Circular dichroism kinetics of acid denaturation of hemoglobin and of its β-subunits

Human hemoglobin and its isolated β-subunits were denatured by addition of HCl so as to reach final pH values ranging from 2.0 to 3.2. The β-subunits were alkylated in both the β93 and β112 cysteines; this treatment makes the β-subunits monomeric. The kinetics of acid denaturation of the two proteins was followed spectropolarimetrically in the millisecond time range, measuring the changes in circular dichroism at 225 nm. At all pH values, in both systems, the decay of ellipticity could be simulated by two exponentials. The initial ellipticity values of the solutions, obtained by extrapolation at zero time, were those expected for the native proteins. The rates of denaturation were lower in the hemoglobin system than in the isolated monomeric β-subunits. The data suggest that in the tertiary structure of hemoglobin and βIAA there are different domains which unfold at different rates upon exposure to acid.