[RNA的杂交酶切割。III. 利用紫外光谱研究大肠杆菌核糖核酸酶的动力学性质]

[Hybridase cleavage of RNA. III. Use of UV-spectroscopy for studying the kinetic properties of E. coli RNAase properties].

作者信息

Krynetskaia N F, Tashlitskiĭ V N, Belkov V M, Metelev V G, Shabarova Z A

出版信息

Biokhimiia. 1991 Apr;56(4):687-93.

Abstract

A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.

摘要

已开发出一种用于估算大肠杆菌核糖核酸酶H活性的一步法。该方法基于在酶促反应过程中对底物紫外线吸收增量的连续记录。发现异源双链体Am.dT20（m = 18 - 24）是该酶的最佳底物。对通过紫外光谱法和离子对高效液相色谱法测定的酶促反应速率进行了比较分析。首次测定了大肠杆菌核糖核酸酶催化的Am.dT20中Am水解的动力学参数（Km = 44 +/- 11 nM，Vmax = 0.0363 +/- 0.0053 E）。该方法的灵敏度为0.01 - 0.05 E，这使得在0.5 - 2.5 u./ml的浓度范围内测定核糖核酸酶H成为可能。