A spectrophotometric study of ionizations in methaemoglobin

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Abstract
The spectrophotometric acid-base character of horse methemoglobin was compared with that of horse metmyoglobin. All the results are consistent with the view that the ionizations of the 4 hematin-bound water molecules in methemoglobin are single and independent. Unlike the case of metmyoglobin, no other ionizing groups appear to affect this ionization above pH 9. The ionization of acid methemoglobin, indicates that its "ionic strength valence" per hem does not exceed -3 even at pH 10. Approx. thermodynamic constants for this ionization were detd. by extrapolation from the data obtained at the lowest ionic strengths. The ionization constant PK3 (20[degree]) = 8.86 [plus or minus] 0.02, [DELTA]H = 3.91 [plus or minus] 0.49 kcal./mole, [DELTA]S[degree] (20[degree]) = -27.2 [plus or minus] 1.8 cal./mole/degree. The spectral characteristics of ewe methemoglobin are similar to those of horse methemoglobin. The values of the ionization constant K''3 were found to be substantially the same in the 2 species, and no differences attributable to possible dissociation of the hemoprotein into subunits could be detected. Small reversible changes in the absorption spectrum of acid methemoglobin were attributed to 2 ionizations, probably of hematin-linked groups. At 20[degree] and Ionic strength = 0.01 the following approx. values for these ionization constants were obtained: pK''1 = 5.1, pK''2 = 6.33. At all values of I > 0.2, the spectrum of acid methemoglobin corresponds to the conjugate acid of pK''2, which is identical with the conjugate base of pK''1. The effect of neutral salts on the hematin-linked ionizations is discussed with reference to possible structure for methemoglobin.