Deoxythymidine 3', 5'-di-p-nitrophenyl phosphate as a synthetic substrate for bovine pancreatic deoxyribonuclease

Bovine pancreatic deoxyribonuclease liberates p-nitrophenol from the 3'-group of deoxythymidine 3', 5'-di-p-nitrophenyl phosphate. A similar hydrolysis occurs with deoxythymidine 3'-p-nitrophenyl phosphate 5'-phsophate, but the rate is less than 2% of that with the di-p-nitrophenyl ester. The rate of formation of the p-nitrophenol, measured spectrophotometrically at 400 nm, varies linearly with DNase concentration. The binding of the substrate is not strong (K-m(app) in the 10 mM range), but the hydrolysis is rapid; 1 mug of DNase (free from other phosphodiesterases) can be assayed in 3 min after addition to a 10 mM substrate solution at pH 7.2, 10mM in MnCl2, and 1mM in CaCl2. All four bovine pancreatic DNases (A,B,C, and D) show the same relative activities toward DNA and toward the di-p-nitrophenyl ester; both activities are lost when DNase is inactivated by iodoacetate or by trypsin. The specificity of DNase toward the di-p-nitrophenyl substrate is different from that which has been established for the enzyme's predominant action on DNA or synthetic oligonucleotides, where a monoesterified phosphate group is formed at the 5'-position.