Differences in spectra of alpha and beta chains of hemoglobin between isolated state and in tetramer.

The absorption spectrum of deoxygenated human hemoglobin differs from that of the isolated alpha and beta chains, and the difference has been ascribed to the conformational changes on which depends the cooperative ligand binding. Studies on hybrid-heme hemoglobins, in which one chain contains protoheme and the other chain mesoheme, showed that the prominent peak in the Soret region of the difference spectrum for deoxyhemoglobin is due to the spectral change in alpha chains and that beta chains show a small trough in the Soret region at the wavelength 10 nm longer than that for the absorption peak. Difference spectra of oxygenated hybrid-heme hemoglobin showed only small peaks which seem to be due to the spectral changes in beta chains. Difference spectra of alpha-NO-beta-unliganded hemoglobin showed a large negative band in the Soret region at the wavelength of the absorption peak for alpha-NO chains, and beta-unliganded chains in this half-liganded hemoglobin showed a small trough corresponding to that of beta chains in fully unliganded hemoglobin. Alpha-Unliganded-beta-NO chains, however, showed only small peaks in the difference spectra. The peaks in difference spectra ascribed to each chain did not completely correlate with the kinetic results reported for hybrid-heme hemoglobin (Nakamura, T., Sugita, Y., Bannai, S. (1973) J. Biol. Chem. 248, 4119-4122) and for half-liganded hemoglobin (Antonini, E., Brunori, H., Wyman, J., and Noble, R.W. (1966) J. Biol. Chem. 241, 3236-3238). Spectral change when alpha and beta chains were mixed was a second order reaction for deoxygenated hemoglobin and was a first order reaction for oxygenated and alpha-NO-beta-unliganded hemoglobin, suggesting the different rate-limiting steps.