Multiple conformations of deoxyribonuclease A. Their separation at alkaline pH and low ionic strength in the presence of Ca2+.

Gel filtration on Sephadex G-100 at pH 9.0 in 1 mM Tris buffer produces denaturation and inactivation of pancreatic DNAase A. Limiting concentrations of Ca2+ in the suspension and elution buffer, reactivates some of the enzyme molecules in an amount proportional to the calcium added. Stable active and inactive forms were separated on Sephadex columns. A model for the conformational role of Ca2+ on DNAase A demonstrates that at least one Ca2+ is involved (Kapp = 8.3 . 10(-5) M) in the correct folding of the polypeptide chain. Na+ was unable to reactivate the enzyme.

The role of Ca2+ on pH-induced hydrodynamic changes of bovine pancreatic deoxyribonuclease A.

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.

A study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease I and to deoxyribonucleic acid by electron paramagnetic resonance.

EPR studies of Mn2+ binding to bovine pancreatic deoxyribonuclease I show that the enzyme can bind three Mn2+ ions at pH 7.5 and 2 degrees. Two sites bind Mn2+ strongly, with a Kd of 10(-4)M, and the third binds Mn2+ weakly, with a Kd of 10(-3)M. Ca2+ competes with the two strong sites, whereas Mg2+ competes only with one of them, indicating that both sites are not equivalent. Mn2+ binding to DNA has been confirmed by EPR measurements. Two types of sites, with different affinities for Mn2+ binding, were found on DNA molecules, one with a Kd of 1.2 times 10(-4)M and the other with a Kd of 10(-3)M. Mg2+ ions can displace Mn2+ from the high affinity sites, but not from the low affinity sites. These results suggest the Mn2+ binds not only to the phosphate groups, but also to the electron donor groups of the base rings.