Modulated excitation of singly ligated carboxyhemoglobin

We have extended the method of modulated excitation, a small perturbation kinetic method, to study ligand binding and conformational change of hemoglobin tetramers with a single ligand bound. To restrict the excitation to the first ligand, only 1% of the hemes have bound CO, and the remainder are kept unliganded. A detailed theory is presented which agrees well with the experimental observations. This method of observing ligand recombination also provides a novel and simple method for determination of hemoglobin concentration. Additional relaxation processes are also observed. By fitting independently determined spectra to the spectra associated with the relaxations, these processes are assigned as thermal excitation and thermally driven protonation/deprotonation reactions. These added relaxations arise from the deoxy-Hb portion of the samples, and demonstrate that modulated excitation can be used effectively for temperature perturbation in the absence of photodissociation. The spectra observed are not well described by the spectra of allosteric change, however, and we conclude that there is no significant mixing of quaternary states at the first ligation step. In an appendix we present a derivation of the particular features seen in thermally modulated protonation reactions.