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 K_d, k_on, and k_off for O₂ binding.