The ionization of acidic metmyoglobin. Ionic-strength charge effects

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Abstract
The ionization constant of the iron-bound water molecule in acidic metmyoglobin was measured from about pH 7 to 10 at eight ionic strengths of buffer solution ranging from 0.40 to 0.002. The values confirm the conclusion suggested by the measurements reported by George and Hanania, 1952, that the ionization constant is independent of pH only in the presence of appreciable concentrations of neutral salt. At I = 0.40 at 20[degree], pK" is 8.98 throughout the pH range 7.5-9.8, but at I <0.02, pK" decreases as pH is decreased below 8.6, and it increases as pH is increased above 9.3. The extrapolation of pK" values plotted against [image]l(1 + [image]I) to zero ionic strength gives pK values of 8.81, 9.07 and 9.25 at pH 7.8, between pH 8.6 and at pH 9.8 respectively. It is suggested that this variation of the free energy of ionization with pH is due to an unspecific interaction between charged groups on the protein moiety and the hematin iron atom, which is observable only at low I because of the screening effect of neutral salt ions. In contrast, the effect of a specific heme-linked ionizing group is observable at all ionic strengths. The limiting slopes of the pK" plots are approximately +0.5, -2.5 and -4.5 at pH 7.8, between pH 8.6 and 9.3 and at pH 9.8, which, on the basis of a simple Debye-Huckel model may be attributed to the effective charge of the haematin iron atom changing upon ionization from +1 to 0, from -2 to -3 and from -4 to -5 respectively.
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