Effect of Sulfate on the Activity and the Kinetics of Deoxyribonucleic Acid Degradation by Porcine Spleen Deoxyribonuclease

Abstract

Abstract Divalent anions such as sulfate, oxalate, and malonate can inhibit porcine spleen deoxyribonuclease completely. At anion concentrations 10- to 100-fold below those which inhibit, a reproducible 20% increase in activity is observed in the hyperchromicity assay of DNase II. For sulfate, half-maximal activation occurs at 6 x 10-5 m and half of complete inhibition occurs at 10-2 m. Sulfate, oxalate, and malonate also protect DNase II completely from inactivation by iodoacetate, which alkylates an essential histidine residue. The concentration of anion needed to reduce by half the rate of inactivation with iodoacetate is approximately equal to the concentration of anion needed to achieve half maximal activation. Concentrations of sulfate which activate DNase II also change the mode of cleavage of the DNA. In the presence of 5 x 10-4 m sulfate the rate of decrease in molecular weight of single-stranded, alkali-denatured DNA is increased dramatically while the rate of decrease of double strand molecular weight is unaffected. The more rapid decrease in single-stranded molecular weight with sulfate corresponds to a 15-fold increase in total phosphodiester bond cleavages. The fraction of total breaks which result in a double strand break declines from 0.80 in the absence of sulfate to 0.04 in 5 mm Na2SO4.