Abstract:
In a recent publication authors claim that a continuous generation of superoxide via NADH oxidase is essential for the growth of Mycobacterium smegmatis (Yeware et al., 2017). The major issue with this report is extremely high superoxide estimation which is contrary to a previous study that records a negligible production of superoxide in M. smegmatis cells using the same method. To address this controversy, I did a careful investigation and concluded that dihydroethidium (DHE)-HPLC profiles were misrepresented which has led to an overestimation of superoxide production. Therefore, I present the case that the authors have failed to show evidence for superoxide generation from NADH oxidase/cell and presumed its physiological role in growth of M. smegmatis. This conclusion is supported by following reasons. Stoichiometric ratio of superoxide and oxyethidium formation is 1:1 in superoxide reaction with DHE (Zielonka et al., 2008). Yeware et al. (2017), showed ~8 mu M 2-OH-E sup(+) per 15 mu g of cell protein in 30 min which translate into ~0.5 mM 2-OH-E sup(+) per mg protein, which is extremely high concentration, almost 1,000 times more in comparison to the maximum reported in biological system. In general physiological consequence of such high concentration should be deleterious as it is a potent disrupter of biomolecules. To date superoxide estimation in living organism was found to be in the range of 10 sup(-9) to 10 sup(-12) mole/mg of protein. In cells and tissues, DHE reacts specifically with superoxide to form 2-OH-E sup(+), whereas other cellular components non-specifically react with DHE to form E+. As a result E+ is produced in much higher concentration than 2-OH-E sup(+) in biological systems. Based on previous reports, Table 1 represents the approximate ratio of sup(+) and E sup(+) to emphasize the higher concentration E sup(+) among the two in biological samples. Whereas, the HPLC profile presented by Yeware et al., shows a tiny peak of E+ which is negligibly smaller than 2-OH-E sup(+) on an arbitrary scale. Furthermore, authors observed constant increase in 2-OH-E sup(+) but no significant change in E sup(+) for the period of 3 h in their study. This discrepancy could have been avoided if authors ensured that some detectable amount of unoxidized DHE remain in their assay while standarizing the balance between cell number and incubation time with dye. Importantly, the authors should present standard chromatographic profile of DHE, 2-OH-E sup(+), and E sup(+) with positive (menadione or pyrogallol) and negative control (SOD or TEMPOL) to justify the exceptional HPLC profile obtained in the experiment.
