A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP + /NBT/PMS (b-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H 37 Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H 37 Ra PGI was 226 mg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H 37 Ra PGI inhibition by PEP.

The quest for new techniques for screening inhibitors of phosphoglucose isomerase is crucially important owing to therapeutic control of chronic bacterial infections associated with the biosynthesis of bacterial biofilm. According to the new method, yellowish zones against the purple background could be visually observed where phosphoglucose isomerase activity was inhibited. The new protocol with NADPH/NBT/PMS staining for TLC-autographic method was able to detect PGI inhibition by pure reference substance as mercury(II) chloride.

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