I'm studying a bacterial protein containing 2 cysteine residues. I subject the bacterial culture to either mock treatment, ROS treatment, and ROS+b-ME treatment, then harvest the culture to run an SDS-PAGE gel then anti-his western.
The mock-treated culture has a single band at the monomer size, the ROS-treated sample has three bands around the monomer size, one at the same position as the mock treatment, one slightly above, and one slightly below. The ROS+ b-ME sample shows up at the same single band position as the mock-treated sample.
I understand that the slightly lower band can be from a intra-molecular disulfide bond, but what about the slightly higher band? It's very close to the monomer size, no where close to the dimer size, and it is Cysteine-derived as I don't see that on the oxidized double-cysteine variant of my protein. And since it goes away in the ROS+b-ME sample, it is a reversible oxidation.
I've seen papers with similar patterns of ROS-treated cysteine-containing proteins migrating to a slightly higher position, but it's almost never talked about (people focus more on the intra-molecular disulfide, dimers and oligomers formed), any idea what kind of cysteine oxidation this is? Glutathionylation? Sulfenylation? Something else?
It depends on the "ROS" treatment you have used. I assume it was H2O2 (in micromolar range) or a nonlethal dose of HOCl.
If you were using Gram-negative bacteria like E. coli, then endogenous NO and its PTMs can be ruled out. Bacteria themselves produced quite a lot of superoxide when stressed (such as an exogenous H2O2 treatment), which typically turns to endogenous H2O2.
My guess is that it is likely to be protein cysteine sulfenylation. Cysteines can be oxidized "three times" at the sulfur. The first oxidation (sulfenylation) is reversible (reducible). The second and third oxidations (sulfinylation and sulfonylation, respectively) are not. That is, as what the textbooks and reviews say.
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