Most of the papers that I've found limit the incubation time to a maximum of ca. 2h, and even the manufacturers recommend incubation times of 90 min or less. I wonder if we can extend the duration of the incubation with the addition of more cofactors?
Dear Paulo,
the incubation time in microsomal assays depends on the enzyme(s) you want to study. Co-factors are typically added in large excess (supra-physiological concentrations) or in reconstituting systems and do not represent a limiting factor.
If you're studying cytochrome P450 enzymes, these are capable of transferring 2 electrons per catalytic cycle. Both are not necessarily transferred onto the substrate but can intoxicate the enzyme itself by forming H202 which under physiological conditions is taken care of by catalase.
Anyway, in case you want to study enzyme kinetics, you'll need to establish linear conditions for the catalytic reaction you want to study. In case the linear range of your assay comes with a limited incubation time that is producing too little product, you need to trim the sensitivity of your assay or add more enzyme protein.
Adding protein however, can lead to a reduction of the free concentration of the educt in your incubation mix and thus needs to be investigated for linearity, too.
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