I appreciate anyone who has thoughts or commentary about the interpretation of some preliminary P450 activity data, as I am certainly no P450 expert. I've had to resort to using spectrophotometric methods to detect P450 activity as flourometric substrates are very expensive. I am interested in looking at several activities of P450s (as their activities are incredibly variable) so I've used the classic PNOD (O-demethylation) as well as a more obscure method I found (N-demethylation) using 4-chloro-N-methylaniline. It is difficult, but possible, to detect PNOD activity with my insect as that activity is fairly low. However, I can reliably detect product with the N-demethylation assay. So here is where I get to my question. We actually haven't detected compounds in my insect's host that would have this structure (N-CH3; e.g. alkaloids), and we have looked. This doesn't mean there are none, but I don't think it is very likely. So what am I seeing? The insect clearly expresses this activity (much more so than the PNOD activity), am I just detecting some sort of steroid synthesis or other endogenous metabolic process of P450s? Would I actually be safe to refer to this as a "detoxification" activity? Any thoughts would be appreciated, thanks for taking time to read this!
Hello Chad. You can use the biochemical assay development to measure the mixed function oxidase (including the P450 cytochrome) in mosquitoes. This assay is with ,3',5,5'-Tetramethyl-Benzidine Dihydrochloride*(TMBZ [2HCL] or TMBZ, H2O2 and read at 620nm.
You have a mix of P450 enzymes. You are most likely looking at one (or more) of the xenobiotic metabolizing enzymes since the P450 enzymes that work on steroids and other endogenous substrates are normally pretty specific. It is difficult to know which one unless you do more experiments. These xenobiotic metabolizing enzymes are non-specific and you do not need to have any equivalent type of compound that is endogenous. N-demethylation reactions are one of the easiest reactions and DMA is metabolized by a number of enzymes in human.
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