Effect of Ionic Strength on the pH Optimum, Specificity, and Mechanism of Action of Acid DNase from Mature Eggs of the Sea Urchin Strongylocentrotus intermedius

Yu. T. Sibirtsev^* and V. A. Rasskazov

Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, pr. 100-letiya Vladivostoka 159, Vladivostok, 690022 Russia; fax: 7 (4232) 314-050; E-mail: piboc@stl.ru

^* To whom correspondence should be addressed.

Received November 1, 1999; Revision received January 10, 2000

---

The effect of ionic strength of the reaction medium on the pH optimum, specificity, and mechanism of action of the acid DNase isolated from mature eggs of the sea urchin Strongylocentrotus intermedius was studied. Changes in ionic strength of the reaction medium caused a displacement of the pH optimum of the enzyme to acidity or alkalinity. The region and range of this displacement depended on the buffer used and on the substrate structure. For single-stranded, duplex, and supercoiled forms of DNA, the pH optimum displacements were 1.0, 1.4, and 2.0 pH units, respectively. The pH optimum displacement changed the mechanism of action of the enzyme. Under optimum pH conditions, the enzyme cleaved supercoiled DNA only by the double-hit mechanism, and fragments of duplex DNA resulted due to the coincidence of breaks in opposite chains. On pH displacement to acidity, the enzyme acted by the mixed mechanism (single- and double-hit). And the quantitative ratio of products of the enzymatic hydrolysis of supercoiled DNA was significantly changed depending on the pH displacement to acidity or to alkalinity. The findings are explained by the effect of salt-dependent electrostatic interactions during the formation of a nonspecific DNA-enzyme complex.

---

KEY WORDS: acid DNase II, DNA-enzyme complex, electrostatic interactions, action mechanism, specificity

---