lastname: Duce
keywords: Ceruloplasmin; Ferroxidase; Iron; Oxidation; Transferrin