Best Basic parts

Part name:

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.

Protocol

Exp1. 4-aminoantipyrine/potassium hexacyanoferrate (III) function test
↓ prepare a 96 well plate, solution I, solution II, phenol (8.3g/100ml)
↓ add 22μl phenol solution, add 49.5μl solution I, 148.5μl solution II to column 1
↓ other wells add 45μl solution I, 135μl solution II
↓ serial dilution 10X (200μl)(control : phenol serial dilution 10X with water)

Exp2. Best concentration ratio between solution I and solution II
↓ prepare a 96 well plate, solution I, solution II, benzene, protein 5-aminolevulinic acid (ALA)+ FeSO4
↓ add 11μl solution I and 99μl solution II to column 1
↓ other wells add 90μl solution II
↓ serial dilution 10X (100μl)
↓ each well add 40μl protein and 0.2μl benzene(1.8*10ˆ-10 g/1L)
↓ shake for 30 min
↓ measure OD and compare
↓ find the best ratio*(control : 100μl water, 40μl protein , 0.2μl benzene (1.8*10ˆ-10 g/1L) last column : blank)

Exp3. Algae degrade phenol ability efficiency measure
↓ prepare a 96 well plate, algae, phenol(8.3g/100ml)
↓ add 198μl algae 22μl to column 1
↓ others column add 180μl algae
↓ serial dilution 10X
↓ shake for 30 min
↓ add 10μl solutionT, solution II 90μl
↓ measure OD*(control : same with Exp3 last column : blank)

Exp4. CYP2E1 function efficiency measure
↓ prepare a 96 well plate, CYP2E1 in five different express ways respectively : Thiamine, FeSO4, Fe2(SO4)3, 5-aminolevulinic acid (ALA), and 5-aminolevulinic acid (ALA)+FeSO4, solution I, solution II, benzene
↓ add 50μl solution I, 150μl solution II, 0.4μl benzene to column 1
↓ other wells add 45μl solution I, 135μl solution II
↓ serial dilution 10X (200μl)
↓ each role add one protein (Thiamine, FeSO4, Fe2(SO4)3, 5-aminolevulinic acid (ALA), and5-aminolevulinic acid (ALA)+FeSO4) 50μl
↓ shake for 30 min
↓ measure OD (all)(control : solution I 50μl, solution II 150μl, 0.4μl benzene, 50μl water last column : blank )

Exp5. Algae and CYP2E1 coaction
↓ prepare a 96 well plate, CYP2E1 in five different express ways respectively : Thiamine, FeSO4, Fe2(SO4)3, 5-aminolevulinic acid (ALA), and 5-aminolevulinic acid (ALA)+FeSO4, solution I, solution II, algae, benzene
↓ add 200μl algae to column 1
↓ other well add 180μl algae
↓ add 0.4μl benzene
↓ serial dilution 10X (200μl)
↓ each role protein (Thiamine, FeSO4, Fe2(SO4)3, 5-aminolevulinic acid (ALA), and+5-aminolevulinic acid (ALA)FeSO4) 50μl
↓ shake for 30 min
↓ add solution I 50μl, solution II 150μl
↓ measure OD(control : sample of Exp.4 last column : blank)
*Note : If the result isn’t clear enough, add Potassium hydroxide to made the pH of solution is around 9.4~10.2

Reference

1. Physiology and Pathology of Chloride Transporters and Channels in the Nervous System-Ion Transport in Choroid Plexus. Peter D.BrownSarah L.DaviesIan D.Millar
2. Human cytochromes P450: evolution and cDNA-directed expression. Gonzalez FJ, Gelboin HV.
3. CYP2E1-dependent benzene toxicity: the role of extrahepatic benzene metabolism. Bernauer U, Vieth B, Ellrich R, Heinrich-Hirsch B, Jänig GR, Gundert-Remy U.
4. Comprehensive Toxicology-Halogenated Hydrocarbons. H.M.Mehendale
5. Selvakumar, Paulraj Mosae. “Phenol Sensing Studies by 4-Aminoantipyrine Method-A Review.” Organic & Medicinal Chemistry International Journal, vol. 5, no. 2, 2018, doi:10.19080/omcij.2018.05.555657.
6. Kim, Hong Yeong, et al. “Reaction-Based Colorimetric Signaling of Cu2 Ions by Oxidative Coupling of Phenols with 4-Aminoantipyrine.” Talanta, vol. 132, 2015, pp. 625–629., doi:10.1016/j.talanta.2014.09.048.
7. Kim, Hong Yeong, et al. “Reaction-Based Colorimetric Signaling of Cu2 Ions by Oxidative Coupling of Phenols with 4-Aminoantipyrine.” Talanta, vol. 132, 2015, pp. 625–629., doi:10.1016/j.talanta.2014.09.048