Spectroscopic properties of the hemes of human adult hemoglobin and its subunits

Abstract

The circular dichroism (CD) and difference spectra of human adult hemoglobin (Hb A) and its subunits in the deoxy-, oxy- and CO-forms are presented. All four absorption bands of the hemes were optically active in the hemoglobins. The positions of the CD bands observed roughly agreed with those of the corresponding absorption bands. Generally, the α-subunit had more positive ellipticity than the β-subunit, and the CD spectrum of Hb A could be roughly composed by taking the arithmetic mean of those of the subunits. Similarly, the difference spectra of the subunits, between oxy- or CO- and deoxy-forms, differed from each other. These facts suggest that the local milieu around the hemes was not the same in the subunits. Human fetal hemoglobin showed the same CD spectrum as Hb A. In the visible region, two additional bands around 520 and 580 mμ were observed in the CD spectra, but these bands were indiscernible in the absorption spectra. A positive CD band around 580 mμ of the deoxy-form appeared in the α-subunit and Hb A but not in the β-subunit. On the basis of the difference spectra, the difference in the absorption of the deoxy-hemes in the separated subunits and in the tetrameric Hb A was confirmed. In the ultraviolet region, the CD peak positions of a band around 310 mμ differed markedly from each other in the subunits, while those of a band around 260 mμ did not. The CD band around 260 mμ of the deoxy-forms was probably degenerated. A negative CD band around 280 mμ was assigned to the aromatic amino acid residues of the protein moiety, and the band was indiscernible in the α-subunit.