Establishment of an effective TLC bioautographic method for the detection ofMycobacterium tuberculosisH37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

Abstract

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.

Antibody-free colorimetric detection of chlorothalonil in cucumberviathe inhibition of an enzyme-triggered reaction

“…38,41 The simultaneously generated NADH participates in a cycling reaction that exhausts NBT yielding a purple coloured formazan (l max : 560 nm) with PMS as an electron carrier. [42][43][44][45] The absorbance intensity is proportional to the concentration of NADH. Whereas, previous investigations 39 have conrmed that inhibition of GAPDH by CTL was via forming a co-valently bond with amino acid residue Cys 149 in the active pocket ( Fig.…”

Recent Advances in Effect‐directed Enzyme Assays based on Thin‐layer Chromatography

“…The auxiliary enzyme glucose‐6‐phosphate dehydrogenase catalyses glucose‐6‐phosphate into gluconolactone‐6‐phosphate by using NADP + as coenzyme. The reduced NADPH can convert the phenazine methosulphate to its reduced form, which subsequently acts as an electron carrier to convert nitroblue tetrazolium (NBT) to purple formazan (Paradowska et al ., ).…”

“…A TLC bioautographic PGI assay was established recently (Paradowska et al , ). The enzyme was isolated from M. tuberculosis H37Ra strain, the commonly used surrogate for virulent M. tuberculosis H37Rv strain.…”

“…An example of such an enzyme is phosphoglucose isomerase (PGI, ÀEC 5.3.1.9) that plays a key role in the metabolic pathways of phosphorylated sugars, such as glycolysis and gluconeogenesis (Mathur and Garg, 2007;Anand et al, 2010). A TLC bioautographic PGI assay was established recently (Paradowska et al, 2016). The enzyme was isolated from M. tuberculosis H37Ra strain, the commonly used surrogate for virulent M. tuberculosis H37Rv strain.…”

A new colorimetric DPPH• scavenging activity method with no need for a spectrophotometer applied on synthetic and natural antioxidants and medicinal herbs

“…In the search for the reduced costs for bioactivity testings, new strategies have been developed. For such purposes, biological activity studies are made using flat surfaces, such as TLC plates or filter papers, to make substitution possible for spectrophotometers 19 . Studies that determine the antioxidant activity using TLC plates are available with names bioautography, spot assay etc 20 .…”