The reactivity of globins towards gaseous ligands is an important indication for the function these proteins might fulfill in vivo, and depends for a large part on the ligation state of the heme Fe-atom. The ligand binding affinities and kinetics can be determined by flash photolysis and stopped flow.
In flash photolysis experiments, a short laser pulse (5-7 ns) is used to dissociate the bound ligand from the heme iron atom. The use of a laser for sample excitation gives the technique the specificity of single wavelength excitation and nanosecond time resolution. Photo-dissociated ligands first migrate into open spaces within the globin interior and then diffuse back to the distal pocket, where they either rebind to the heme iron atom, a process called geminate recombination, or escape to the solvent. The ligand can then rebind to the heme iron atom from outside the protein matrix, called bimolecular rebinding.
Our lab is equipped with the LP920 laser photolysis system (Edinburg Instruments) coupled to a frequency-doubled Q-switched Nd:YAG laser (Spectra Physics Quanta-ray).
In stopped flow experiments, two reagents are rapidly mixed together and then ‘stopped’ in an observation cell. The sample cell is irradiated with (usually) monochromatic light and as the reaction proceeds the change in the recorded signal, usually a fluorescence signal or the absorbance at a specific wavelength, is recorded as a function of time.
Our lab is equipped with a SX20 Stopped Flow Reaction Analyser from Applied Photophysics.