The pH-dependent Soret difference spectra of the deoxy and carbonmonoxy forms of human hemoglobin and its derivatives

Abstract

The transition from deoxy to oxystructure of HbA is accompanied by the breaking of the salt bridges formed by C-terminal residues in deoxy-Hb. This, in turn, changes the state of the heme. The switch between these different allosteric forms can be followed by changes in the optical absorbance spectra. Using difference spectroscopy in the Soret region, pH dependent spectral changes of Hb and its derivatives (carbamylated at both the .alpha.-NH2 groups, .alpha.2c.beta.2c; N-ethylsuccinimide Hb, NES-Hb) in their deoxy and carbonmonoxy forms were measured. From these measurements, the pK values of histidine-146.beta. and valine-1.alpha. in deoxy-Hb were determined to be 8.6 .+-. 0.2 and 7.7 .+-. 0.1, respectively. In carbonmonoxy-Hb a pK value of 6.3 .+-. 0.1 found.