Factors Affecting the Release by Dilute Acid of Hidden Prototropic Groups in Carbonylhemoglobin

Abstract

Abstract A ferrohemoglobin, horse COHb, titrated with acid and back-titrated with base in the absence of air and with more rapid mixing (⅕ sec) than in earlier work, exhibits much more complex unmasking behavior (prototropic groups) than previously reported for the ferri form, Hb+. The numbers and kinds of groups liberated by exposure to acid depend not only on pH, but on salt concentrations, and on time of exposure as well. Under mild conditions of denaturation, all the groups unmasked appear to be remasked so rapidly at higher pH that a meaningful back titration curve has not been obtained. With more severe treatment, regeneration and remasking are manageably slower, and it is clear that masked basic groups, including (but not solely) imidazoles, appear. Loss of α helix, when brought about by mild acid treatment, is recovered at higher pH values which depend on the severity of the denaturation process. With the most severe treatment, about 24 groups titrating at pH g 3 are unmasked; about half of these appear to be imidazole. The number is in fair agreement with that recently calculated by Tanford and Nozaki for ferrihemoglobin. Evidence is presented that exposure of the ferroprotein to low pH (below 3) produces results (unmasked groups, regenerability of Cotton effects, and Soret band absorption) which are quantitatively different from those accompanying the maximum attainable degree of unfolding at longer times at higher pH. The pH and time of exposure to acid may determine whether unfolding of the ferroprotein occurs with or without detachment of the prosthetic group.