The rates of acid denaturation for human and horse ferrihemoglobin followed by the disappearance ofthe Soret absorption band with the Gibson stop-flow apparatus have been extended to pH 1.8 and denaturation half-periods down to 25 msec. The results are essentially a linear extrapolation of the log velocitypH profiles which prevail at higher pH values, maintaining a constant velocity differencebetween the species down to pH about 2.5. At lower pH the denaturation velocity for both human and horse proteins levels off to the same rate; the differences in stability of the two proteins at higher pH values may be due to the known differences in their content of carboxylic acids. Thefast measurements at low pH make it possible to distinguish between several steps in the spectral changes: A → B, a small exceedingly fast reaction, accompanied by a red shift of the Soret band; B → C, expulsion of the heme from the protein (blue shift of the Soret band); C → D, the dimerization reaction of the free heme (disappearance of the Soret band). The same kinetic analysisof other spectral changes at λ 230 mμ, and measurements of the optical rotatorydispersion at this wavelength, lead to a demonstration that the unfolding of the protein (loss of helix) and the expulsion of the heme occur at the same rate over a very wide pH range above about 2.2. Thus, at pH below about 3, rates of changes in e of the Soret band do not suffice to measure unfolding, because these changes are due to a complex series of events, one of which (dimerization of the heme) occurs outside the protein. © 1969, American Chemical Society. All rights reserved.
