Cytochrome P450 – Isolation, Expression, Purification and Assay

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Dr. D. Victor A. Doss

Dr. D.Victor A.Doss

Cytochrome P450 is  a set of enzymes that coverts Xenobiotics (drugs and toxins  that are not native to the body) into water-soluble so that they are excreted. The fact that the expression and functioning of these enzymes differ from person to person, explains why people vary in their response to drugs and toxins. Thus the duration and the intensity of  the beneficial or harmful effects of drugs  vary from person to person.

History

Cytochrome P450 (CYP) was first discovered in 1954 by Martin Klingenberg 1  and David Garfinkel 2 .   However, Minor J. Coon is one of the leading authorities in this field. Though only a dozen or so papers  were  published in 1966, total number of papers published till this date stands to  52, 832 (PubMed 3 ). This explains the importance of the CYP in the modern drug discovery Process.

Human CYP (PDB Code 2D09)

Human CYP (PDB Code  2D09)

Biochemistry of CYP

It is a 550 amino acid long Protein (Monooxygenase) with a heme pigment (haem group)  at its active site. It absorbs maximum light (λ max ) at 450 nm when the heme iron is reduced (with sodium dithionite) and combined with Carbon Monoxide. 

From bacteria to plant and in mammals more than 6000 cytochrome P450 sequences are known and named by the CYP Nomenclature Committee ( http://drnelson.utman.edu 4 ). Mammalian/Plant origin Cytochrome P450 Oxidases are abbreviated  CYP and the bacterial enzymes as  P450 .  The human  Genome Project has identified about 63 human genes (57 full genes and 5 pseudogenes).  The example given below explains the system of Nomenclature followed ;

Image

Thus CYP2E1 is the Cytochrome P450 Oxidase of Mammalian or Plant origin belonging to Genome family 2 and subfamily E and enzyme number one . (enzyme involved in the metabolism of acetamenopen).

Function

CYP is present mainly in the Liver and in other parts of the body including mucosa of the gastrointestinal tract.. It is important for the Phase I metabolism  (modification or degradation of chemicals such as carcinogens, alkanes, terpenes, aromatic compounds, herbicides, insecticides  and endogenous compounds such as eicosanoids, fatty acids, fat soluble vitamins, bile acids.  Besides it is important in the bio-synthesis of Steroid hormones and in Vitamin D metabolism.  The broad range of reactions catalyzed by CYP are carbon hydroxylation, heteroatom oxygenation, dealkylation, epoxidation, aromatic hydroxylation, reduction and dehalogenation (Bernhardt 5 ).

Drug Metabolism

What makes the CYP very important in drug research is that it can be induced or inhibited by drugs. Thus when two drugs are taken together as is often the case, one drug may activate the metabolism of another drug, thus making it less available than what is required.  On the contrary, if one drug inhibits the metabolism of another drug by CYP, the accumulation of the second drug may reach the “overdose” level that it sometimes may become toxic or lethal. For eg., Fluoxetine slows down the metabolism of Desipramine which leads to its toxicity ending up in prolonged arrhythmia or even death.

Besides, individuals differ in their genetic makeup (guengerich 6 et al ., 1986) and will not have uniform drug response (Pharmacogenomics). Thus in future,  the drug-drug interaction may necessitate  the correct dose adjustment and different choice of  drug combinations for different people.

Methods in CYP Research

Detailed monitoring of any drug metabolism, its kinetics and the dose response of the drug are studied using the following techniques;

Preparation of Liver Microsomes

As the hepatocyte microsomal membranes anchor these cytochrome p450 enzymes most of the drug metabolic studies are carried out   in vitro   by purifying the microsomes from liver cells using ultracentrifuge (Wang   et al ., 1980 7 , 1983 8 ).

Purification of Human Liver P450 enzymes

The general methods for purification of P450 enzymes  involves solubilization of liver microsomes from 400 gms of liver with sodium choleate, followed by ultracentrifugation at 10 5  * 8  for 60 minutes and elution through n-octylamine Sepharose 4B Column (Guengerich 9 and Martin, 1980).

Recombinant enzyme preparation          

Since 6000 sequences of CYP 450 genes are known,, any gene of interest can be cloned into a suitable plasmid as fusion protein, its presence in the construct checked and transformed in to E. coli DH5α cells (manjunath s. Shet 10 et al ., 1993).  Cell disruption and solubilization of recombinant P450 from the bacterial membranes and purification of it using 2’,5’- ADP Sepharose affinity chromatography (Fisher 11 , C.W., et al .,1992) may yield 150 – 200 nmol of P450 per litre of bacterial growth medium.

Biotransformation of drugs

Biotransformation of any drug by human liver can be investigated in vitro by incubating the drug with liver microsome, hepatocyte or precision cut liver slices in the presence of NADPH or NADH regenerating system. This is a very common system of study in which formation of drug metabolite in vitro , its elimination and the possible effects of the drug etc., can be studied (Thierry createil 12 et al. , 1994). Amount of metabolized drug can be determined  by  analysis of the purified reaction mixture using GC-MS (Gas chromatography – Mass Spectroscopy) method (Gross 13 et al ., 1977)  or by HPLC method (Yanyan Cui 14 et al ., 2004).

Pharmacokinetics and Pharmacodynamics

Pharmacokinetic parameters such as C max,  t max,  t ½ , AUC, CL 0 , F, A e , MRT CL R , t u  etc., can be analyzed by withdrawing blood samples (from indwelling canula) in the forearm veins of volunteers right from 0.25 to 15 hours of time in fixed intervals.

Pharmacodynamic measurements

Pharmacodynamic effect can be assessed in each volunteer before drug administration and at the time of blood sampling for 5 hours post dose.  For eg.,  pharmacodynamics of enantiomers of the calcium antagonist can be studied by

 measuring vital parameters such as blood pressure, heart rate using automated sphygmomanometer, ECG and  peripheral blood supply to both legs using automatic venous occlusion plethysmograph. In addition to these parameters  concentration of drug metabolites in blood and urine samples can be analysed using GC-MS after administering the radio-labeled drug  (for eg., using 2 H 2 ).

Kinetic model Studies

Kinetic parameters of the metabolism of the drug such as K m , V max  (by graphic analysis of Eadie-Hofste Plots), k mapp  (Apparent K m in the presence of inhibitors), I  the inhibition Constant, CL (Plasma clearance), CL int  (intrinsic clearance) etc., can be studied by incubating the drug with liver microsomes (Wilhem kuhnz 15 and Hille Gieschen, 1998 and Akio Suzuki 16 et al., 1999).

Drug – Drug Interactions

Since the active site of CYP has the capacity to accommodate larger molecules and more than one substrate at a tine in the active site or allosterically.  Drug-Drug interaction leads to inhibition or activation of one drug takes place in the presence of another drug. For eg., Testosterone-Terfennadine Interaction  or Testosterone – Midazolam interaction can be studied by measuring the concentration of the major metabolites using any one of the methods describe above ( Regina 17 et al ., 2000).

Immunoinhibition Assay

The immunoinhibition of Human Liver Cytochrome P450 is carried out by pre-incubation with various anti-CYP antibodies  in phosphate buffer for about  30 minutes in different concentration. This is followed by incubating with the suitable drug and the NADH regenerating system (NADP, Glucose 6-Phosophate, Glucose 6-Phosphate dehydrogenase and MgCl 2.  HPLC assay of the metabolized drug is carried out in the pre-incubated and  free microsomal fractions (Kaoru kobayashi 18 et al ., 2001).

NMR and Computer-assisted Molecular modeling

To understand the structural basics of CYP interaction with any particular drug, the structure of the enzyme and  the substrate binding site have to be determined. This will give an idea of the “pharmacophore” recognized by the CYP.  In the absence of crystal structure for each of the membrane-bound mammalian P450s, the use of series of  indirect methods such as biochemical methods, UV-Visible Spectroscopy, NMR Spectroscopy, computer assisted molecular modeling are used (Sonia Poli-Scaife 19 et al ., 1997).

Summary

Thus every drug metabolized by the CYP and the drug-drug interaction that makes some drugs ineffective or toxic has to be studied in the earlier stage of drug discovery. The  difference in genetic makeup and the expression levels  between the individuals makes it more challenging.  The use of modern analytical instruments   such as HPLC,  GC-MS,  NMR,  and the availability of modern techniques such as Recombinant DNA Spectroscopy  and Computer Assisted Drug Discovery  (CADD) have become important and interesting.

References:

  1. Klingenberg M (1958). Pigments of liver microsomes. Arch.Biochem.Biophys. 75: 376-386.
  2. Garfinkel D (1958) Studies on pig liver microsomes . Enzymes and pigment composition of different microsomal fractions. Arch.Biochem.Biophys.77:493-509.
  3. PubMed ( www.ncbi.nlm.nih.gob/pubmed)
  4. http://drnelson.utmen.edu
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