Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active α and β subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kontani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16°. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studied at 77 K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the α and β chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10 2 times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy α chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy β chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of α and β chains were indistinguishable. These observations suggest that one of the inequivalences between α and β chains might exist near the distal histidine group.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1977|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology