Kinetics of iron hexacyanide release from the ferric hemes of partially oxidized intermediates of human deoxy hemoglobin.

A new experiment is presented in which the kinetics of iron hexacyanide release is measured from partially oxidized intermediates of human deoxy hemoglobin (T state). The intermediates were generated by briefly exposing a solution of hemoglobin and dithionite to various concentrations of ferricyanide at 6 degrees C in 0.1 M phosphate buffer, pH 7. The reaction of the residual dithionite with the partially oxidized intermediates was limited by the release of ferrocyanide and was strongly biphasic with about 75% fast phase and 25% slow and with the two phases differing in rate by a factor of 26. The predominance of rapid over slow phase is the result of a wide (approx. factor of 5) difference in the kinetics of oxidation of the alpha and beta chains by ferricyanide and is not due to heme-heme interaction. This experimental approach appears to offer a clear example of wide kinetic differences between the two types of subunits within the deoxy quaternary structure of human hemoglobin, uncomplicated by strong cooperative effects.