The Kinetics of the Bohr Effect in the Reaction of Human Hemoglobin with Carbon Monoxide

Several years ago we made an attempt to measure the time course of the liberation of the Bohr protons accompanying the oxygenation of hemoglobin by the use of the pH indicator bromthymol blue in a stopped flow apparatus (1). The experiment failed in its original purpose because of the interactions of the indicator with the protein, but led t,o other interesting observations. Since then, the static relation between the binding of proton and the uptake of ligand has been studied in detail in equilibrium measurements in which pH was determined electromotively (2). These recent equilibrium studies, as well as recent results on the kinetics of the ligand binding process in relation to pH and their interpretation in terms of conformational effects (3, 4), reawaken interest in the original problem. The present study is based on the use of another pH indicator, phenol red, which, as is shown by an extensive series of controls, does not significantly interact wit’h hemoglobin under the conditions of the experiments. Its use has made possible the measurement of the time course of the pH change accompanying the reaction of human hemoglobin with carbon monoxide. The procedure used here may find application in the study of the kinetics of many other reactions of hemoglobin, and indeed of proteins generally associated with exchange of protons wit,h the medium. Human hemoglobin, prepared by t’he usual procedure (5), was dialyzed in the cold against COz-free twice distilled water. Hemoglobin concentrations were determined spectrophotometrically and are expressed in heme equivalents (6). The pH indicator, ljhenol red, was obt’ained from Merck Sharp and Dohme and was used without further purification. Static spectroscopic measurements were carried out with a Beckman model DKl recording spectrophotometer. Kinetic measurements were made with Gibson’s stopped flow apparatus (7), equipped with a 2-cm observation tube. The dead time of the apparat,us was 3 msec. The total spectral band width in all the experiments was less than 2 rnp. order to see whether phenol red could be used as a suitable pH indicator in the presence of hemoglobin, the absorption spectrum of the dye was measured in the absence and in the presence of oxyhemoglobin over the pH range 6.53 to 8.21 in 0.08 M phosphate buffers. The hemoglobin