Landers J P, Schuchard M D, Subramaniam M, Sismelich T P, Spelsberg T C
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.
J Chromatogr. 1992 Jun 19;603(1-2):247-57. doi: 10.1016/0021-9673(92)85368-4.
This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.
本研究突出了高效毛细管电泳（HPCE）在监测酶活性方面的潜在效用。自由区毛细管电泳用于快速且可重复地分析细菌酶氯霉素乙酰转移酶（CAT）的活性，该酶可将底物乙酰辅酶A（CoA）和氯霉素转化为乙酰氯霉素和CoA。本研究结果表明，HPCE可能是研究酶活性的一种出色工具，因为它相较于标准单参数测定法具有多个优势，最显著的是能够同时监测底物的消耗和产物的生成。已确定了从产物（乙酰化衍生物）中最佳分离底物（氯霉素）的条件。这展现了HPCE在分析酶促反应方面的独特潜力，可应用于目前利用酶促反应的分析研究领域。一种这样的分析方法是用于分析基因启动子活性的CAT测定法。在本研究中，HPCE被证明在短得多的时间内使用非放射性标记底物就能产生类似的定量结果。HPCE相较于标准技术具有多个优势，包括分析速度快、无需放射性标记底物、样品体积小、灵敏度/分辨率高以及出色的定量能力。
