Acid denaturation of horse carbonylhemoglobin in the absence of oxygen.

Unlike ferrihemoglobin (Hb+), denatured carbonylhemoglobin (COHb) has almost the same absorption spectrum in the visible and near ultraviolet region as the native form; the very slight difference’appears only in the Soret band. Kinetic and equilibrium experiments under anaerobic conditions show that COHb is considerably more resistant to acid denaturation at 25’ than is Hb+ although the rates of denaturation of both proteins depend on pH in much the same way. These conclusions are based on assays of denatured protein by precipitation at the isoionic point, by the measurements of unmasking of imidazole groups inaccessible in the native protein, and by changes in optical rotatory dispersion. Air, when present, oxidizes the denatured protein practically instantaneously, but it has no discernible effect on the denaturation rate. It does, however, predictably shift the equilibria by removing the initial product, unoxidized aciddenatured protein. COHb, unlike Hbf, is not stabilized by formate, but another stabilizer of Hb+, azide, produces complex effects. Bromphenol blue greatly accelerates the rate of denaturation and shifts the denaturation equilibrium to higher pH. The possible significance of the absence of a marked spectral change is discussed in terms of a possible nonbonded clathrate structure for the prosthetic group in COHb. Changes in the Cotton effects in the Soret band differ in acid-denatured COHb and Hb+, and there are differences in the regenerability of the denatured proteins. These facts add support to the evidence that conformation differences exist in the native forms; thus the difference in stability between COHb and Hb+ need not be solely the direct consequence of the positive charge on the prosthetic group of the latter as previously postulated.