Abstract
Abstract 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 acid-denatured protein. COHb, unlike Hb+, 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.
Highlights
The first (“Comparison of Spectral with other Criteria of Denaturation”) is concerned with establishing that the denaturation of COHb by acid in the absence of air is not accompanied by the drastic spectral changes that accompany denaturation of methemoglobin; we first present, as a function of pH, the rates of denaturation determined by several distinct criteria other than light absorption, and the range of pH values in which an equilibrium occurs. These data are of interest in themselves in that they show the large extent to which COHb is more resistant to acid denaturation than Hb+
COHb and Hb+ were based on (a) changes in spectrum; (b) increases in acid-binding due to the liberation of imidazole groups which are inaccessible to acid in the native protein (these increases were estimated from time-dependent changes in the titration curve [9]) ; and (c) time-dependent losses in solubility
With complete identity of spectra, one might be driven to assume that with COHb no chemical bond is involved in the heme linkage at either pH, but that the prosthetic group exists as a clathrate, a caged but unbound molecule, within a protein which can be disorganized by acid
Summary
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+ 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. More unexpectedly, to the realization that COHb may be completely denatured by any of a number of critical criteria and still undergo only minor changes in its visible andnear ultraviolet absorption spectrum
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