SIGNIFICANCE OF INITIAL FAST REACTION IN ACID DENATURATION OF FERRIHEMOGLOBINS

Acid denaturation measurements of both horse and human ferrihemoglobins (Hb+) at 2.5° show that the initial red shift of the Soret band at low pH is slowed to a rate measurable by stop-flow techniques. Parallel changes occur in the visible absorption bands of Hb+; the bands at 500 mμ and 630 mμ disappear and a new band appears at 525 mμ (horse Hb+) or 531 mμ (human Hb+). These changes are suppressed by the presence of formate ion only, among several common anions tried. These spectral events, which occur only at pH < 3, although acid denaturation occurs at much higher pH, are interpreted as changes in the environment of the heme group which affect the iron orbitals coordinating with the ligands. The changes are probably due to the effect of the high-charge density acquired by the protein at low pH values prior to heme separation; contrary to an earlier report, the initial fast reaction is probably not important to the over all acid denaturation scheme of Hb+ previously described; it is a manifestation of an alternative pathway, superimposed on the basic mechanism. In contrast to the result at 25°, the pH-denaturation rate profiles for the two species at low temperature do not approach the same limiting rate; the difference corresponds to the known difference in the final products at high and low temperatures at least with horse ferrihemoglobin. © 1969, American Chemical Society. All rights reserved.