{Reference Type}: Journal Article
{Title}: Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins.
{Author}: Damodaran AR;Bhagi-Damodaran A;
{Journal}: Methods Mol Biol
{Volume}: 2648
{Issue}: 0
{Year}: 2023
暂无{DOI}: 10.1007/978-1-0716-3080-8_1
{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 (Kd) 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 Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.