Kinetics and mechanism of hemoglobin denaturation in alkali

The denaturation of oxy, deoxy, CO and met derivatives of human hemoglobin A at pH 11.7 and 25 degrees C was followed by three assay methods: chromophore absorbance (which indicates changes in the heme), and the amount of precipitation in 24% ammonium sulfate neutral buffer or 0.1 M NaCl neutral buffer (which indicates degree of destabilization of the protein structure). We find that oxyhemoglobin denatures in two parallel reaction sequences. The rate of sequence I is increased when the sulfhydryl groups in the alpha 1 beta 1 subunit interface have been modified by binding p-hydroxymercuribenzoate (which forces monomer formation) and is decreased by the binding of -HgOH (which, unlike the sulfhydryls, is not ionized at pH 11.7). These results support a mechanism in which the net rate of monomer formation is rate limiting and is enhanced in alkali by the ionization of sulfhydryls in the alpha 1 beta 1 subunit interface. In subsequent rapid reactions, ferric hemoglobin and low-salt-precipitable protein are formed. The formation of an oxidant, such as superoxide, is indicated by the kinetics of sulfhydryl oxidation. The same oxidant would be available to initiate the second sequence by oxidizing some of the unreacted oxyhemoglobin to methemoglobin. During methemoglobin denaturation, as in sequence II, a low-salt-soluble ferric hemochrome is formed. In both reactions, this intermediate becomes low-salt-precipitable at the same rate. Deoxyhemoglobin denatures to ferrous hemochrome at the same rate as oxyhemoglobin denaturation in sequence I, and provided oxygen is excluded, the denaturation is fully reversible on neutralization. In the absence of oxygen, CO-hemoglobin does not denature to any detectable extent. The destabilization of hemoglobin structure that was indicated by precipitability occurred only for ferriheme derivatives and was independent of disulfide formation.