Liao T
J Biol Chem. 1975 May 25;250(10):3721-4.
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.
牛胰脱氧核糖核酸酶能从脱氧胸苷3',5'-二对硝基苯磷酸酯的3'-基团上释放出对硝基苯酚。脱氧胸苷3'-对硝基苯磷酸酯5'-磷酸酯也会发生类似的水解反应，但反应速率不到二对硝基苯酯水解反应速率的2%。通过在400nm处进行分光光度测定，对硝基苯酚的生成速率与脱氧核糖核酸酶浓度呈线性关系。底物的结合力不强（表观米氏常数在10mM范围内），但水解反应迅速；在pH 7.2、MnCl2浓度为10mM、CaCl2浓度为1mM的10mM底物溶液中加入1μg脱氧核糖核酸酶（不含其他磷酸二酯酶）后，3分钟内即可进行测定。所有四种牛胰脱氧核糖核酸酶（A、B、C和D）对DNA和二对硝基苯酯都表现出相同的相对活性；当脱氧核糖核酸酶被碘乙酸盐或胰蛋白酶失活时，这两种活性都会丧失。脱氧核糖核酸酶对二对硝基苯底物的特异性不同于其对DNA或合成寡核苷酸的主要作用所确定的特异性，在对DNA或合成寡核苷酸的主要作用中，5'-位会形成单酯化磷酸基团。
