Gite S, Shankar V
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
J Mol Recognit. 1995 Sep-Oct;8(5):281-9. doi: 10.1002/jmr.300080502.
A simple procedure, involving heat treatment, gel filtration on Sephadex-G 100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37 degrees C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde medicated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the epsilon-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of the only available cysteine residue (Cys25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA, 3'AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5'AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.
我们开发了一种简单的纯化程序，包括热处理、在Sephadex-G 100上进行凝胶过滤，随后在与琼脂糖结合的抗S1核酸酶抗体上进行层析，可将S1核酸酶纯化至同质，总产率为72%。在邻苯二甲醛存在下，pH 6.0和37℃时，S1核酸酶迅速失活。邻苯二甲醛介导的失活动力学分析表明，该反应遵循准一级动力学，酶活性的丧失是由于每分子酶形成了一种单一的异吲哚衍生物。吸光度和荧光分光光度数据也给出了类似的结果。已知邻苯二甲醛处理导致的异吲哚衍生物形成是通过天然酶中紧密相邻的半胱氨酸硫醇基团和赖氨酸ε-氨基的交联发生的。由于唯一可用的半胱氨酸残基（Cys25）的修饰不影响酶的催化活性，邻苯二甲醛介导的S1核酸酶失活是由于赖氨酸的修饰。S1核酸酶的底物，即单链DNA、RNA、3'-AMP，可以保护酶免受邻苯二甲醛介导的失活。此外，修饰后的酶（催化活性非常低）与S1核酸酶的竞争性抑制剂5'-AMP结合的能力显著降低，这表明修饰发生在底物结合位点。上述结果表明半胱氨酸25位于底物结合位点附近。
