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

Y Sugita

doi:10.1016/s0021-9258(19)41806-1

Abstract

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.

Highlights

Kempsey,’ and Hb-Rainier [5], gave the lower peak in the Soret region in the deoxygenated forms, lyhereas oxygenated forms showed about the same spectrum as that of normal hemoglobin. All of these modified hemoglobins have a high affinity for ligands and show no heme-heme interactions. These results suggest that the difference in spectra is due to the alteration of conformational changes in the deoxygenated hemoglobin on which depends the cooperative ligand binding, It has been shown that the Q( and @ chains within tetrameric hemoglobin are nonequivalent in the reaction with oxygen and carbon monoxide (6-g), and it is possible that the two kinds of chains contribute different.ly to the absorption spect,ra
Deoxygenated protohemoglobin shows the spectrumsimilar to that reported by Brunori et al [16], giving positive peaks at 430, 553, and 588 nm
Oxygenated hemoglobins showed no large extremum in both the Soret region and the visible region (Fig. 1)

Summary

Methods

The absorption spectrum of cleoxygenated human hemoglobin A differs from that of a number of modified hemoglobins. It was found that the isolated LYand p chains of human hemoglobin (a), the hemoglobin-haptoglobin complex [3], hemoglobin digested with carboxypeptidase A [4], and abnormal human hemoglobins, namely, Hb-Yakima, Hb-. Preparation of Hemoglobin--Isolated 01and p chains containing protoheme were prepared from human hemoglobin by the method of Bucci and Fronticelli [14]. Mesohemoglobin was prepared by the method previously reported [15], and the chains containing mesoheme were isolated from the mesohemoglobin as described previously [6]. The nitric oxide derivatives of hemoglobin chains were made as follows. The solution of oxygenated chains, about 1 mM in 0.1 M phosphate buffer, pH 7.0, was placed in a Thunberg tube and evacuated. The NO derivative of the chains was stable in the

Results

Discussion

Conclusion