Purification and characterization of a novel earthworm DNase.
A new deoxyribonuclease (DNase), referred to as EWDNase, was isolated from earthworm tissues. The purification protocol included acetone precipitation, chromatography on CM-Sepharose, and gel electrophoresis. The overall purification was 73-fold with a recovery rate of 2.3% and a final specific activity of 2039 U/mg. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested a molecular mass of 30 kD for EWDNase, with an isoelectric point of approximately 7.0. Maximum activity was detected at a pH of 5.6 and a temperature of 40°C. Addition of Mg(2+) and Ca(2+) ions promoted enzyme activity strongly, while Zn(2+) and ethylenediamine tetraacetic acid (EDTA) acted as inhibitors. Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis indicated that there was no known matching sequence. The properties of EWDNase were sufficiently different from previously reported enzymes to suggest that it is a new enzyme requiring further confirmation and characterization.
a Capital Institute of Pediatrics , Beijing , China.
Zhiqiang
Z
Zhu
Qi
Q
Yang
Baofeng
B
Yu
Qiu
Q
Xie
Jianhua
J
Wang
Xiuwei
X
Wang
Zhen
Z
Guan
Guannan
G
Li
Xu
X
Han
Bo
B
Niu
D000818:Animals; D002853:Chromatography, Liquid; D003851:Deoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009835:Oligochaeta / Q000201:enzymology*; D053719:Tandem Mass Spectrometry; D013696:Temperature
D008670:Metals; D003851:Deoxyribonucleases
activity assay; deoxyribonuclease; earthworm; isolation and purification