1. Abstract
The expression of Cytochrome P450 enzymes determine an individual’s ability to
metabolize xenobiotics (Ioannides, 2008) which is why these enzymes are significant in
forensic toxicology. Current methods for obtaining Cytochrome P450-containing
microsomes require the use of an ultracentrifuge which is not commonly used in forensic
facilities. Super-paramagnetic microspheres are able to be used in the extraction of proteins
and structures from crude biological samples (Safarik & Safarikova, 2004) and
microspheres have been previously used for the immunoprecipitation of microsomes
(Fujiwara & Itoh, 2014). Here a method is proposed for the application of super-
paramagnetic microspheres with anti-CYP2D6 IgGs to extract microsomes from human
liver homogenate. Recovery of the beads from the liver homogenate proved to be difficult
initially due to the viscosity of the fluid. A method was developed to overcome fluid
viscosity to recover the beads. Anti-CYP2D6 IgG binding to CYP2D6 was assessed using
liquid chromatography mass spectrometry and flow cytometry.
1. Introduction
1.1. Cytochrome P450
Cytochrome P450 (CYP) is a large family of proteins that preform oxidation and reduction
reactions for the purpose of metabolizing different endogenous and foreign compounds
(Kawakami et al., 2011). This superfamily of haem-thiolate enzymes are found in all five
kingdoms of life and can be found in the membranes of the Endoplasm reticulum and
Mitochondria for eukaryotic systems. Within bacteria, CYPs can be found in the cytosol instead
of microsomes (Ioannides, 2008).
1.1.1. Protein Structure and Function
It is hypothesized that CYPs with exogenous roles developed via co-evolutionary process
in animals to counteract toxins of plants that were partially synthesized using CYP-mediated
pathways. In comparing the enzyme structure between species, enzymes of mammals and
bacteria show relatively few structural differences. Despite few structural differences between
species, CYPs are able to bind to a variety of different endogenous and exogenous substrates.
Furthermore, genetic polymorphisms alter the metabolic capacity of these enzymes for different
substrates (Ioannides, 2008).
Cytochrome P450 enzymes are most alike to triangular prisms with the haem group
located approximately in the center of one triangular face (Ioannides, 2008). An anchor peptide,
about 20-40 hydrophobic residues long, binds these enzymes to phospholipid bilayers. This
peptides is believed to be helical in structure and stretches across the thickness of the
phospholipid bilayer. The way in which the rest of the enzyme is embedded in the membrane is
unclear. Within a membrane, CYPs may move laterally, transversely and rotate about an axis
that is perpendicular to the membrane plane. It is suggested that about 20% of the enzyme is
embedded in the phospholipid membrane which is due to belief that a portion of the enzyme
between 35 Å and 45 Å is located above the microsomal membrane.

2. It is known that lipophilicity plays a significant role in the binding of microsomal CYPs
to substrates (Ioannides, 2008). Substrates that CYPs bind to include fatty acids, eicosanoids,
vitamins, steroids and xenobiotics. The selectivity and binding capacity for different substrates is
linked to the enzyme’s active site region. Compound molecular, acid/base characteristics and
propensity for forming hydrogen bonds also affect the substrate selectivity of different CYPs.
Some drug-metabolizing CYPs show distinct but overlapping selectivities.
1.1.2. The Role of Cytochrome P450 in Drug Metabolism
CYP enzymes are involved in the metabolism of xenobiotics (Kawakami et al., 2011).
There are different CYPs located in the human liver such as CYP1A2, 2A6, 2B5, 2C8, 2C9,
2C19, 2D6, 2E1, and 3A4, which contribute to the pharmacological effect of drugs depending on
their expression and activities (Kawakami et al., 2011). For example, CYP2D subfamily has
been shown to take part in the demethylenation of MDMA (Kreth et al., 2000).
CYP 2D6 is found in relatively low levels in the human liver samples (Ioannides, 2008).
In the study by Kawakami et al. (2011), it was found it was in the range of 11.5 +/- 0.3 pmoles
per mg of human liver microsomal protein in a pooled sample. These levels vary depending on
the type of metabolizer (Ioannides, 2008). There are four categories of metabolizer phenotypes.
These phenotypes are poor metabolizers (PM), intermediate metabolizers (IM), extensive
metabolisers (EM), and ultrarapid metabolizers (UM). In the case of CYP 2D6 IM individuals
can have around 1 pmoles per mg of microsomal protein while other phenotypes such as EM or
UM can have around 30 or more pmoles per mg of microsomal protein. There are over 60
different alleles with a portion of them affecting the enzyme’s ability to metabolize different
xenobiotics. Despite low levels, this enzyme is significant in drug metabolism as it has the
highest ratio of drug/non-drug interactions compared to all other CYPs. This enzyme is able to
metabolize over 50 clinically used drugs.
1.1.3. Extraction Methodologies of Microsomal Cytochrome P450
One method of extracting CYP enzymes is the use of differential centrifugation
(Kawakami et al., 2011). In differential centrifugation, the tissue sample is homogenized using a
buffer then centrifuged in stages, with each next stage centrifuging at higher velocities until a
microsomal fraction is obtained (Kawakami et al., 2011). CYP enzymes have been quantified
from microsomes using Western blot analysis using monoclonal and polyclonal antibodies to
CYP 2D6 enzymes (Ioannides, 2008). Another method used to extract microsomes from
homogenate is the use of sucrose density-gradient centrifugation to separate the microsomes
from other subcellular components (Amar-Costesec et al. 1981).
1.1.4. Antibody Interaction with Cytochrome P450
A current difficulty in the use of antibodies for quantification is the preparation of specific
antibodies because of the high sequence similarity in CYP isoforms (Kawakami et al., 2011).
This study attempts to use this difficulty as an advantage at the extraction stage.

3. 1.2.Microsomes
Microsomes are composed of endoplasmic reticulum vesicles, plasma membranes, fragments
of the Golgi apparatus as well as other subcellular components (Amar-Costesec A., 1981). There
are smooth and rough microsomes. Smooth microsomes have an average size of about 200 nm in
diameter (Kawajiri et al., 1976) while rough microsomes have an average size of about 120 nm
(Leskes et al., 1971).
1.3.Magnetic microsphere Affinity Extraction
Antibody coupled microspheres have been used for various immunoassay (Roos & Skinner,
2003). The use of super-paramagnetic microspheres allows for the separation of beads from a
solution in immunoassays. These microspheres have also been used for the isolation of
organelles and subcellular structures (Safarik & Safarikova, 2004). The benefits of using these
magnetic microspheres is the procedure simplicity, applicability in crude samples, steps can be
automated and the separation process is usually fairly gentle. These beads are available in
different sizes which are suited to different applications.
1.4.Study Objective
The purpose of this study is to develop a method that uses super-paramagnetic microspheres
to extract microsomes containing CYP enzymes to be analyzed by mass spectrometry in forensic
laboratories. Tosylactivated super-paramagnetic microspheres have been chosen for this study
due to their simple protocol for coupling proteins to the surface and the ability to magnetically
separate the beads from their surrounding solution. CYP2D6 was chosen as the target protein
because of its significance of metabolizing many drugs. The use of super-paramagnetic
microspheres could also provide a less expensive means for obtaining microsomes from different
samples.
2. Materials and Methods
2.1. Bead Coupling Procedure
Volumes of reagents used were determined based on scaled down proportions used in the
manufacturer’s protocol for 5 mg of M-280 Tosylactivated Dynabeads (Cat. No 14203). The
concentration of contents of the Buffers used can be found in the Table below (Table 1).
Table 1: Buffers used for protein-bead coupling
Buffer Name Contents
Buffer B 0.1 M Na-phosphate buffer, pH 7.4
Buffer C 3 M ammonium sulphate, 0.1 M Na-phosphate buffer, pH 7.4
Buffer D1 Added 0.88 g NaCl then filled to 100 mL with 0.1 M Na-
phosphate buffer, pH 7.4
Buffer D PBS with 0.5% BSA, pH 7.4
Buffer E PBS with 0.1% BSA, pH 7.4

4. All incubation steps except for bead coupling used a Fisher Scientific Isotemp®
Incubator
(Model 630D) which henceforth will be referred to as the incubator. A rotator device with
unknown make and model was used. This device rotated a horizontal beam at a rotation speed
that resulted in one (360º) rotation every 5 seconds. No dial was available to change the rotation
speed for this device.
2.2.Determination of protein coupled onto bead surface
For this experiment, Buffers D and E were substituted for Buffer D1 which contained the
same concentration of reagents except it contained no BSA. The reason for this is that it would
interfere with determining the quantity of protein coupled to the surface of the beads as the BCA
assay would not differentiate between protein coupled during incubation and protein coupled
during the subsequent washing stages meant to block any unreacted tosyl groups on the surface
of the beads. A stock solution of BSA at a concentration of 2.224 μg/μL was prepared.
2.2.1. BSA coupling to Bead surface
BSA was used as a substitute for the IgG in this experiment. Thus the mass of BSA used
for this experiment was calculated from the equivalent amount of moles that would be used for
the IgG coupling experiment to come. From the mass of BSA used, the volume of beads and
reagents was also determined.
BSA was coupled to the surface of the beads using a similar manner as described in the
manufacturer’s protocol provided (Catalog nos. 14203, 14204; Dynabeads®
M-280
Tosylactivated, Rev. 010) but scaled down from 5mg of beads. Briefly, the beads were vortexed
for 70 seconds. Pipetted 18.56 μL of beads to a 500 μL microcentrifuge tube (W1). Pipetted
18.56 μL of Buffer B then suspended beads. Affixed quarter-sized neodymium magnet to W1,
left for 1 minute then removed supernatant. Removed magnet then resuspended beads in 50 μL
of Buffer B again. Transferred beads to new tube of same size (C1). Affixed same magnet to C1
and left for 1 minute then discarded supernatant. Resuspended beads in 5.01 μL of stock BSA
(2.224 μg/μL) and 11.70 μL of Buffer B. Pipetted 11.14 μL of Buffer C into the C1 then
incubated at 37ºC while mixing on a rotator device (parallel to rotating beam) for 21 hours
(minimum of 18 hours due to the reduced volume of the reaction). The tube was placed parallel
to the rotating beam of the device to ensure that the droplet would move around thus mixing the
solution. Affixed quarter-sized magnet to beads and left it for over 2 minutes then removed
supernatant. Removed magnet, added 500 μL of Buffer D1 to C1 then placed C1 on rotator
device (parallel to rotating beam) while incubating at 37ºC for 1 hour. Tube C1 was affixed to
the magnet to collect the beads then the supernatant was removed. Then the beads were subjected
to two more washes by vortexing in 500 μL of Buffer D1 for 10 seconds then removing the
supernatant after leaving the C1 attached to the quarter-sized magnet for 2 minutes. The beads
were then used reacted with the BCA reagent
2.2.2. Bovine Serum Albumin standard preparation and BCA assay
measurements using spectrophotometry
In the BCA assay experiment, 6 standards were prepared at concentrations of 0, 5, 25, 50,
125 and 250 μg/mL from a BSA stock solution (2.224 μg/ μL). Also two replicate tubes were

5. prepared for each of the solutions used (Buffer B, Buffer C and Buffer D1). However, this
experiment was repeated to include a blank set of beads (Cnp) as a control for this experiment.
Thus in the second BCA assay experiment, a set of beads coupled exactly the same way
as described in the previous section (2.2.1) was prepared as well as a set of beads prepared in the
same manner but without any BSA included in the protein coupling step. Thus no protein should
be detected from a BCA reaction with these beads. Six standards of concentrations 0, 5, 25, 35,
50 and 125 μg/mL were prepared from a stock solution of BSA (2.224 μg/μL). These standards
were separated into two sets of test tubes containing 0.1 mL of each of these standards. For the
protein coupled beads (Cp) and beads without protein coupled (Cnp) 95 μL of ddH2O was added
to give a volume of about 0.1 mL then added to a test tube.
Working reagent of the BCA assay was prepared as per the protocol provided by the
manufacturer (Pierce TM
BCA Protein Assay Kit, No. 23227). Briefly 50 parts of BCA reagent A
was mixed with 1 part BCA reagent B to give a final volume of 51 mL of working reagent.
To each tube prepared, 2 mL of BCA working reagent was added to the tube then
incubated together in a hot bath (temperature 54ºC) for 30 minutes. Then the tubes were
removed from the hot bath and allowed to cool in another bath at room temperature until the
tubes reached room temperature. This was not done for the previous BCA experiment. The
previous experiment used an oven to heat the samples which likely introduced error into the
absorbance measurements. This experiment used the hot bath method for heating the samples as
it was recommended by the manufacturer and was found not to introduce the same level of error.
The absorbances at 562 nm were measured on a Cary 100 Bio UV-vis Spectrophotometer
(Varian) with average time set to 3.000 seconds and SBW set to 4.0 nm. The instrument was
blanked on both replicates of the 0 μg/mL solutions in glass cuvettes. This instrument contained
two lanes for measurement so one blank was left in to ensure the instrument gave absorbance
values relative to the blank. Then each of the solutions were pipetted into the other cuvette one at
a time and measured 3 times. Between each standard and sample tested, the cuvetttes were
washed first with ddH2O twice then acetone solution once to dry the cuvette. Measurements were
recorded then analyzed in Excel to determine concentration of the samples tested via use of a
standard curve.
2.3. Co-Immunoprecipitation of microsomal CYP2D6 from Human Liver
Homogenate
The purpose of this experiment was to determine if the IgG-coupled microspheres could be
used recovered from the liver homogenate and if the recovered microspheres had pulled out
microsomes from the liver homogenate. Human liver homogenate samples were provided by
Brigitte Desharnais from the Montreal Forensic Laboratory. The Sample ID #s were 2015-A-01,
2015-A-02 and 2015-A-03. This method used acid denaturation to help in eluting the proteins off
of the beads. Therefore a solution of 0.01 HCl and 0.04 NaOH were created. The purpose of the
NaOH solution is to neutralize the HCl acid in the eluate prior to trypsin digest. The magnets
used in this study are neodymium rare earth magnets. The large magnet was 7.5 cm x 0.8 cm x
3.7 cm, in the shape of a bar. The small magnets were 1.2 cm in diameter and 0.3 cm thick, in the

6. form of disks. The “quarter-sized” magnet was 2.0 cm in diameter and 0.3 cm thick, in the form
of a disk. The rotator device had no make or model number printed on it.
2.3.1. Antibody Purification and aliquoting
Since the reaction that couples the beads to the protein require the IgG solution to contain
no carrier protein and sodium azide, the goat anti-CYP2D6 N-20 obtained from Santa Cruz
Biotechnology (sc-23690) had to be purified prior to coupling to the beads.
Purification of the antibodies was performed using the PearlTM
Antibody Clean up Kit
(Cat #786-803) from Geno Technology Inc. and using the manufacturer’s protocol with some
modifications. From the original stock solution of IgG, 250 μL of solution was purified
(corresponding to 50 μg of IgG).
For the desalting steps, a “Safety Centrifuge” (115 V 50/60 CV.) by Fisher Scientific was
used to centrifuge the IgG samples. Because this device could only centrifuge at about 200 xg
the time for each centrifuge step was increased by 5X to obtain the same to obtain the same level
of separation as per Stokes’ law. So all centrifuge step requiring a time of 2 minutes in the
centrifuge were centrifuged for 10 minutes instead. For the IgG Purification steps, no alteration
to the Manufacturer’s protocol was made.
The sample was then transferred to a 30 k NMWL Millipore centricon filter device and
filtered as per the manufacturer’s protocol in Buffer C. A final volume of 123.78 μL was
obtained for the purified IgG solution in Buffer C. This was separated into three aliquots. Two
contained precisely 41.2 μL while one aliquot contained about 41.38 μL. Two of the aliquots
were placed in the -80ºC Freezer for long term storage. The IgG was put into Buffer C because it
would reduce the need for adding Buffer C separately prior to the incubation step.
2.3.2. IgG coupling to magnetic microspheres
The anti-CYP2D6 IgG was coupled to the magnetic microspheres using the same method
mentioned in section 2.2.1. The volume of beads used for coupling was 27.78 μL (corresponding
to 833.3-
μg of beads). Prior to the incubation step, the bead were washed with 50 μL of Buffer
B, then in 100 μL of Buffer B in the second wash prior to incubation. Each wash was performed
by resuspending the beads in the given volume of buffer, affixing the quarter-sized magnet to the
tube for 1 minute then discarding the supernatant. Also instead of adding Buffer C and the
protein solution separately, a full IgG aliquot was added (at least 41.2 μL) to the beads and no
Buffer B was added. The reason Buffer B was not included in this step prior to incubation was
because the concentration of IgG in the solution was already too dilute and thus Buffer B was not
needed for this step to dilute the protein.
After an 18 hour incubation (39-40ºC) with rotation (360º rotation every 5 seconds) for
18 hours, the quarter-sized magnet was affixed to the side of the tube then left for 2 minutes.
After 2 minutes, the supernatant was removed then the beads were resuspended in 500 μL of
Buffer D. The tube was placed in the incubator and rotator for 1 hour at 37-38ºC. The tube was
affixed to the same magnet for 2 minutes then the supernatant was removed. The beads were
then washed in 450 μL of Buffer E twice. To resuspend the beads during each wash, the beads
were vortexed for 10 seconds. Lastly, the beads were resuspended in 40 μL of Buffer E then