Abstract The reaction of hemoglobin with oxygen allows oxygenation of the body, ensuring homeostasis and cell survival. Although it occurs in four reversible, successive, and cooperative steps, the reaction is usually treated as a one-step reaction. This article aims to point out experimental and theoretical aspects relevant to the kinetic study of this reaction, but which are generally not taken into account. Just the oxyhemoglobin deoxygenation is discussed, since this step is easier to perform experimentally when compared to oxygenation. Oxyhemoglobin was obtained from the reduction of meta-hemoglobin by size exclusion liquid chromatography using sodium dithionite. Deoxygenation of oxyhemoglobin was performed by rapid mixing with interrupted flow, also using sodium dithionite, but in the absence of oxygen. From the experimental results, the dissociation constant was determined considering the pseudo-first order model. Subsequently, experimental questions were discussed, such as the contribution of at least two hemoglobin conformations in the measured absorbance and theoretical questions, such as limitations of the first order pseudo model. The results indicate that a more complete model is needed to describe oxyhemoglobin deoxygenation as well as a more sophisticated treatment in the experimental data, considering that more than one hemoglobin conformation has a significant absorbance value at a given wavelength.
Keywords oxyhemoglobin; deoxygenation;rapid mixing method; pseudo-first order kinetics.