Figure 4

Experimental analyses of ancient and extant Mbs. The values of aMbWp, aMbWb’, aMbWb, and swMb are indicated in diamonds (light brown), squares (light green), triangles (light blue), and circles (blue). The data for horse (hsMb) and cow (bvMb) apoMbs are indicated by black and gray circles, respectively, for comparison. (a) Solubility dependence of holo-forms of ancient and sperm whale Mbs on the concentration of PEG-6000. Log solubility values in mg/mL (S) are plotted against the precipitant concentration. (b) Dependence of the absolute scattering intensity at q = 0 on the protein concentration in small angle X-ray scattering experiments (see also Fig. S6). (c) Estimated second virial coefficients (A₂) indicating repulsive interaction between Mb molecules. A₂ calculated presuming that Mb is a hard sphere is shown as a red, dashed line. (d) Chemical denaturation profiles of apoMbs. The unfolded fractions estimated by the CD signal intensity at 222 nm were plotted against the Gd-HCl concentration. The inset shows the ΔG_fold (kcal mol⁻¹) of the proteins. The data for hsMb and bvMb are presented in black and gray, respectively, for comparison.