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I often see statements about adding reducing agents like cysteine to anaerobic medium to decrease the amount of the dissolved oxygen.

However, I am not sure why.

I am wondering if it is because under the low redox potential conditions, dissolved molecular oxygen is easily reduced, is that correct? If so, what is the oxygen reduced to? H2O?

For example, I am not sure of the rationale for the following statement?

The higher the redox potential is, the higher the oxygen dissolved in the media will be.

https://biology.stackexchange.com/a/52439/72932

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  • $\begingroup$ Thank you very much for your comments. I did not quite understand the original purpose of adding reducing agents (to lower the redox potential) and the concomitant results (to reduce the contaminated oxygen). $\endgroup$
    – Beginner
    Commented Sep 27, 2022 at 6:32
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    $\begingroup$ @Beginner - I think that lot of people have applied different amounts of reducing agents to their culture and have found the best concentration enabling best viability simply by trial and error. One can speculate why certain concentrations are toxic while others support growth, however most of the time, culturing conditions are configured empirically. Usually people are simply happy that it just works for once. Here, we did lay out somewhat of a basis that there might be a "redox-sweet-spot" that is capable of reducing O2 in culture medium while minimizing H2O2 production. $\endgroup$
    – markur
    Commented Sep 27, 2022 at 11:27

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You‘re partially correct. Adding reducing agents will cause $O_2$ to be converted to $H_2O_2$, as thiol groups are turning from $SH$ to $S$-$S$ (forming dimers). In a second reaction, further thiols may reduce $H_2O_2$ to $H_2O$, (but H2O2 also may react with $SH$ to form toxic byproducts like thiooxides $SO$).

Intermediates (like $H_2O_2$ or $O_2^-$) might get reduced to $H_2O$ by subsequent reactions with thiol groups, Enzymes or spontaneous dismutation (Kiley & Stolz 2004, Aires et al. 2008)

  1. $O_2$ + 2 $SH$ -> $H_2O_2$ + $S$-$S$

  2. $H_2O_2$ + 2 $SH$ -> 2 $H_2O$ + $S$-$S$ (see NG et al. 2007, DTT Assay Wang et al. 2018)

However, while the first reaction is very probable, the second reaction is less probable (some even argue that it is too unlikely to have an effect)

The second reaction might depend on concentration. Usually cell culturing protocols are designed by trial and error. So it’s based on testing multiple concentrations to find out the amount that yields the highest culture viability. I can only speculate that e.g. the right concentration hits a sweet-spot. That sweet-spot might lie between production of toxic thiooxide ($SO$) and oxygen reduction. But that depends on a lot of factors like organism and other culturing conditions.

Cell Cultures are in Generally too Oxidative; Reducing Agents might Help no Matter the Exact Reaction Path

Oxygen is a general problem in many cell cultures (Jagannathan et al. 2017). Even for human cell cultures, 20% $O_2$ is not physiological and introduces a background of oxidative stress (it even increases mutation rate).

To describe aggressive oxidative features of oxygen, it's useful to use the more generalized term „reactive oxygen species“ (ROS) instead of "oxygen", since redox potential doesn‘t care which exact compound acts as an electron donor. By referring to ROS, it‘s not necessary to keep track of exact oxygen-compounds.

The main point is that in the end, some biomolecule gets damaged by oxidation and the probability for that increases with high $O_2$ -levels. Hence, reducing agents could increase viability by scavenging any kind of oxidative species from the medium. And it makes sense to assume that one of the end products might be $H_2O$.


„the higher the potential, the higher the dissolved oxygen in medium“.

I think the wording is misleading, since high redox potential is a consequence of oxygen being present, not the other way around (as that would imply something like high redox potential attracting atmospheric oxygen into water). I speculate that the author meant that dissolved (organic) compounds tend to release $O_2$ (or rather any reactive oxygen species (ROS)) more often in an oxidative milieu.

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  • $\begingroup$ I added "ultimately" in the first line to imply a possible reaction chain. Also, I don't think that sophisticated biochemistry is necessary, since in the presence of excessive thiol groups each oxygen-intermediate should be reduced subsequently by further thiol groups. It's not stated explicitly in the literature, but in my own experience, adding DTT to reverse transcription reactions is good practice to protect RNA from oxidation damage. No other biochemistry needed. $\endgroup$
    – markur
    Commented Sep 25, 2022 at 16:57
  • $\begingroup$ first: O2 + 2 SH -> H2O2 +S2, second: H2O2 + 2 SH -> 2 H2O +S2 ... Where's the problem? $\endgroup$
    – markur
    Commented Sep 25, 2022 at 20:36
  • $\begingroup$ @user338907 - how probable is H2O2 + 2 SH leading to -SOH compared to 2 H2O +S2? In fact, thiol groups do remove hydrogen peroxide NG et al. 2007 ... I think you might have gotten sidetracked here... $\endgroup$
    – markur
    Commented Sep 25, 2022 at 20:52
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    $\begingroup$ Thank you very much for your detailed explanation and discussions! $\endgroup$
    – Beginner
    Commented Sep 27, 2022 at 3:42
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    $\begingroup$ No problem. Remember to accept your favorite answer, if it answered your question. $\endgroup$
    – markur
    Commented Sep 27, 2022 at 5:52

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