NMR was used to study the quarternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the α and β subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the α subunit 10 times more strongly than it does to the β subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5x10-3 s-1 from the β subunit and at 5x10-4 s-1 from α subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the α than the β subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both α and β subunits were oxidized at the same rate.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1979|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology