The pulse-radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced a or p subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH-values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second-order on-rate constants measured for the binding of oxygen to Hb3+ are 14 k 3 mM-' ms-' and 56 f 9 mM-' ms-I, respectively. The relative contribution of the faster fraction is about 0.63 f 0.08 of the total oxygenation process.A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret-region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on-rate constants.The intrinsic association and on-rate constants for the successive binding of oxygen to hemoglobin tetramers increase at each ligation step. This property of the hemoglobin molecule leads to the well-known sigmoid shape of the saturation curve for oxygen binding, indicating a positive mode of interaction between the subunits of the hemoglobin tetramer [1,2]. Monod et al. [3] proposed that the existence of a low affinity state for unliganded hemoglobin (T-state) and for a high affinity state of liganded hemoglobin (Rstate) can explain the positive mode of interaction. These two states of the MWC-model correspond to the fully liganded and unliganded X-ray structures observed by Perutz et al. [4].Recently it has been suggested by several investigators [5 -81 that the cooperative interactions of hemoglobin are not determined exclusively by the two quaternary conformations. The observed differences Abbreviations and Symbols. MetHb, methemoglobin; Hb4', Hb3+, Hb", €Ib+ : the superscripts refer to the number of oxidized subunits of the hernoglobin tetramer; Hb, deoxyhemoglobin; Hb*, rapidly reacting hemoglobin (R-state); HbOz, oxyhemoglobin; Bistris, [bis(2-hydroxyethyl)amino]tris (hydroxymethy1)methane.in the ligation and the redox reaction properties of the a and B subunits indicate that the predominance of one type of subunit may introduce an apparent slight negative cooperativity into the hemoglobin tetramer. It is evident that the non-equivalence of the subunits will be observed most favourably when the hemoglobin molecules are in the same quaternary conformation. The purpose of this paper is to establish the contribution of the a-p chain heterogeneity to the partial reduction of methemoglobin to Hb3+ and the kinetics of oxygen binding to this species.It is more difficult to determine the intrinsic reaction rate constants for the combination of oxygen with hemoglobin than for carbon monoxide to ...