Probing heme protein-ligand interactions by UV/visible absorption spectroscopy.
Ultraviolet/visible (UV/vis) absorption spectroscopy is a powerful tool for steady-state and time-resolved studies of protein-ligand interactions. Prosthetic groups in proteins frequently have strong electronic absorbance bands that depend on the oxidation, ligation, and conformation states of the chromophores. They are also sensitive to conformational changes of the polypeptide chain into which they are embedded. Steady-state absorption spectroscopy provides information on ligand binding equilibria, from which the Gibbs free energy differences between the ligated and unligated states can be computed. Time-resolved absorption spectroscopy allows one to detect short-lived intermediate states that may not get populated significantly under equilibrium conditions, but may nevertheless be of crucial importance for biological function. Moreover, the energy barriers that have to be surmounted in the reaction can be determined. In this chapter, we present a number of typical applications of steady-state and ns time-resolved UV/vis absorption spectroscopy in the study of ligand binding to the central iron in heme proteins.
Nienhaus
Karin
K
Department of Biophysics, University of Ulm, Ulm, Germany.
Nienhaus
G Ulrich
GU
D000818:Animals; D001665:Binding Sites; D005914:Globins / Q000737:chemistry / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D000078787:Neuroglobin; D010782:Photolysis; D011485:Protein Binding; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics
D006420:Hemeproteins; D006454:Hemoglobins; D008024:Ligands; D009211:Myoglobin; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D005914:Globins