Measurement of O2 Binding by Sensory Hemeproteins.

The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.