High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.
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.
Landers
J P
JP
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905.
Schuchard
M D
MD
Subramaniam
M
M
Sismelich
T P
TP
Spelsberg
T C
TC
D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology; D004586:Electrophoresis / Q000379:methods*; D004795:Enzyme Stability / Q000187:drug effects; D013696:Temperature
D015500:Chloramphenicol O-Acetyltransferase; D004229:Dithiothreitol