Heterogeneity in the Kinetics of Oxygen Binding to Partially Reduced Human Methemoglobin

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Abstract
The pulse-radiolysis technique was introduced because it permits a rapid reduction (in a few microseconds) of 1 heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the O2 binding to this particular valence intermediate (Hb3+) with 1 reduced .alpha. or .beta. subunit was studied. The hydrated electrons apparently preferentially reduced 1 type of subunit of methemoglobin at acid and neutral pH-values as was shown by the biphasic behavior of Hb3+ on oxygenation. The 2nd order on-rate constants measured for the binding of O2 to Hb3+ were 14 .+-. 3 mM-1 ms-1 and 56 .+-. 9 mM-1 ms-1, respectively. The relative contribution of the faster fraction was about 0.63 .+-. 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 decrease 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.