Purification and characterization of squid liver nuclease

• Main Authors: Chiung-Ru Chen, 陳瓊如
• Other Authors: Ta-Hsiu Liao
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/60929372612335425418

碩士 === 國立臺灣大學 === 生化學研究所 === 87 === A Deoxyribonuclease I ( DNase I ) was purified from the crude extract of the squid (Nototodarus sloani) liver. The purification protocol involved an ammonium sulfate precipitation step and chromatography on Macro-Prep High Q, Phenyl Sepharose, Sephadex G-100 and hydroxyapatite columns. The purified enzyme was homogeneous as evidenced by a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ). Purified squid DNase I was shown to be a protein of Mr 61 kDa by SDS-PAGE and 47 kDa by gel filtration on Superose 12. By isoelectric focusing on thin-layer polyacrylamide gel, the enzyme exhibited a single band with a low pI value of 3.6. The squid DNase can not be bound to a Con A Sepharose, indicating that it is not a glycoprotein. Squid DNase I has an alkaline pH activity optimum and is heat stable. It still retained 20% activity after heating at 100 °C for 5 min. The heated sample after 3 days at room temperature could regain the activity up to 53%. When squid DNase was heat at 100 °C for 1 hr, the enzymatic activity was completely lost. The gel filtration and SDS-PAGE analyses showed that the lost of activity was due to unfolding of the polypeptide chain. CD spectra for the native and the denatured proteins are completely different. Squid DNase I contained high Asp and Glu, consistant with the low pI for the enzyme. The acidic nature of the enzyme resembled that of shrimp DNase I. Squid DNase I processes an intrinsic RNase activity as analyzed by the RNase zymogram method. Thus, the enzyme is a nuclease as is shrimp DNase I. The N-terminal sequencing of the enzyme failed to show any PTH amino acid, indicating a blocked N-terminus.