Activation of Deoxyribonucleases by Divalent Cations

Abstract

The activation ofDNase I by Mg, Mn, Co, Ni, Fe, Cd, Zn, Ba, Sr, Ca, and Cu ions has been studied by several methods, at different pH and salt concentrations. Mg, Mn, and Co are the best activators for initial stages of degradation. A synergistic effect is shown only by the pair Mg-Ca. Optimal pH of action is always situated at 6.5. DNase II is activated to about the same degree by alkaline earths and Mn ions. Cd and Cu are strong inhibitors. Optimal pH is always 4.6. By titration of liberated secondary phosphate groups, two stages in the hydrolysis of DNA by DNase I are evidenced: a rapid phase activated most by Mg and a slow phase activated by Ca. Some possible mechanisms of action of both enzymes are outlined and the general influence of metal ions is discussed.Two main categories of deoxyribonucleases specifically hydrolyze the phosphoester bonds in deoxyribonucleic acid (DNA): the first one (DNase I), extracted and crystallized from pancreas by Kunitz (1950), requires divalent cations (in particular Mg); the second one, DNase II, extracted from thymus or spleen and purified by chromatography (Koerner and Sinsheimer, 1957; Shimomura and Laskowski, 1957;Fredericq and Oth, 1958), is already active in the absence of metallic cations but is activated by several divalent cations.Wiberg (1958) showed that Mg and Mn are the most powerful activators of DNase I. He also reported a very interesting synergistic effect of Ca ions when added to Mg ions, although they had themselves very little activity. His method of determination was, however, restricted to the earlier stages of hydrolysis and under conditions in which DNA was partially denatured. The synergistic effect of Ca and Mg was confirmed by Feinstein (1960) using a viscometric assay and by one of us (Fredericq, 1959), using a titration of secondary phosphate; differences in the activation of hydrolysis of DNA and oligonucleotides were also observed.In the present paper we describe a study of the activation of DNase I by a variety of divalent cations and its extension to subsequent stages of DNA 93

Automation of the Differential Digestion Process of Sexual Assault Evidence,

“…The addition of DNase to the differential digestion process required changing the detergent in the lysis buffer from SDS (ionic detergent) to a nonionic detergent. The sodium ions in SDS inhibit DNase by competing with divalent ions (such as Mg 2+ and Ca 2+ ) for a site that either directly or indirectly affects the active site on DNase . Three nonionic detergents, Triton X‐100, Tween 80, and IGEPAL CA 630, were selected and evaluated for the following criteria: compatibility with DNase, cell lysis efficiency as compared to SDS, and compatibility with microscope slide sample preparation steps.…”

Synthesis and Characterization of Dendronized Polymers

“…FT-IR (cm À1 ): hydroxyl band disappearance (2500-3300, n OH) and a new signal at 2252 (n CN) appearance. 13 FT-IR (cm À1 ): nitrile band disappeared (2252) and a new signal appeared at 1740 (n C¼O). 13 C-NMR (CDCl 3 ): (d ppm) ¼ 172.0 (C¼O); 72.7 (H 2 NCOCH 2 ); 66.8 (OCH 2 CH 2 ); 56.0 (CNH 2 ); 51.5 (CH 3 ); 34.8 (CH 2 CO).…”

“…13 FT-IR (cm À1 ): nitrile band disappeared (2252) and a new signal appeared at 1740 (n C¼O). 13 C-NMR (CDCl 3 ): (d ppm) ¼ 172.0 (C¼O); 72.7 (H 2 NCOCH 2 ); 66.8 (OCH 2 CH 2 ); 56.0 (CNH 2 ); 51.5 (CH 3 ); 34.8 (CH 2 CO). To obtain diacidchloride, 5, SOCl 2 (10 mL) was added to diacid (0.4000 g, 16 mmol) in THF (10 mL) anhydro under nitrogen and allowed to react for 4 h under reflux.…”

“…The carbonyl absorption band of ester appeared at 1736. 13 C-NMR (CDCl 3 ): (d ppm) ¼ 173.1 (C¼O ester); 163.3 (C¼O amide); 148.3 (CNO 2 ); 136.9 (C 3 and 5 aromatic); 131.7 (C 4 aromatic); 124.1 (C 2 and 6 aromatic); 80.9 (CH 3 CO); 58.4 (CNHCH 2 ); 30.3 (CH 2 CH 2 CO); 29.9 (CH 2 CH 2 CO); 27.9 (OCCH 3 ).…”

“…Intrinsic viscosities, [h], were determined using a capillary type Desreux-Bischoff [13] viscometer and their were calculated according to the classical empirical relations [14] in DMSO at 25 8C.…”