The use of dextran-coated charcoal for kinetic measurements: Interaction between rifampicin and DNA-dependent RNA polymerase ofEscherichia coli
✍ Scribed by Erika Wyss; Walter Wehrli
- Publisher
- Elsevier Science
- Year
- 1976
- Tongue
- English
- Weight
- 382 KB
- Volume
- 70
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
Rifampicin inhibits bacterial DNA-dependent RNA polymerase by forming a l:l complex with the enzyme. In previous studies the extent of this binding was determined by chromatographic isolation of the complex on Sephadex columns with t4C-labeled rifampicin as tracer, To measure the kinetics of the enzymeantibiotic interaction, however, this procedure is too slow and laborious. In this paper we describe the use of dextran-coated charcoal, which proved to be ideal for isolating the complex rapidly and quantitatively. The free [t4C]rifampicin is adsorbed on the charcoal and can be removed by centrifugation, whereas the [~4C]rifampicin-enzyme complex remains in the supernatant and can be easily measured. An account is given of the application of this procedure in measuring the dissociation and association kinetics of the RNA polymerase-rifampicin complex.