Protonation of Tyrosyl Residues in the Carboxy Derivatives of Hemoglobin, Its Isolated Subunits, and Apohemoglobin
Yum K. Yip 1 and Enrico Bucci 1
From the
1 From the Indiana University School of Medicine, Department of Biochemistry, Indianapolis, Indiana 46202
The tyrosines were titrated adding KOH or HCl to the protein solutions in 0.25 m NaCl at 20°. Their ionization was followed by differential spectrophotometry at 245 mµ against a reference solution at pH 8.5. The titrations were reversible only up to pH 10.5. The back titrations from pH 12 indicated that the denaturation of the protein produced a change in the absorption spectrum of the heme in the ultraviolet region. Making correction for this transition, the 
per tyrosyl residue was calculated to be approximately 10,000 for all of the proteins investigated. Considering that only 2 out of 3 tyrosyl residues per chain ionize in the native proteins and neglecting the correction for the electrostatic interaction, the reversible part of the titrations was shown to be consistent with pK near 10.4 for hemoglobin, apohemoglobin, and the ß chains, with pK near 9.9 for the 
chains. This difference was too large to be accounted for on the basis of the electrical charge of the various proteins. It could indicate a difference in the ionization of the tyrosyl residues of the chains when these are isolated or polymerized with the ß chains in the hemoglobin molecule.
Submitted on June 28, 1968
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