Isolation of Peroxisomes
The investigation of unique functional and structural
aspects of peroxisomes (PO) requires the preparation
of highly purified fractions of this organelle.
This is, however, hampered by two serious problems:
(1) the relative paucity of PO (2% of total liver protein)
and (2) their considerable fragility. Thus, mild homogenization
conditions minimizing mechanical, hydrostatic,
and osmotic stress have to be sustained.
In general, the isolation of PO is accomplished in
three steps: (a) homogenization of the tissue or disruption
of the cells; (b) subfractionation of the
homogenate by differential centrifugation usually
according to the classical scheme of de Duve et al.
(1955); and (c) isolation of purified peroxisomes by
density gradient centrifugation of the so-called light
mitochondrial (λ) fraction.
Homogenization is commonly carried out in an isotonic
medium (e.g., see Section III,A,1) at low salt
concentrations to avoid aggregation. Addition of
a chelator (e.g., EDTA), however, is feasible, as it
prevents the aggregation of microsomes that may
contaminate PO. Moreover, the homogenization
buffer (HB) should be supplemented with antioxidants
(dithiothreitol, DTT), as well as protease
inhibitors (phenylmethylsulfonyl fluoride, PMSF; ε-aminocaproic
acid) to block oxidative and proteolytic
activities in the course of the isolation procedure.
For the purification of PO by density gradient centrifugation,
three approaches have been developed. In
the classic procedure (Leighton et al.
, 1968), sucrose
gradients and the specialized type Beaufay rotor were
employed. A self-generating Percoll gradient in conjunction
with a vertical rotor is used for the isolation
of PO under isotonic conditions (Neat et al.
, 1980). The
most straightforward approach to obtain highly purified
PO makes use of iodinated gradient media such
as metrizamide, nycodenz or Optiprep in combination
with a vertical rotor (Völkl and Fahimi, 1985; Hartl et
al., 1985; van Veldhoven et al.
, 1996). In the latter
media, PO band at the quite high density of 1.24 g/cm3
because of their permeability to low molecular weight
compounds (van Veldhoven et al.
, 1983), well separated
from lysosomes as well as from mitochondria
The method described in this article is a modification
of the protocol established for the isolation of
highly purified (>98%) PO from normal rat liver (Völkl
and Fahimi, 1985). Meanwhile, it has been applied to
livers and kidneys of several other mammalian species
(Fahimi et al.
, 1993; Zaar et al.
, 1992), as well as for the
isolation of PO from cell cultures (Schrader et al.
With an additional differential centrifugation step,
even peroxisomal subpopulations may be isolated
according to this protocol, as has been demonstrated
by Lüers et al.
II. INSTRUMENTATION AND
- Perfusion device (self-made)
- A 30-ml Potter-Elvehjem tissue grinder (Cat. No.
1931 05145) with a loose-fitting Teflon pestle (Cat.
No. 1931 05155; clearance 0.10-0.15mm) and a
motor-driven homogenizer (Cat. No. 5308 50001)
are from Migge, 69123 Heidelberg, FRG.
- Refrigerated low- and high-speed centrifuge
(e.g., Beckman TJ-6 and J 2-21); ultracentrifuge (e.g.,
Beckman L90) with corresponding rotors (e.g.,
Beckman JA-20; VTi 50).
Morpholinopropane sulfonic acid (MOPS, Cat. No.
29 836), PMSF (Cat. No. 32 395), and DTT (Cat. No.
20 710) are from Serva (Heidelberg, FRG). Ethanol
(Cat. No. 8006) and NaCl (Cat. No. 0277) are from
J. T. Baker (Deventer, Netherlands). EDTA (Cat. No.
E 2628-2) is from Max Keller (Mannheim, FRG), ε-aminocaproic acid (Cat. No. 62 075) is from Riedel-de
Haen (Seelze, FRG), sucrose (Cat. No. 4621) is from
Roth (Karlsruhe, FRG), and metrizamide (Cat. No. 10o
1983N) is from Life Technologies (76344 Eggenstein-
Female rats of 220-250g body weight, starved
III. PROCEDURES (FIG. 1)
A. Perfusion and Homogenization
|FIGURE 1 Flowchart for the isolation of highly purified (>98%) peroxisomes from rat liver.
- Homogenization buffer (HB): To make 1 liter, dissolve
85.56g of sucrose (250mM), 1.046g of MOPS
(5mM), 0.372 g of EDTA-Na2 (1mM), and 1 ml of
ethanol (0.1%) in distilled water, adjust pH to 7.2 with
NaOH, and add water up to 1 liter. Store at 4°C. Prior
to use add per 100ml: 0.2µl of 0.1M PMSF, 0.1ml of
1M ε-aminocaproic acid, and 20µl of 1M DTT.
- Saline (0.9%): To make 1 liter, dissolve 9g of
NaCl in distilled water and adjust to a total volume of
B. Subcellular Fractionation
- Anesthesize the animal (e.g., by ip injection of
- Weigh the animal, open abdominal cavity, and
perfuse liver with 0.9% saline via the portal vein until
all blood is drained away.
- Remove liver, dissect connective tissue, and
weigh and cut liver into small pieces collected in a
Potter tissue grinder held in an ice bath containing
3 ml/g (wet liver weight) of ice-cold HB.
- Homogenize tissue with a loose-fitting pestle,
applying a single down-and-up stroke at 1000rpm.
- Pour homogenate into a 50-ml centrifuge tube.
HB as described in Section III,A.
C. Metrizamide Density Gradient
- To remove debris, unbroken hepatocytes, and
blood cells, as well as most of nuclei, centrifuge the
total homogenate at 70g for 10min in a refrigerated
- Carefully pour off the supernatant (loose pellet
!), resuspend pellet in 2ml/g of ice-cold HB, rehomogenize,
and spin again under the same conditions.
- Pour off the second supernatant and combine it
with the first one (postnuclear supernatant); discard
- Centrifuge postnuclear supernatant at 1950g for
10min in a refrigerated high-speed centrifuge.
- Decant supernatant (firm pellet), resuspend
pellet manually in 1 ml/g of ice-cold HB using a glass
rod, and spin again at 1950g. The final pellet (heavy
mitochondrial fraction) contains the majority of mitochondria,
large microsomal sheets, and some remaining
nuclei. The combined supernatants represent the
- Subject the latter to 25,300g for 20min; remove
supernatant, including the reddish fluffy layer by
suction; resuspend pellet in about 10 ml of ice-cold HB
using a glass rod; and recentrifuge at 25,300g for
15min. Resuspend the final pellet in 5ml of ice-cold
HB again by means of a glass rod, which comprises the enriched heavy peroxisomal = light mitochondrial (λ)
fraction. The corresponding supernatant may be either
used directly to prepare a microsomal fraction and a
final supernatant (soluble proteins mostly of cytosolic origin) or processed further for the isolation of "light
- To this extent it is centrifuged at an integrated
relative centrifugal force (RCF) of 4.47 × 105gmin (gmax = 39,000). Resuspend the pellet thus obtained in 5 ml
of ice-cold HB using a glass rod; this pellet represents
the enriched light peroxisomal fraction (Luers et al.,
Preparation of a Metrizamide Gradient
- Gradient buffer (GB): To make 1 liter, dissolve
1.046g of MOPS (5 mM), 0.372g of EDTA-Na2 (1 mM),
and 1 ml of ethanol (0.1%) in distilled H2O, adjust with
NaOH to pH 7.2, and add H2O up to 1 liter. Store at
4°C. Prior to use add per 100ml: 0.2ml of 0.1M PMSF,
0.1 ml of 1M ε-aminocaproic acid, and 20 µl of 1M DTT.
- Metrizamide solutions (MS)
- 60% (w/v) stock solution: Dissolve 60g of
metrizamide in GB by stirring. Add GB up to
100ml. Store at 4~
- Gradient solutions: To prepare one gradient,
take 3.78, 3.38, 3.53, 2.06, and 3.2 ml of the 60%
stock solution. Add up GB to 10, 7, 6, 3, and
4ml using a refractometer to definitely adjust
densities to 1.12, 1.155, 1.19, 1.225, and
1.26 g/ml, respectively.
- Layer sequentially 4, 3, 6, 7, and 10ml, respectively,
of MS (1.26-1.12g/ml) in a 40-ml centrifuge tube
(e.g., Quick-seal polyallomer, Beckman) to form a
- Immediately freeze the gradient in liquid nitrogen
and store it at -20°C.
- Thaw the gradient quickly at room temperature
using a metallic stand, thus transforming the step
gradient into one with an exponential profile.
IV. OBSERVATIONS AND
- Layer 5ml of either the light or the heavy
enriched peroxisomal fraction (corresponding to one
liver of approximately 5-6 g) on top of the thawed gradient
and seal the tube.
- Centrifuge gradients in a vertical-type rotor (e.g.,
Beckman VTi50) at an integrated force of 1.256 × 106g min (gmax= 39,000) using slow acceleration/deceleration
modes. Under the conditions employed, highly
purified heavy peroxisomes band at 1.23-1.24g/ml
and light peroxisomes band at 1.20-1.21 g/ml.
- Recover the peroxisomal fraction by means of a
fraction collector; alternatively, the gradient tube can
be punctured and the fraction aspirated by a syringe.
Store fraction at -80°C.
- To remove metrizamide, which interferes with
the determination of some peroxisomal enzymes (e.g.,
urate oxidase) or of protein (Lowry method), dilute the
peroxisome fraction about 10-fold with HB followed
by centrifugation at 25,000 and 39,000 g, respectively,
to pellet the organelles.
The properties of the heavy peroxisomal fraction are
listed in Table I. Estimated by the specific peroxisomal
reference enzymes, it shows a purification rate of about
38-fold over the original homogenate. More than 95%
of the total protein content of this fraction is contributed
by peroxisomes (Völkl and Fahimi, 1985), with
mitochondria and microsomes accounting for about
2% each and lysosomes for less than 1%. This is confirmed
by electron microscopy, which shows that peroxisomes
make up 98-99% of the fraction (Figs. 2 and
3). Many peroxisomes contain the typical inclusions of
urate oxidase in the matrix (Fig. 2), but some extruded
free cores are also found between the organelles. The
electron-dense cytochemical reaction product of catalase
after the incubation of filter preparations in the
alkaline 3,3'-diaminobenzidine medium (Fahimi, 1969)
is seen over the matrix of the majority of peroxisomes (Fig. 3), demonstrating their integrity and the absence
of leakage of catalase.
|FIGURE 2 Electron microscopic appearance of
peroxisomes after fixation in
glutaraldehyde and osmium. The fraction
almost exclusively of peroxisomes (PO) with only a rare
mitochondrion (M). Many peroxisomes contain urate
(arrowheads). Bar: 1µm.
|FIGURE 3 A preparation comparable to that in Fig. 2
with alkaline 3,3'-diaminobenzidine for
localization of catalase
(Fahimi, 1969). Note the
electron-dense reaction product of
catalase over the
matrix of most peroxisomes. This illustrates the
of catalase leakage, confirming their integrity. Bar: 1µm
The polypeptide pattern (SDS-PAGE) of rat liver
heavy peroxisomes is shown in Fig. 4 (lane R) confirming
their high degree of purity because of the
absence of bands typical for mitochondria and microsomes.
It also shows distinct differences in protein
composition of hepatic peroxisomes between rat (R) and guinea pig (Fig. 4, lane G). The selective induction
of specific peroxisomal proteins, such as the trifunctional
protein (PH) in rats treated with hypolipidemic
fibrates (lane Bz) with the concomitant reduction of
catalase (Cat) and urate oxidase (UOx), is apparent
in Fig. 5.
|FIGURE 4 SDS-PAGE of highly purified heavy peroxisomes
from rat (R) and guinea pig (G) liver. A 10-12.5% resolving gel was
used, and the amounts of protein loaded per lane were (R) 2.4µg
and (G) 5.4µg. Silver staining of polypeptide bands. Mr standards:
BSA (66kDa), ovalbumin (45kDa), and trypsinogen (24kDa). Note
the distinct differences in the polypeptide patterns between rat and
guinea pig peroxisomes.
|FIGURE 5 SDS-PAGE of highly purified peroxisomes from
control (Co) and bezafibrate-treated (Bz) rat liver. A 10-15% resolving
gel was used, and 5.0µg of protein was loaded per each lane.
Silver staining of bands. Peroxisomal polypeptides indicated by an
arrowhead are PH, trifunctional protein; Cat, catalase; and UOX,
urate oxidase. Note the induction of PH and the concomitant reduction
of Cat and UOX in peroxisomes of Bz-treated rats. Also note
that the control preparations, R in Fig. 4 and Co in Fig. 5, are not
identical and that different resolving gels have been used.
The extended procedure outlined here has been successfully
employed to isolate heavy and light peroxisome
subpopulations from normal and regenerating
rat liver differing in density, size, shape, and enzymatic
composition (Lüers et al.
, 1993). Moreover, peroxisome
subsets as divergent as the former have been
also obtained from the human heptoma cell line
HepG2 (Schrader et al.
, 1994). However, further subpopulations,
which are also present in those tissues,
escape purification by conventional gradient centrifugation,
most probably because their sedimentation
properties are close to those of other subcellular
organelles, particularly microsomes. Nevertheless,
their purification has become an essential task in view
of the functional significance of PO in humans in
general (Wanders et al.
, 1995) and the putative importance
of peroxisomal subpopulations in the biogenesis of this organelle in particular (Erdmann et al.
Titorenko et al.
As an alternative to density gradient centrifugation,
immune free flow electrophoresis (IFFE) has been
introduced and applied successfully for the purification
not only of regular PO (ρ = 1.22-1.24 g/cm3
a light mitochondrial fraction of rat liver, but also of
PO from heavy and postmitochondrial fractions (Völkl et al.
, 1997, 1999). IFFE combines the advantages of
eletrophoretic separation with the high selectivity of
an immune reaction. It makes use of the fact that the
electrophoretic mobility of a subcellular particle,
complexed with an antibody directed against the cytoplasmic
domain of one of its integral membrane proteins,
is diminshed greatly, provided the pH of the
electrophoresis buffer is adjusted to pH-8.0, which is
the pI of IgG molecules. The PO isolated by IFFE from
the diverse hepatic subcellular fractions differed in
their composition of matrix and membrane proteins
as has been revealed by immunoblotting, thus supporting
the view that IFFE is a valid method for the purification of distinct subsets of the peroxisomal
The original work in the laboratory of the authors
has been supported by grants of the Deutsche
Forschungsgemeinschaft, Bonn, FRG (Fa 146/1-3; Vo
317/3-1; SFB 352 and Vo 317/4-1) and Landesforschungsschwerpunkt-
Programm of the State of
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