TEMPERATURE TITRATION - A NEW APPROACH TO THE THERMODYNAMICS OF OXYGEN-BINDING TO HEMOGLOBIN

A cell was constructed in order to study hemoglobin's reaction with ga seous ligands. The temperature of the hemoglobin sample is systematica lly altered within a given temperature range (275-310 degrees K), whil e the percentage of oxygen in the equilibrating gas is kept constant. The equilibration time of the sample at each temperature step depends on sample concentration, ligand affinity, and absolute temperature; in most cases, the equilibration time is on the order of minutes. The co nstruction of the optical compartment allows the experimenter to vary the optical pathlength using specially designed spacers, thus making i t possible to study hemoglobin-ligand interactions over a wide range o f protein concentrations (0.1-200 mg/ml). Optical glass is used in the construction of the cuvette in order to optimize its optical stabilit y over a long period of time. At equilibrium the absorption spectrum o f the sample is collected and decomposed into the relative contributio ns of oxy-Hb, deoxy-Hb, and ferric-Hb, thus revealing the fraction of oxyhemoglobin as well as any baseline drifts and protein degradation. Temperature steps of 1 degrees K are already sufficient to change the absorption spectra in a significant way. This type of setup is also ad vantageous in that the experimenter can change the sample at any point (temperature) without having to restart the entire experiment. This m akes it possible to study the oxygen binding characteristics of unstab le hemoglobins. Analyses of the binding curves obtained with this tech nique immediately yield the overall oxygen binding constants beta(i) t ogether with the respective standard enthalpies Delta H-i. (C) 1998 Ac ademic Press.