ABSTRACT
DNase A studied by gel filtration on Sephadex G-100 at pH 7.4 in 40 mM Tris-HCl buffer, behaves hydrodynamically as a spherical monomeric macromolecule of around 31,000 molecular weight, with a Stokes radius = 24.7 A, f/fo = 1.19, and D20,W = 8.69. Similar results were obtained by analytical dialysis using zinc chloride-modified cellophane membranes. The elution volume of DNase A decreases as the pH increases between pH 4.7 and pH 9.5. This effect has been attributed to a change in the tridimensional structure of the protein and interpreted as a modification in the axial ratio due to unfolding of the polypeptide chain with increase in the apparent Stokes radius. The addition of Ca2+ produce reversion of the pH-induced changes at pH 9.5. The transition occurs when Ca2+ binds to at least two binding sites (n = 1.66 in a Hill plot) with a Kd = 8.9 X 10(-5) M and the effect appears to be cooperative. These findings support the hypothesis that Ca2+-binding to DNase A causes a conformational change that maintains a more active structure of the enzyme, especially when the pH-induced unfolding reduces its activity.
