Iron is the most prevalent metal in biology. Its chemical and redox versatility allows it to direct activity of many Fe binding proteins. While iron's biological applications are diverse, challenges inherent in having Fe(II) present at high abundance means cells must ensure delivery to the correct recipient, while also ensuring its chemistry is regulated. Having a detailed understanding of the biophysical characteristics of a protein's iron binding characteristics allows us to understand general cellular metal homeostasis events. Unfortunately, most spectroscopic techniques available to measure metal binding affinity require protein be in a homogeneous state. Homogeneity creates an artificial environment when measuring metal binding since within cells numerous additional metal binding biomolecules compete with the target. Here the authors investigate commercially available Fe(II) chelators with spectral markers coupled to metal binding and release to determine their utility as competitors while measuring aspects of metal binding by apoproteins, during a metal binding competition assay. Adding chelators during apoprotein metal binding mimics heterogeneous metal binding environments present in vivo, and provides a more realistic metal binding affinity measurement. Ferrous chelators explored within this report include: Rhod-5N, Magfura-2, Fura-4F, Fura-2, and TPA (Tris-(2-byridyl-methyl)amine. These chelators were used to calibrate binding affinities for yeast and fly frataxin (Yfh1 and Dfh, respectively).

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