Effects of p-hydroxymercuribenzoate binding on the visible absorption spectrum of methemoglobin.

The binding of p-hydroxymercuribenzoate to human methemoglobin causes a perturbation of the visible heme abosrption spectrum which is expressed by an increase in absorbance in the high spin band regions, 480 to 510 nm and 590 to 640 nm, concomitant with a decrease in absorbance in the alpha- and beta-band absorption regions. The pH dependence of the p-hydroxymercuribenzoate-induced difference spectrum can be accounted for quantitatively by a 5% shift toward higher spin of the aquo form of methemoglobin, a 15% shift toward higher spin of the hydroxide form, and a shift in the apparent pKa for the water to hydroxide transition from 7.92 to 8.04 when mercurial is bound. The rate of these heme abosrbance changes is consistent with the rapid second order formation of the beta93 cysteine, mercury-mercaptide bond and does not represent a change due to the dissociation of methemoglobin tetramers into dimers, even though the latter, slow process does follow mercurial binding. The observation of an increase in spin produced by the binding of a reagent which also promotes dimer formation argues strongly against any direct correlation between an increase in spin and the appearance of deoxyhemoglobin-like conformations.

Binding of inositol hexaphosphate to human methemoglobin.

The observed static difference spectrum produced by inositol hexaphosphate binding to methemoglobin is the sum of a very fast and a slow spectral transition. The more rapid absorbance change is too fast to be measured by stopped flow techniques, whereas the slow change exhibits a half-time in the range 1 to 6 s. From the pH dependence of the rapidly formed difference spectrum and from a series of heme ligand binding studies, the rapid phase is interpreted to reflect a localized tertiary conformational change which immediately accompanies inositol hexaphosphate binding and results in a selective increase in spin and reactivity of the beta chain heme groups. In contrast, the slow phase appears to reflect a first order isomerization process which involves only a small portion (less than 10%) of the hemoglobin molecules and results primarily in a marked alteration of the spectral properties of the alpha chains with little change in spin. While the rapid spectral transition cannot be directly related to the overall quaternary transition which occurs during oxygen binding to ferrous deoxyhemoglobin, the slow spectral transition may represent the abortive formation of a deoxyhemoglobin A-like conformation which is inhibited in both rate and extent by the presence of water molecules bound to the heme iron atoms.