Association-dependent absorption spectra of oxyhemoglobin A and its subunits.
A number of factors which lower the oxygen affinity of hemoglobins are also known to produce shifts of the absorption band maxima of the oxyheme. We have studied the variation of the absorption spectra of oxygenated alpha SH and beta SH subunits of human Hb A as a function of their state of association (i.e. alpha, alpha 2, beta, beta 4, alpha beta, or alpha 2 beta 2), and have attempted to correlate the spectral changes with changes to O2 affinity. These studies were carried out under solution conditions (0.1 M Tris, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4, 10 degrees C) where detailed thermodynamic data for subunit association and oxygen binding are available (Ackers, G. K. (1980) Biophys. J. 32, 331-346). Concentration-difference spectra reveal that the visible and Soret absorption band maxima of beta O2 are slightly red shifted relative to beta 4O8. A unique feature of this spectral change is that the red shift is accompanied by an increase in the ratio of the peak absorbances of the visible alpha and beta spectral bands. By measuring the spectral change as a function of concentration, an association constant of 6.4 +/- 1.9 X 10(15) M-3 was determined for the 4(beta O2) in equilibrium beta 4O8 equilibrium. In contrast, no spectral differences were found between alpha O2 and alpha 2O4 or between oxy alpha beta dimers and oxyHb. Mixing experiments show that the spectrum of oxyHb differs from the average of either alpha O2 + beta O2 or alpha O2 + beta 4O8, but is closer to the former. A comparison between these spectral data and the reported O2 affinities of these species shows that affinity and oxyheme spectra are not correlated.
D002263:Carboxyhemoglobin / Q000378:metabolism; D006441:Hemoglobin A / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010108:Oxyhemoglobins / Q000378:metabolism*; D013053:Spectrophotometry; D013816:Thermodynamics
D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D006441:Hemoglobin A; D002263:Carboxyhemoglobin