Abstract
Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (K(d)) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of K(d) of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate K(d) determination using Cs H-NOX (K(d) = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.