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IUBMB Life, 49: 451 – 456, 2000
Copyright ° c 2000 IUBMB
1521-6543/00 $12.00 + .00
Original Research Article
Ferritin Oxidation in Vitro: Implication of Iron Release
and Degradation by the 20S Proteasome
M. Rudeck,1 T. Volk,2 N. Sitte,1 and T. Grune1
1
Clinics of Physical Medicine and Rehabilitation and 2 Clinics of Anaesthesiology and Operative Intensive
Medicine, Medical Faculty (Charité), Humboldt University Berlin, Schumannstr. 20/21,
D-10098 Berlin, Germany
Summary
Ferritin, the major iron storage protein in mammalian cells,
was treated with various concentrations of different oxidants: xan-
thine/xanthine oxidase, Sin-1 (3-morpholinosydnonimine , pur-
chased from Alexis, Grunberg, Germany), DEA-NO (Diethylamine
NONOate, purchased from Calblochem-Novabiochem, Schwal-
bach, Germany), and hydrogen peroxide. The proteolytic suscepti-
bility towards the isolated 20S proteasome of untreated ferritin and
oxidized ferritin was measured in parallel with the iron liberated
by these oxidants. With increasing hydrogen peroxide, Sin-1, and
xanthine oxidase concentrations, the measured proteasomal degra-
dation of ferritin also increased. At higher oxidant concentrations,
however, the proteolytic susceptibility began to decrease. The oxi-
dation of ferritin by DEA-NO was accompanied by a lesser increase
of proteolytic susceptibility in comparison with the effects of the
other oxidants. Addition of DEA-NO to Sin-1 suppressed the in-
crease in proteolytic susceptibility of ferritin, whereas adding xan-
thine/xanthine oxidase had no additional effect. Iron was liberated
readily from ferritin as a result of the oxidation process, although
the increase in proteolytic susceptibility was not always correlated
to the iron release. In fact, the degradation of oxidatively damaged
ferritin was not accompanied by a further increase of free iron.
Therefore, we conclude that the proteasome is a secondary antiox-
idative defense system that degrades only nonfunctional ferritin.
IUBMB Life, 49: 451 – 456, 2000
Keywords Ferritin; iron; proteasome; protein degradation; protein
oxidation; proteolysis.
Reactive oxygen species are constantly formed in an oxidiz-
ing environment (1 – 3). Primarily, several relatively nonreactive
compounds such as the superoxide radical and hydrogen perox-
ide are formed; however, the presence of reactive iron potentiates
Received 10 January 2000; accepted 22 March 2000.
Address correspondence to Dr. Tilman Grune. Fax: +49 30 2602
3190; E-mail: tilman.grune@charite.de
the danger of these substances (2, 3). In fact, most of the oxida-
tive damage in tissues requires the participation of catalytically
active iron (2, 3). Free iron can catalyze the decomposition of
hydroperoxides, including hydrogen peroxide, resulting in the
formation of highly reactive substances that promote lipid perox-
idation and other oxidative damage reactions (4, 5). An increase
of this reactive iron is believed to be a pathophysiological factor
in several diseases that are accompanied by oxidative damage
(5 – 7). To prevent this oxidative damage, a system has evolved to
limit the concentration of reactive iron but also to provide iron for
biochemical synthesis (8, 9). The main intracellular iron-storage
protein, ferritin, binds iron in a catalytically inactive manner; ac-
cordingly, oxidative reactions cannot be promoted by this iron.
Because the main iron pool of most cells is located within the
ferritin, it is important to know under what circumstances, if any,
this pool may be a source of iron. Ferritin has been shown to
release iron on contact with activated microglia and neutrophils
by a superoxide-dependen t mechanism (10, 11).
On the other hand, oxidative damage to ferritin that results
in a physiological malfunction, e.g., the release of iron, might
be accompanied by damage to the apoferritin molecule. Pro-
tein oxidation in mammalian cells is generally accompanied by
the degradation of oxidized protein molecules (12). As previ-
ously reported, hydrogen peroxide – induced protein oxidation
enhances the proteolytic susceptibility of the damaged protein
(13 – 15). The intracellular proteolytic system responsible for
the degradation of oxidized proteins is the proteasomal system
(15 – 17). Therefore, it would be interesting to know whether
degradation of ferritin by the proteasomal system has the poten-
tial to enhance any oxidative damage caused by the release of
iron. We tested here the effect of various oxidants on the pro-
teolytic susceptibility of ferritin and the release of iron from
the protein. Furthermore, we tested whether the degradation
of oxidized ferritin was accompanied by an enhanced iron
release.
451
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452 RUDECK ET AL.

METHODS removed by dialysis for 16 h at 4 ±C versus 2 l dialysis buffer

Proteasome Isolation. Proteasome was isolated from eryth-
rocytes of outdated human blood conserves according to
Hough et al. (18). Erythrocytes were lysed in 1 mM dithio-
threitol. After membranes and nonlysed cells were removed by
centrifugation, the proteasome was isolated by DEAE-chroma-
tography, sucrose-density gradient ultracentrifugation, and
separation on a Mono Q column of a fast protein liquid chro-
matography (FPLC; Pharmacia) system. The purity of the prepa-
ration was controlled by both one-dimension sodium dodecyl
sulfate – polyacrylamide gel electrophoresis and nondenaturing
polyacrylamide electrophoresis. Nondenaturing electrophoresis
was followed by overlaying the gel with the  uorogenic sub-
strate suc-LLVY-MCA to detect peptidase activity. The pro-
teasome preparation was diluted to a Ž nal concentration of
0.35 mg/ml.
Ferritin Treatment with Oxidants. We treated ferritin (1 mg/
ml) with various concentrations of hydrogen peroxide, xanthine/
xanthine oxidase, Sin-1, and DEA-NO in a 20 mM phosphate
buffer (pH 7.2) for 2 h at 25 ±C. Afterwards, the oxidants were
(10 mM potassium phosphate containing 10 mM KCl, pH 7.4).
When evaluating xanthine/xanthine oxidase, the amount of xan-
thine oxidase used was varied (from 2.23 to 64.1 ng/ml) and
xanthine was used at 0.4 mM. The activity of the xanthine oxi-
dase was assessed by measuring uric acid formation, which was
determined from absorbance at 292 nm (e = 12.5 mM ¡ 1 cm ¡ 1 ).
The reaction was stopped by adding allopurinol ( 2.5 l M).
Measurement of Protein Degradation. The degradation of
native and oxidized ferritin (1 mg/ml) was measured by deter-
mining acid-soluble free amines, for which we used  uorescence
detection after  uorescamine derivatization. Substrate protein
(40 l g) was incubated with 0.6 l g of proteasome in a prote-
olysis buffer containing 50 mM Hepes (pH 7.8), 20 mM KCl,
5 mM magnesium acetate, and 1 mM dithiothreitol for 2 h at
37 ±C. The reaction was stopped by adding an equal volume of
trichloroacetic acid (20%). After centrifugation (15 min, 12 000
£ g) the supernatants containing the primary amines were neu-
tralized with 1 M Hepes (pH 7.8). Fluorescamine (0.3 mg/ml in
acetone) was added with rigorous shaking, and the  uorescence

Figure 1. Proteolytic degradation of ferritin by the proteasome after oxidation. Ferritin was treated with the indicated concentra-
tions of the oxidant and extensively dialyzed. Xanthine oxidase (XO) was used at concentrations as great as 64.1 ng/ml, liberating
as much as 3 nmol of uric acid in 2 h of incubation. The amount of proteolytic degradation was determined after 2 h of incubation
with isolated proteasome. The values shown are the means of 6 independent experiments. X/XO, xanthine/xanthine oxidase system.

FERRITIN DEGRADATION AND IRON RELEASE
was determined at 470 nm emission (390 nm excitation). Leu-
cine was used as a calibrator. Proteolysis rates were calcula-
ted as the difference between the measured value and the blank
value (determined by summing the result for incubated subst-
rate protein without protease and the result for incubated
protease only).
Measurement of Iron Release. Nitriloacetate (10 mM) was
added to ferritin (1 mg/ml) after oxidation. The released iron
bound to nitriloacetate was Ž ltered through a membrane (Am-
icon) and used for determining iron by the Ferrozine method
(19). That determination was performed by a spectrophotomet-
ric measurement at 562 nm. Fe(NH4 )2 (SO4 )2 was used as the
calibrator.
RESULTS
Proteolytic Susceptibility of Ferritin after Oxidation
To determine the proteolytic susceptibility of ferritin after
oxidation by various oxidants, we analyzed the degradation of
the protein by the isolated 20S proteasome, the cytosolic pro-
tease responsible for the removal of oxidatively damaged pro-
teins. Given that the major reactive oxygen species released by
macrophages and neutrophiles are known to be the superoxide
anion and nitric oxide, we selected both substances as oxidants.
Because of the high likelihood of the combinatory reaction of
both the compounds, forming peroxynitrite in 1:1 stoichiome-
try, we used also Sin-1 as an oxidant. Hydrogen peroxide, an-
other major biologically occurring oxidant, was used also in our
experiments. The treatment of ferritin with hydrogen peroxide
increased the proteolytic susceptibility of the ferritin molecule
dramatically (Fig. 1). Additionally, the oxidants (mainly super-
oxide) produced by the xanthine/xanthine oxidase system and
the treatment with Sin-1 increased the proteolytic susceptibility
of ferritin towards the 20S proteasome >3.5-fold. Interestingly,
the release of NO from DEA-NO increased the proteolytic sus-
ceptibility of ferritin by 60%. This seems to be a speciŽ c effect
on the treatment of ferritin with NO, which was not found in ear-
lier studies that used aconitase as a substrate (20). Nevertheless,
the maximal proteolytic degradation of ferritin achieved by the
20S proteasome is comparable using all three major oxidation
systems.
With hydrogen peroxide, xanthine/xanthine oxidase, Sin-1,
and to a lesser extent DEA-NO, a decrease of the proteolytic sus-
ceptibility at higher concentrations of the oxidant was measured.
This is in accordance with earlier results from our laboratory (14,
15, 17, 21) and from others (13, 16, 22, 23).
Because the release of NO and superoxide anion in cellular
systems is not necessarily equimolar, we decided to use an addi-
tional experimental setup to determine the effect of a high or low
NO/O2¡ ratio. As Fig. 2 demonstrates, excessive amounts of NO
can prevent the increase of proteolytic susceptibility of ferritin,
whereas the combination of the xanthine/xanthine oxidase sys-
tem and Sin-1 did not change degradation of ferritin by the 20S
proteasome in comparison with Sin-1 alone. Given that one of
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453
Figure 2. Proteolytic degradation of ferritin treated with com-
bined oxidizing systems. Ferritin was treated with various ox-
idants and combination of oxidants. The following concentra-
tions were used: Sin1, 5 mM; DEA-NO, 1 mM; xanthine oxidase,
16.0 ng/ml. After extensive dialysis, the proteolytic degradation
was determined after 2 h of incubation with isolated proteasome.
The values are the means of 6 independent experiments.
the major factors in the oxidative damage of biological factors
is the concentration of free iron, it is important to know what
happens to the iron storage function of ferritin during oxidation
and proteolytic degradation.
Iron Release from Ferritin as a Result of Oxidation
First we measured the release of iron from ferritin after oxi-
dation with the already described oxidation systems. As demon-
strated in Fig. 3, all the oxidation systems used were able to
release iron from ferritin when oxidized, and in all cases iron
release reached a maximum. In our system DEA-NO increased
the iron concentration to > 11 l M, or » 10% of the total iron
bound to ferritin. Therefore, DEA-NO, a compound that does
not greatly increase the proteolytic susceptibility of ferritin, can
release iron from the protein.
For hydrogen peroxide and Sin-1, iron was released before
the maximal possible degradation of oxidized ferritin had oc-
curred (see arrows in Fig. 3). The superoxide-generating xan-
thine/xanthine oxidase system and DEA-NO, however, released
iron only at concentrations higher than the concentration of the
oxidant that resulted in the greatest proteolytic susceptibility.
Therefore, NO and the superoxide radical seem to increase the
proteolytic susceptibility of ferritin before the iron is released,
whereas the combined use of the radicals in equimolar amounts
is followed by a parallel process of iron release and increased
proteolytic susceptibility.
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454 RUDECK ET AL.

Figure 3. Release of iron from ferritin during treatment with various concentrations of oxidant. The release of iron was
measured as described in Methods. The concentration of xanthine oxidase ranged up to 2.7 l g/ml. The values are the means of
6 independent experiments. The arrows indicate the concentration of the oxidant at which the maximal proteolytic susceptibility
of ferritin is achieved.

An increase of the NO/O2¡ ratio was followed by a decline of

the iron release in comparison with the equimolar ratio produced
by Sin-1 (Fig. 4). On the other hand, including more superoxide
had no further effect on the release of iron from ferritin.

Degradation-Mediated Iron Release from Ferritin

Because the oxidation of ferritin by hydrogen peroxide, xan-
thine/xanthine oxidase, and Sin-1 is followed by an dramatic
increase in proteolytic degradation of the protein (Fig.1), we
wanted to know whether degradation of the substrate protein
would be followed by an enhanced iron release. When we tested
the in uence of the proteasome on the release of iron from fer-
ritin, as demonstrated in Fig. 5, we found that the addition of pro-
teasome had no effect on the iron concentration. Therefore, we
conclude that the proteasome recognizes only ferritin molecules
that are already depleted of iron, which are nonfunctional.

Figure 4. Release of iron from ferritin treated with com-
bined oxidizing systems at the following concentrations:
Sin-1, 5 mM; DEA-NO, 1 mM; xanthine oxidase, 16.0 ng/ml.
The iron liberation was measured as described above. The
values are the means of 6 independent experiments.
DISCUSSION
Previous studies have discussed the cytosolic proteasomal
system as one of the major defense mechanisms of the cell
against oxidative protein accumulation (12 – 17, 20 – 23). The role
of this proteolytic system is underlined by the ability of the
20S “core” proteasome to recognize speciŽ cally and degrade
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FERRITIN DEGRADATION AND IRON RELEASE 455

Figure 5. Release of iron from ferritin during proteasomal degradation. Ferritin was treated with Sin1 at 5 mM, H2 O2 at 10 mM,
or xanthine oxidase at 16.0 ng/ml. The iron liberated was measured directly after oxidation and after as much as 6 h of degradation
by the proteasome (PS). The values are the means of 6 independent experiments.

oxidized proteins (12, 14). In various in vitro studies using the
isolated proteasome and several oxidized protein substrates have
demonstrated the degradation of oxidized model proteins (12 –
17, 20 – 23). Such studies have used a variety of oxidizing agents,
including hydrogen peroxide (14, 15, 17, 20, 21), superoxide
(13), peroxynitrite (20), and hypochloric acid (24), and other
sources of oxidative damage (25, 26). Failure of the degrada-
tion of oxidized proteins has been proposed as one of the major
events promoting the age- or disease-dependent accumulation of
lipofuscin/ceroid-like  uorescent material (12, 27). Therefore,
the degradation of oxidatively damaged proteins seems to be the
major strategy of mammalian cells to deal with oxidized and
inactivated proteins.
Because cells are exposed to oxidative stress continuously or
at least most of the time, investigating the fate of the protein-
bound active compounds seems important. This is especially true
for iron because catalytically active free iron may enhance the
oxidative damage of the cell. Ferritin is the major iron storage
protein of the cell and is readily oxidized by hydrogen perox-
ide with an accompanying increase in proteolytic susceptibil-
ity (14). The increase of proteolytic susceptibility of ferritin by
other oxidants was also tested. The results with Sin-1 and xan-
thine/xanthine oxidase are in agreement with previous results
achieved by oxidation of other substrate proteins (13, 20); how-
ever, NO increases the proteolytic susceptibility of ferritin only
moderately. In a previous study using aconitase as a substrate,
we reported (20) no increase of degradation resulting from NO
treatment. Therefore, we think it is likely that, in the case of high
iron content and release from ferritin, several more-reactive ox-
idizing species are formed by NO treatment that are able to
increase the proteolytic susceptibility of the protein. NO and the
superoxide anion may react in a diffusion-limited reaction and

456 RUDECK ET AL.
form the highly reactive peroxynitrite. This reaction is stimu-
lated by the liberation of equimolar amounts of both radicals
from Sin-1. The formation of NO and superoxide may be not
be balanced in an in vivo situation, however, and various other
oxidants may be formed through further reactions (28 – 30).
When we tested here the effects of excess amounts of NO or
superoxide on proteolytic susceptibility of proteins, we clearly
demonstrated that a high amount of NO reduces the proteolytic
susceptibility of ferritin, whereas the addition of xanthine/xan-
thine oxidase to Sin-1 has no effect. Therefore, we conclude that
NO prevents protein oxidation—which is in accordance with
the liberation of iron from ferritin. NO suppresses the Sin-1 –
mediated iron liberation, whereas the xanthine/xanthine oxidase
system has no effect. We found that no iron is released because
of the degradation of damaged ferritin; rather, it is released as a
result of the oxidation process and the malfunction of ferritin.
In conclusion, we have demonstrated that the proteasome
degrades nonfunctional ferritin, which does not contain iron.
This Ž nding is in accordance with our previous work (14, 15,
17, 20, 21) and that of the group of Stadtman (23, 31, 32), which
demonstrated that enzymatically inactive oxidized proteins are
degraded. Because the proteasome is not involved in the further
liberation of iron from feritin through degradation of the protein,
we can assume that the degradation of oxidized nonfunctional
ferritin is an essential part of the antioxidant defense of the cell.
ACKNOWLEDGMENTS
This work was supported by a grant of the Deutsche Forsch-
ungsgemeinschaft SFB 507/A7. We thank Dr. O. Ullrich and
Mrs. G. Schrötter for their help in proteasome isolation.
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