Monitoring FtsZ Protofilament Disruption

FtsZ polymerization results in the formation of protofilament and monitoring the protofilament with a beam of monochromatic light causes scattering of the rays [42 (link)]. Right angle (90°) light scattering assay was used to study the ability to disrupt the protofilament formation by the selected plant extracts / D- Pinitol at 1.5 x MIC. This was monitored with JASCO Spectrofluorometer with both excitation and emission set at 400 nm. The reactions were carried out using a polymerization buffer (50 mM MoPs, 100 mM MgCl₂ & 50 mM KCl). Mtb-FtsZ (12 μM) is first incubated with / without plant extracts and D- Pinitol and monitored for 300 seconds to establish the baseline (zero condition). Then, 50 seconds later 1 mM GTP was quickly added by interrupting the measurement and the scatter intensity is measured continuously for 1000 seconds. For all treatments, the time lag addition of GTP was maintained between 5 and 6 seconds [12 (link),43 ].