Characterization of ligand migration mechanisms inside hemoglobins from the analysis of geminate rebinding kinetics

The presence of internal hydrophobic cavities and packing defects has been demonstrated for several small globular proteins, including hemoglobins. The reduced thermodynamic stability appears to be compensated for by the capability of controlling ligand diffusion through the protein matrix to the active site, possibly by stocking more than one reactant molecule in selected sites. Photolysis of carbon monoxide complexes of hemoglobins encapsulated in silica gels leads to multiphasic geminate rebinding kinetics at room temperature, reflecting rebinding also from different temporary docking sites inside the protein matrix. A careful analysis of the ligand rebinding kinetics allows the determination of the microscopic rates for the underlying reactions, including those governing the migration to and from the docking sites. This chapter describes the experimental approach used to characterize the ligand rebinding kinetics for heme proteins in silica gels after nanosecond laser flash photolysis and the computational methods necessary to retrieve the kinetic parameters.