Rushizky G W, Whitlock J P
Biochemistry. 1977 Jul 12;16(14):3256-61. doi: 10.1021/bi00633a033.
A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.
通过丙酮沉淀以及在二乙氨基乙基纤维素和羧甲基纤维素上进行色谱分离，米曲霉α淀粉酶商业制剂中存在的一种脱氧核糖核酸酶（DNase）得到了纯化，纯化倍数为1550倍，产率为25%。该酶分离后不含污染性的核糖核酸酶和脱氧核糖核酸酶。通过在葡聚糖凝胶G - 100上进行凝胶过滤测定，该酶的分子量为48000，而在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳时观察到的单一条带的分子量为58000。该脱氧核糖核酸酶的等电点为9.2。该酶仅水解DNA，最适pH为8.2，被Co2 +激活，在较小程度上被Mg2 +和Mn2 +激活。天然DNA是比热变性DNA更好的底物。小牛胸腺DNA和大肠杆菌DNA的酶促消化产物产生链长范围为10至200的寡聚物，只有当大量（100毫克）DNA在7M尿素中通过二乙氨基乙基 - 葡聚糖凝胶A - 25柱色谱分离时，才检测到单核苷酸和小寡核苷酸（链长小于5）。消化产物含有5'-末端磷酸基团，3'端大多为腺苷，5'端为鸟苷和腺苷。
