An enzymatic method for precise oxygen affinity measurements over nanomolar-to-millimolar concentration regime

Research output: Contribution to journalReview article

Abstract

Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source. Using this method, we measure thermodynamic and kinetic oxygen affinities (Kd and KM) of different classes of heme and non-heme metalloproteins involved in oxygen transport, sensing, and catalysis. The method enables oxygen affinity measurements over a wide concentration range from 10 nM to 5 mM which is unattainable by simply diluting oxygen-saturated buffers. In turn, we were able to precisely measure oxygen affinities of a model set of eight different metalloproteins with affinities ranging from 48 ± 3 nM to 1.18 ± 0.03 mM. Overall, the Cld method is easy and inexpensive to set up, requires significantly lower quantities of protein, enables precise oxygen affinity measurements, and is applicable for proteins exhibiting nanomolar-to-millimolar affinity values. Graphic abstract: [Figure not available: see fulltext.].

Original languageEnglish (US)
Pages (from-to)181-186
Number of pages6
JournalJournal of Biological Inorganic Chemistry
Volume25
Issue number2
DOIs
StatePublished - Mar 1 2020

Keywords

  • Dissociation constant
  • Heme
  • Metalloproteins
  • Non-heme iron
  • O affinity

PubMed: MeSH publication types

  • Journal Article
  • Review

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