KINETICS OF THE REACTIONS OF HEME PROTEINS WITH GASEOUS LIGANDS : STUDIES WITH STOPPED-FLOW SPECTROPHOTOMETRY : Ⅰ. THE REACTION OF MYOGLOBIN AND THE ISOLATED HEMOGLOBIN CHAINS WITH OXYGEN

A simple procedure for the determination of the dead time of the stopped-flow spectrophotometer was developed using the reducing reaction of 2, 6-dichlorophenolindophenol (DCIP) by L-ascorbic acid. The use of this simple irreversible reaction system made it possible to measure the dead time by a simple graphical analysis. Two other methods reported previously were also employed for the measurement. The values determined by the present method were in good agreement with those by the previous methods. Since the isolated hemoglobin chains as well as myoglobin exhibit no cooperativity in the O₂ equilibria, their reactions with O₂ are expected to be described by a single-step reaction mode. The kinetics of the reaction with O₂ of freshly prepared myoglobin from chicken gizzard and isolated chains from human adult hemoglobin (αᴬ and βᴬ) were studied with the stopped-flow method. The rate constants of O₂ association and dissociation were determined and the O₂ affinity, expressed as the O₂ pressure at half-saturation (P₅₀), was calculated therefrom. The values of P₅₀ calculated from the rate constants agree well with those obtained directly from the O₂ equilibrium measurements. I conclude that the reaction with O₂ of myoglobin and isolated hemoglobin chains is a single-step reaction