The IC50 for inhibition of gyrase supercoiling is visually assessed as the concentration of compound which leads to a 50% reduction of the supercoiled band and the appearance of a spread of slower migrating topoisomers above. This is then verified using gel documentation software and subsequent statistical analysis.
The IC50 for inhibition of relaxation is obtained in a similar way. 50% inhibition is determined visually as being the compound concentration at which the relaxed band is reduced by 50% and a supercoiled band of topoisomers becomes apparent. Gel documentation software and statistical analysis is then used to confirm the result.
Potential problems which may occur in gel based inhibition studies
High IC50 values
If the level of enzyme used is too high, the IC50 obtained will be inaccurate because the assay is not sensitive enough to show the correct level of inhibition. For accurate IC50determinations the enzyme concentration should first be established by titration and the amount used should be the lowest concentration which shows 100% activity (it is sometimes acceptable to use slightly less to obtain maximum sensitivity to the drug)
Retardation of DNA
This can be caused by the drug binding strongly to the DNA. Normally it is sufficient to stop the reaction and extract the protein with a mixture of EDTA and chloroform/iso-amyl alcohol. However, if the drug binds strongly to the DNA it can be removed with a butanol extraction before the chloroform.
If the drug is not soluble or poorly soluble in the preferred solvent there may be problems obtaining an accurate result. Solubility can also be an issue when the drug is dissolved in a solvent but is titrated into an aqueous reaction mix. Results have to be carefully assessed in these cases. Controls should also be carried out to determine the effects of a particular solvent under the assays conditions used as too high a concentration may inhibit the activity producing a false positive result. DMSO, a commonly used solvent, causes inhibition of supercoiling at levels above 10% (v/v).
Resolution of Bands on gels
Better resolution can be obtained with lower voltages and hence longer running times (except in the case of decatenation gels which can be run very quickly). Avoid overnight gels as this can lead to the bands being very diffuse and difficult to quantitate with conventional gel documentation systems.