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Rabbit CYP2E1
Part: BBa_K2932003
Cytochrome P450 are family of enzyme containing heme and all of them use O2 and electrons to oxidize substrates(2).
They plays an important role in human liver cells metabolism, it can also degrade benzene and chloroform which affect human a lot indoors. The following is the pathway that CYP2E1 breaks down the benzene:
It will produce phenol while CYP2E1 breaking down benzene, so it is a proof for benzene broke down by CYP2E1.(4-aminoantipyrine/potassium ferricyanide (lll) Emerson reaction) Both oxidative and reductive pathways of chloroform metabolism are described in the following. Carbon dioxide is the major metabolite of chloroform generated by the oxidative pathway of metabolism in vivo(4).
We got the sequence CYP2E1 in rabbit from KEGG-Oryctolagus cuniculus-CYP2E1, and it was optimized to bacillus subtilis by IDT-Codon Optimization Tool. Twist Bioscience synthesized it to us.
CYP2E1 efficiency measure
Introduction:
CYP2E1 has the ability to break down benzene, one of the reaction will produce phenol, which can be degraded by algae. To measure the efficiency of CYP2E1, we use “Emerson reagent” as our way to detect the decomposition of benzene.
Emerson reagent
Emerson reagent is made from two solutions:
Solution I : g 4-aminoantipyrine in 100 ml 80% ethanol.
Solution II: 4g potassium hexacyanoferrate (III) in 20 ml water 80ml ethanol.
The purpose of this reagent is to estimate the existence of phenol, phenolic compounds were determined by buffering the sample to an alkaline oxidant in a solution of high pH to prevent the formation of quinonoid substitution product and adding 4-aminoantipyrine to produce a red or orange colored complex in the presence of potassium hexacyanoferrate (III). By measuring OD, we can infer the efficiency of CYP2E1. The maximum yield of chromogen is expected around the absorption at 420 nm to 500nm. The highest stability of the color producing appeared to be in the pH range of 9.4-10.2.