MODULATED EXCITATION OF SINGLY LIGATED CARBOXYHEMOGLOBIN

We have extended the method of modulated excitation, a small perturbat ion kinetic method, to study ligand binding and conformational change of hemoglobin tetramers with a single ligand bound. To restrict the ex citation to the first ligand, only 1% of the hemes have bound CO, and the remainder are kept unliganded. A detailed theory is presented whic h agrees well with the experimental observations. This method of obser ving ligand recombination also provides a novel and simple method for determination of hemoglobin concentration. Additional relaxation proce sses are also observed. By fitting ir dependently determined spectra t o the spectra associated with the relaxations, these processes are ass igned as thermal excitation and thermally driven protonation/deprotona tion reactions. These added relaxations arise from the deoxy-Hb portio n of the samples, and demonstrate that modulated excitation can be use d effectively for temperature perturbation in the absence of photodiss ociation. The spectra observed are not well described by the spectra o f allosteric change, however, and we conclude that there is no signifi cant mixing of quaternary states at the first ligation step. In an app endix we present a derivation of the particular features seen in therm ally modulated protonation reactions.