BIOCHEMICAL
Vol. 66, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SINGLET OXYGENAND SUPEROXIDEDISMUTASE (CUPREIN) Ulrich
Wulf Paschen and Maged Younes
Weser,
Physiologisch-Chemisches Institut der Universitat Tiibingen, D-74 Tiibingen, Hoppe-SeylerstraRe 1, Germany Received
June 27,1975
SUMMARY: Singlet oxygen of elongated lifetime was generated in the presence of molecular photochemically in 2H O-solutions oxygen and methylene ii lue. The oxidation of l,+iiphenylisobenzofuran by singlet oxygen was both significantly and specifically inhibited by superoxide dismutase. No such inhibition was observed using the apoprotein, CuSO4, Cu(tyr)2, Cu(his)2, Cu(lys)2, Cu-serum albumin, hemocyanin, ceruloplasmin, &amine oxidase, horseradish peroxidase, catalase and hemoglobin. Cyanide and azide added in approximately equimolar concentrations proved to be potent inhibitors of this biochemical action of cuprein. The extraordinary stability of superoxide dismutase before and after singlet oxygen treatment was demonstrated by the identical parameters obtained from circular dichroism, electron paramagnetic resonance and X-ray photoelectron spectroscopy (see also ref.1). There was also no detectable change in the electron absorption spectra compared to the untreated copper protein.
The search for a genuine biochemical dismutase (erythrocuprein) the exclusive valentlg
reaction
the specificity
oxide dismutation Cu(I1)
(3-7).
It
dismutation
occurring
is favoured
even in cellular
compared to 1.3 x IO9 M
S-l
as the protein
Furthermore,
The rate
in the presence of Cu(tyr12 -1
considerable
of the enzyme catalysed
the treatment
chelator.
While
(1,2).
super-
has been shown by pulse radiolysis
dismutase.
complex survived
naturally
going on
dismutase with the mono-
alone was twice as active
found in superoxide Cu(tgr)2
of superoxide
of superoxide
(21, we presented some data indicating
doubts regarding
that
is continuously
charged oxygen radical
biochemistry
function
calculated
bound Cu(I1)
the hydrophobic
of serum albumin, constant was
1.0
a
for superoxide x
IO9
per equivalent
Mm1
s-l
of en-
Vol. 66, No. 2, 1975
BIOCHEMICAL
zyme-bound Cu(II) solution
(4,5).
prise native singlet
photochemically for this
epecific
were active
generated singlet
excited
manner. In this
less than 0.2% (7).
of CrOg3- in aqueous solutions
sur-
oxygen saturated
blue
(10-12).
source
0 in water g2 2 one order of magnitude H20 was employed. Uncon-
avoided by irradiating to the addition
the solutions
system the reactivity
of superoxide
residues
agent
was
one hour prior
a powerful
and
Using this
(13-15).
dismutase to supress singlet
was examined and compared with the reactivity
metal proteins
diamine oxidase,
'A
for at least
of 'l,j-diphenylisobensofuran, oxygen trapping
oxygen formation
of
oxygen by histidine
singlet
action
we considered
was produced by irradia'* go2 'HZ0 solutions in the presence of methylene
scavenging of singlet
established
Due to the
oxygen as a convenient
In order to prolong the lifetime
by at least
system
oxygen species.
ting
tory
To our great
dismutase was capable to supress the
agents in a very
complex behaviour
of other
between a reducing
oxygen induced chemiluminescence in the absence of che-
model Cu-chelates
trolled
of the reaction
oxygen was shown (6,7,9).
superoxide
miluminescent all
With the decay of CrOg3- in aqueous
the complex nature
agent and molecular
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
including
ceruloplasmin,
and some hemoproteins.
of CN-, N -, 3
hemocyanin,
Furthermore
the inhibi-
EDTA and NH3 was studied.
MATERIALS AND METHODS or All chemicals employeg were of reagent grade quality better. Na02H, 2H3P04 and H20 Merck, Darmstadt. l,+diphenylisobenzofuran Aldrich, Milwaukee. Dibenzoylbensene EGA-Chemie, Steinheim. Methylene blue Serva, Heidelberg. Hemocyanin and diamine oxidase Calbiochem, San Diego. Catalase, horseradish peroxidase and hemoglobin Boehringer, Mannheim. Superoxide dismutase was isolated from yeast following the procedures given in (16,171. Apo-superoxide dismutase and ceruloplasmin were a ift from Dr.H.-J. Hartmann, Physiol.-them. Inst. Tiibingegl an oxygen saturated solufA g02 was generated by irradiating tion of methylene blue in 2H20 using a tungsten light source (Osram 100 W, Krypton lamp). To avoid the direct oxidation of
770
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1,3-diphenylisobenzofuran by light (18) only light above 600 nm was allowed to enter the cuvette. The assay was performed in a 23 mmlight path quartz cell. The concentrations of the different components were: phosphate buffer 100 mM, p2H 7.4, methglene blue 89 pM, 1,3-diphenylisobenzofuran 34 PM (added as a concentrated stock solution in 50 pl acetonitrile), copper chelates 36 pN. The total volume was 8.2 ml. The assay solution was irradiated 1 h before the reaction was started by adding 1,3-diphenylisobenzofuran. One ml aliquots were removed at zero time and after 3, 10, 20, 30 and 40 min. The formation of dibenzoylbenzene was monitored at 420 nm in a Unicam SP 1800 spectrophotometer. For control 1,3-diphenylisobenzofuran and dibenzoylbenzene were detected using thin layer chromatography (19). Histidine was assayed following the method given by Jori et al. (20). EPR spectra were run on a Varian V-4502-11 spectrometer. The circular dichroism was measured on a JASCO 20 unit. X-ray photoelectron spectroscopy was performed on a Varian VIEE spectrometer equipped with an on line Varian 620~, 8K computer. RESULTS Due to the strong is roughly stead. ted
quenching effect
in the order of
Indeed,
-1
M
IO5
the half&lifetime
of H20 on 'A
*-I
0 which l32 2H 0 was used in2
(13)
of the photochemically
genera-
'0
0 was high enough to detect a significant oxidation 432 of 1,3-diphenylisobenzofuran. Dibenzoylbenzene was characterized by thin
layer
chromatography
of 1,3-diphenylisobenzofuran tion
the cuvette
After
this
for
Attributable oxidation Cu(I1)
with
concentration
dismutase-Cu(II)
neither
histidine
(0.8
x IO9
detected
resi-
(see also 20).
of 1,3-diphenylisobenzofuran
M-’
s")
(13)
at least
the same
was added. In the presence of superoxide
a significant
and rather oxidation
specific
was even more distinct
771
inhibition
can be seen (Fig.
was seen when apo-superoxide
albumin and some copper or iron proteins The specificity
of the experiment.
nor the histidine
could be chemically
the 1,3-diphenylisobenzofuran such inhibition
to the start
to the high rate 'Ago,
oxidation
by 'A
1 h prior
pretreatment
dues in the apoprotein
the successful
0 The uncontrolled inhibig 2’ process by histidine was avoided by irradi-
of the oxidation
ating
indicating
dismutase,
1).
No
serum
were employed (Table when Cu(I1)
of
1).
was added as
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1: Time course of 'A 0 induced formation of dibenzoylbenzene in the presence and a&a&e of superoxide dismutase. ~NO addition, @+ 36 nM superoxide dismutase-Cu(II), a+ 36 PM superoxide dismutase-Cu(I1) + 40 PM cyanide. To minimize dilution effects all additions were made in less than 150 ~1. 23 mm light path quartz cells were irradiated with a 100 W tungsten light source. Light filters were: a concentrated solution of methylorange and a Kodak 29 filter. After irradiation for 1 h prior to the start of the measurements the 'l,3-diphenylisobenzofuran was added. Readings were taken from In11 aliquots at 420 nm in a 10 mm light path glass cell.
Cu(ly~)~,
Cu(hisj2,
molecular
weight
the
singlet
Cu-chelates
oxygen
successful
Cu(I1)
pletely
damaged
treatment
'A
was
investigated.
and
was consistent
copper
ion
proved
to
is
(1,2).
actually
be almost
NH3 caused
could
successfully
not (Table
Cu(II)
of
involved as active no significant
Apart
from
analysis
before
some established
metal
the
most
reactivity
Indeed,
2).
potent
known
this
would
in
the
(42% inhibition). inhibition.
772
for
these
low
with
the
un-
was comand after
chelators
agent
(Fig.
other
superoxide
indicate
catalytic
All
compete
histidine
proved its
complex.
bound
by chemical
Cyanide
assays
aquo
the
0 8 2’ action
with
or as the
agent
catalysis
inhibitory
dismutase
trapping
as shown
with
The
EDTA or
Cu(tyr)2
that
the
process. The
I)
Aside
presence
A parallel
could
of
Vol. 66, No. 2, 1975
Metal
BIOCHEMICAL
Protein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
uMoles benzene
Dibenzoyl-, formed x 1
% Inhibition
None
26.2
2 1.0
0.0
t4
native superoxide dismutase
12.0
+
0.6
54.0
+
5
the
21.4
+
0.9
18.3
+
4
23.1
+
0.9
12.2
+
4
23.1
2
0.9
12.2
+
4
hemocyanin
22.8
2
0.9
13.0
+
4
ceruloplasmin
22.8
2
0.9
13.0
t
4
diamine
23.1
+
0.9
12.2
_+
4
22.8
2
0.9
13.0
+
4
hemoglobin
23.1
+
0.9
12.2
+
4
catalase
21.8
+
0.9
16.8
+
4
apoprotein
Cu4-serum serum
albumin
albumin
oxidase
horseradish
peroxidase
Table 1: Dibenzoylbenzene formation in the presence of different copper or iron proteins after 40 min. irradiation. The concentration of the respective protein bound metal ion was 36 pM. For further experimental details see caption to fig. 1.
be drawn
to
plexing of
species
the
were
nativity
dismutation
also
of
was ascertained
mica1
properties.
oxide
dismutase
single The
superoxide
unable
where
to
affect
the
last
two
the
enzymic
after
'AgO2
com-
activity
cupreins. The
ment
the
At
the
end
was separated
chemical
peak
parameters
described
(20).
sorption
spectra,
dismutase
by examining
homogeneous
paramagnetic
superoxide
All
spectral
resonance
the
in
properties
and
the
773
and
irradiation
in
the
accordance
photoelectron data
chemical
on a Sephadex
was monitored were
X-ray
of
the
treat-
physicoche-
experiment G-25
column.
elution with
superOne
diagram.
those
already
including
electronic
spectroscopy,
the
circular
dichroism
abelectron
remained
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cu-complex
pMoles Dibenzoyl- -, benzene formed x 1
% Inhibition
None
26.2 2 1.0
0.0 2 4
native superoxide dismutase
12.0 + 0.6
Cu(tyrJ2
24.8
Cu(lyd2
25.3 2
Cu(his)2 Cu2+aq
54.0
+ 1.0
+ 5
5.3
+4
1.0
3*5
+4
25.5
!: 1.0
2.7
+4
26.0
2 1.0
0.8
,+ 4
Table 2: Dibenzoylbenzene formation in the presence of different mhelates after 40 min. irradiation. Values are calculated on an equivalent basis for Cu(I1). Experimental details as in the legend to Fig. 1.
unchanged throughout.
Regarding the enzymic function
for superoxide
(1,2,6,7)
between the native
dismutase activity
and the irradiated
To exclude possible by 02- solid
K02,
1
oxidation
mMsolutions
in the absence of superoxide phenylisobenzofuran
trapping plete
showed no difference
superoxide
dismutase.
of 1,3-diphenylisobenzofuran of K02 in dimethylsulphoxide
dismutase. No oxidation
was detected.
/ xanthine
agent.
assay
prepared 02- were added to the assay mixture
and electrolytically
the xanthine
either
Enzymically
of 1,3-di-
generated 02- using
oxidase system did not affect
These observations
support
very
the -'a
strongly
0 g2 the com-
absence of superoxide.
DISCUSSION As in the case of the CrOg3' decay in aqueous solutions a significant
and specific
shown, the same specificity nerated
'A
reaction
of superoxide
where
dismutaee was
can be seen in the photochemically
0 system. In fact, I32
the unique reactivity
774
of super-
ge-
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
oxide dismutase is even more pronounced in argued why using 2H20 , this want to say that relevance.
in the living
is not a natural
pulse radiolytic
However, it cell
systems. The solid
H20. It
studies
system? We do not
are of biological
should be kept in mind that
is not exclusively
restricted
phase chemistry,
could be
biochemistry to aqueous
for example, of the alkaline
earth metal phosphates and carbonates
as well as the reactions
in solvents
lipids
of low polarity
the hydrophobic
regions
be pointed
It
out.
need neither taminase"
including
of proteins
is certainly
a superoxide
and polynucleotides
correct
that
completely
different
situation
in concentrations
hydrophobic
many macromolecules are involved..In time of singlet deleterious
action
kable resistance be brought
superoxide
oxygen will
dismutase.
Throughout this
oxida-
bilayers
of
presented
in
increased
and the
oxygen species may proceed
are scavenging enzymes available. dismutaae is of a remar-
An attractive
conclusion
be accepted by the protein
The enzyme itself
will
might
portion
of
be in an excited
given away to adjacent
H20 mole-
process both the metal and the protein
play a major role.
ture is essential
during
The 22 kcal of -'AgO2 above the triplet
and the energy finally
conformation
However, a
these systems the half-life-
superoxide
versus 'ng02.
forward.
ground state
cules.
unless there
has been shown that
regions
be substantially
of these excited
uninhibited It
state
oxygen will
dismutase.
macromolecular lipid
membranes or the above cited
be rapidly
up to 55 M
has to be considered
processes where lipids,
rather
oxygen decon-
in aqueous systems. Both oxygen species will
compared to the nM amounts of superoxide
should
we do not really
dismutase nor a "singlet
scavenged by H20 which is present
tive
and membranes or in
to fulfill
all
An appropriate
tertiary
the requirements
struc-
of singlet
Vol. 66, No. 2, 1975
BIOCHEMICAL
'A 0 4 2
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'A 0 92
Figure 2:
'A 0 92
Proposed decay of a X02 complex
oxygen decontamination. Looking at the reaction component it
between oxygen and a reducing
can be envisaged that
all
sorts
of excited
species may be generated depending on the nature macromolecular
portion
decay are dictated this
(Fig.
2).
The different
by the respective
system superoxide
is just
gen could be formed. The conclusion dismutase prevents may react latter
level.
systems, however, this
ligand.
action
superoxide
In
case where singlet
of singlet
studies
dismutation
oxy-
superoxide oxygen.
oxygen directly.
must be answered by separate
The exclusive
pathways of oxygen
can be drawn that
with X02 complexes or singlet
question
cular
the deleterious
of the surrounding
macromolecular
one special
oxygen
It
This on a mole-
in biological
cannot be regarded as the predominant role
of
copper protein.
ACKNOWLEDGEMENTS This study was aided by DFG grants on metal proteins to U.W. W.P. is a recipient of a predoctoral fellowship of the Land Baden-Wiirttemberg. M.Y. is a recipient of a German Academic Exchange Service (DAAD) fellowship. Special thanks go to Miss R. Prinz and G. Strobe1 for valuable help and discussions. REFERENCES 1)
(1973)
Weser,U.
IJ
Structure
& Bonding (Spr inger
I-65
776
Verlag,
Berlin
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(1974) Advances in Enzymology (Meister, A. ed.) 2) Fridovich,I. 29 35-97 and Weser,U. (1972) 3) Joester,K.E.,Jung,G.,Weber,U. FEBS Letters 3, 25-28 and 4) Brigeliue,R.,Sp~ttl,R.,Bors,W.,Lengfelder,E.,Saran,M. FEBS Letters 47, 72-75 Weser,U. (1974) 5) Brigelius,R.,Hartmann,H.J.,Bors,W.,Lengfelder,E.,Saran,M. and Weser,U. (1975) Z. Physiol. Chem. a, 739-745 Biochim. Biophys. Acta 327, 6) Paschen,W. and Weser,U. (1973)
217-222
Paschen,W. and Weser,U. (1975) Z. Physiol. Chem. s, 727-737 Richter,C.,Wendel,A.,Weser,U. and Azzi,A. (1975) FEBS Letters 2, 300-303 (1974) Biochemistry 1 , 3811-3815 9) Hodgson,E.K. and Fridovich,I. and Wexler,S. (1964) J. Am. Chem. Sot. d IO) Foote,C.S. 3879-3880 and Wexler,S. (1964) J. Am. Chem. Sot. 3: 3880-3881 11) Foote,C.S. (1937) Biochem. Z. 291, 271-284 12) Kautsky,H. and Kearn6,D.R. (1972) J. Am. Chem. Sot. '& 13) Merke1,P.B. 7244-7253
14) 15) 16) 17) 18) 19) 20)
Matheson,I.B.C. and Lee,J. (1970) Chem. Phys. Lett. 4, 475-476 Adams,D.R. and Wilkinson,F. (1972) J. Chem. Sot. Farad. Trans. 68, 586-593 Weser,U.,Prinz,R.,Schallies,A.,Fretzdorff,A.,Krauss,P.,Voelter,W. Z. Physiol. Chem. m, 1821-1831 and Voetsch,W. (1972) and Weser,U. (1975) Z. Physiol. Chem. z, 767-776 Prinz,R. Le Berre,A. and Ratsimbazafy,R. (1963) Sot. Chim. 2, 229-230 Pederson,T.C. and Aust,S.D. (1973) Biochem. Biophys. Res. Comm. 2,
1071-1078
Forman,H.J.,Evans,H.J.,Hill,R.L. Biochemistry l2, 823-827
and Fridovich,I.
777
(1973)
Vol. 66, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SINGLET OXYGENAND SUPEROXIDEDISMUTASE (CUPREIN) Ulrich
Wulf Paschen and Maged Younes
Weser,
Physiologisch-Chemisches Institut der Universitat Tiibingen, D-74 Tiibingen, Hoppe-SeylerstraRe 1, Germany Received
June 27,1975
SUMMARY: Singlet oxygen of elongated lifetime was generated in the presence of molecular photochemically in 2H O-solutions oxygen and methylene ii lue. The oxidation of l,+iiphenylisobenzofuran by singlet oxygen was both significantly and specifically inhibited by superoxide dismutase. No such inhibition was observed using the apoprotein, CuSO4, Cu(tyr)2, Cu(his)2, Cu(lys)2, Cu-serum albumin, hemocyanin, ceruloplasmin, &amine oxidase, horseradish peroxidase, catalase and hemoglobin. Cyanide and azide added in approximately equimolar concentrations proved to be potent inhibitors of this biochemical action of cuprein. The extraordinary stability of superoxide dismutase before and after singlet oxygen treatment was demonstrated by the identical parameters obtained from circular dichroism, electron paramagnetic resonance and X-ray photoelectron spectroscopy (see also ref.1). There was also no detectable change in the electron absorption spectra compared to the untreated copper protein.
The search for a genuine biochemical dismutase (erythrocuprein) the exclusive valentlg
reaction
the specificity
oxide dismutation Cu(I1)
(3-7).
It
dismutation
occurring
is favoured
even in cellular
compared to 1.3 x IO9 M
S-l
as the protein
Furthermore,
The rate
in the presence of Cu(tyr12 -1
considerable
of the enzyme catalysed
the treatment
chelator.
While
(1,2).
super-
has been shown by pulse radiolysis
dismutase.
complex survived
naturally
going on
dismutase with the mono-
alone was twice as active
found in superoxide Cu(tgr)2
of superoxide
of superoxide
(21, we presented some data indicating
doubts regarding
that
is continuously
charged oxygen radical
biochemistry
function
calculated
bound Cu(I1)
the hydrophobic
of serum albumin, constant was
1.0
a
for superoxide x
IO9
per equivalent
Mm1
s-l
of en-
Vol. 66, No. 2, 1975
BIOCHEMICAL
zyme-bound Cu(II) solution
(4,5).
prise native singlet
photochemically for this
epecific
were active
generated singlet
excited
manner. In this
less than 0.2% (7).
of CrOg3- in aqueous solutions
sur-
oxygen saturated
blue
(10-12).
source
0 in water g2 2 one order of magnitude H20 was employed. Uncon-
avoided by irradiating to the addition
the solutions
system the reactivity
of superoxide
residues
agent
was
one hour prior
a powerful
and
Using this
(13-15).
dismutase to supress singlet
was examined and compared with the reactivity
metal proteins
diamine oxidase,
'A
for at least
of 'l,j-diphenylisobensofuran, oxygen trapping
oxygen formation
of
oxygen by histidine
singlet
action
we considered
was produced by irradia'* go2 'HZ0 solutions in the presence of methylene
scavenging of singlet
established
Due to the
oxygen as a convenient
In order to prolong the lifetime
by at least
system
oxygen species.
ting
tory
To our great
dismutase was capable to supress the
agents in a very
complex behaviour
of other
between a reducing
oxygen induced chemiluminescence in the absence of che-
model Cu-chelates
trolled
of the reaction
oxygen was shown (6,7,9).
superoxide
miluminescent all
With the decay of CrOg3- in aqueous
the complex nature
agent and molecular
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
including
ceruloplasmin,
and some hemoproteins.
of CN-, N -, 3
hemocyanin,
Furthermore
the inhibi-
EDTA and NH3 was studied.
MATERIALS AND METHODS or All chemicals employeg were of reagent grade quality better. Na02H, 2H3P04 and H20 Merck, Darmstadt. l,+diphenylisobenzofuran Aldrich, Milwaukee. Dibenzoylbensene EGA-Chemie, Steinheim. Methylene blue Serva, Heidelberg. Hemocyanin and diamine oxidase Calbiochem, San Diego. Catalase, horseradish peroxidase and hemoglobin Boehringer, Mannheim. Superoxide dismutase was isolated from yeast following the procedures given in (16,171. Apo-superoxide dismutase and ceruloplasmin were a ift from Dr.H.-J. Hartmann, Physiol.-them. Inst. Tiibingegl an oxygen saturated solufA g02 was generated by irradiating tion of methylene blue in 2H20 using a tungsten light source (Osram 100 W, Krypton lamp). To avoid the direct oxidation of
770
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1,3-diphenylisobenzofuran by light (18) only light above 600 nm was allowed to enter the cuvette. The assay was performed in a 23 mmlight path quartz cell. The concentrations of the different components were: phosphate buffer 100 mM, p2H 7.4, methglene blue 89 pM, 1,3-diphenylisobenzofuran 34 PM (added as a concentrated stock solution in 50 pl acetonitrile), copper chelates 36 pN. The total volume was 8.2 ml. The assay solution was irradiated 1 h before the reaction was started by adding 1,3-diphenylisobenzofuran. One ml aliquots were removed at zero time and after 3, 10, 20, 30 and 40 min. The formation of dibenzoylbenzene was monitored at 420 nm in a Unicam SP 1800 spectrophotometer. For control 1,3-diphenylisobenzofuran and dibenzoylbenzene were detected using thin layer chromatography (19). Histidine was assayed following the method given by Jori et al. (20). EPR spectra were run on a Varian V-4502-11 spectrometer. The circular dichroism was measured on a JASCO 20 unit. X-ray photoelectron spectroscopy was performed on a Varian VIEE spectrometer equipped with an on line Varian 620~, 8K computer. RESULTS Due to the strong is roughly stead. ted
quenching effect
in the order of
Indeed,
-1
M
IO5
the half&lifetime
of H20 on 'A
*-I
0 which l32 2H 0 was used in2
(13)
of the photochemically
genera-
'0
0 was high enough to detect a significant oxidation 432 of 1,3-diphenylisobenzofuran. Dibenzoylbenzene was characterized by thin
layer
chromatography
of 1,3-diphenylisobenzofuran tion
the cuvette
After
this
for
Attributable oxidation Cu(I1)
with
concentration
dismutase-Cu(II)
neither
histidine
(0.8
x IO9
detected
resi-
(see also 20).
of 1,3-diphenylisobenzofuran
M-’
s")
(13)
at least
the same
was added. In the presence of superoxide
a significant
and rather oxidation
specific
was even more distinct
771
inhibition
can be seen (Fig.
was seen when apo-superoxide
albumin and some copper or iron proteins The specificity
of the experiment.
nor the histidine
could be chemically
the 1,3-diphenylisobenzofuran such inhibition
to the start
to the high rate 'Ago,
oxidation
by 'A
1 h prior
pretreatment
dues in the apoprotein
the successful
0 The uncontrolled inhibig 2’ process by histidine was avoided by irradi-
of the oxidation
ating
indicating
dismutase,
1).
No
serum
were employed (Table when Cu(I1)
of
1).
was added as
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1: Time course of 'A 0 induced formation of dibenzoylbenzene in the presence and a&a&e of superoxide dismutase. ~NO addition, @+ 36 nM superoxide dismutase-Cu(II), a+ 36 PM superoxide dismutase-Cu(I1) + 40 PM cyanide. To minimize dilution effects all additions were made in less than 150 ~1. 23 mm light path quartz cells were irradiated with a 100 W tungsten light source. Light filters were: a concentrated solution of methylorange and a Kodak 29 filter. After irradiation for 1 h prior to the start of the measurements the 'l,3-diphenylisobenzofuran was added. Readings were taken from In11 aliquots at 420 nm in a 10 mm light path glass cell.
Cu(ly~)~,
Cu(hisj2,
molecular
weight
the
singlet
Cu-chelates
oxygen
successful
Cu(I1)
pletely
damaged
treatment
'A
was
investigated.
and
was consistent
copper
ion
proved
to
is
(1,2).
actually
be almost
NH3 caused
could
successfully
not (Table
Cu(II)
of
involved as active no significant
Apart
from
analysis
before
some established
metal
the
most
reactivity
Indeed,
2).
potent
known
this
would
in
the
(42% inhibition). inhibition.
772
for
these
low
with
the
un-
was comand after
chelators
agent
(Fig.
other
superoxide
indicate
catalytic
All
compete
histidine
proved its
complex.
bound
by chemical
Cyanide
assays
aquo
the
0 8 2’ action
with
or as the
agent
catalysis
inhibitory
dismutase
trapping
as shown
with
The
EDTA or
Cu(tyr)2
that
the
process. The
I)
Aside
presence
A parallel
could
of
Vol. 66, No. 2, 1975
Metal
BIOCHEMICAL
Protein
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
uMoles benzene
Dibenzoyl-, formed x 1
% Inhibition
None
26.2
2 1.0
0.0
t4
native superoxide dismutase
12.0
+
0.6
54.0
+
5
the
21.4
+
0.9
18.3
+
4
23.1
+
0.9
12.2
+
4
23.1
2
0.9
12.2
+
4
hemocyanin
22.8
2
0.9
13.0
+
4
ceruloplasmin
22.8
2
0.9
13.0
t
4
diamine
23.1
+
0.9
12.2
_+
4
22.8
2
0.9
13.0
+
4
hemoglobin
23.1
+
0.9
12.2
+
4
catalase
21.8
+
0.9
16.8
+
4
apoprotein
Cu4-serum serum
albumin
albumin
oxidase
horseradish
peroxidase
Table 1: Dibenzoylbenzene formation in the presence of different copper or iron proteins after 40 min. irradiation. The concentration of the respective protein bound metal ion was 36 pM. For further experimental details see caption to fig. 1.
be drawn
to
plexing of
species
the
were
nativity
dismutation
also
of
was ascertained
mica1
properties.
oxide
dismutase
single The
superoxide
unable
where
to
affect
the
last
two
the
enzymic
after
'AgO2
com-
activity
cupreins. The
ment
the
At
the
end
was separated
chemical
peak
parameters
described
(20).
sorption
spectra,
dismutase
by examining
homogeneous
paramagnetic
superoxide
All
spectral
resonance
the
in
properties
and
the
773
and
irradiation
in
the
accordance
photoelectron data
chemical
on a Sephadex
was monitored were
X-ray
of
the
treat-
physicoche-
experiment G-25
column.
elution with
superOne
diagram.
those
already
including
electronic
spectroscopy,
the
circular
dichroism
abelectron
remained
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cu-complex
pMoles Dibenzoyl- -, benzene formed x 1
% Inhibition
None
26.2 2 1.0
0.0 2 4
native superoxide dismutase
12.0 + 0.6
Cu(tyrJ2
24.8
Cu(lyd2
25.3 2
Cu(his)2 Cu2+aq
54.0
+ 1.0
+ 5
5.3
+4
1.0
3*5
+4
25.5
!: 1.0
2.7
+4
26.0
2 1.0
0.8
,+ 4
Table 2: Dibenzoylbenzene formation in the presence of different mhelates after 40 min. irradiation. Values are calculated on an equivalent basis for Cu(I1). Experimental details as in the legend to Fig. 1.
unchanged throughout.
Regarding the enzymic function
for superoxide
(1,2,6,7)
between the native
dismutase activity
and the irradiated
To exclude possible by 02- solid
K02,
1
oxidation
mMsolutions
in the absence of superoxide phenylisobenzofuran
trapping plete
showed no difference
superoxide
dismutase.
of 1,3-diphenylisobenzofuran of K02 in dimethylsulphoxide
dismutase. No oxidation
was detected.
/ xanthine
agent.
assay
prepared 02- were added to the assay mixture
and electrolytically
the xanthine
either
Enzymically
of 1,3-di-
generated 02- using
oxidase system did not affect
These observations
support
very
the -'a
strongly
0 g2 the com-
absence of superoxide.
DISCUSSION As in the case of the CrOg3' decay in aqueous solutions a significant
and specific
shown, the same specificity nerated
'A
reaction
of superoxide
where
dismutaee was
can be seen in the photochemically
0 system. In fact, I32
the unique reactivity
774
of super-
ge-
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
oxide dismutase is even more pronounced in argued why using 2H20 , this want to say that relevance.
in the living
is not a natural
pulse radiolytic
However, it cell
systems. The solid
H20. It
studies
system? We do not
are of biological
should be kept in mind that
is not exclusively
restricted
phase chemistry,
could be
biochemistry to aqueous
for example, of the alkaline
earth metal phosphates and carbonates
as well as the reactions
in solvents
lipids
of low polarity
the hydrophobic
regions
be pointed
It
out.
need neither taminase"
including
of proteins
is certainly
a superoxide
and polynucleotides
correct
that
completely
different
situation
in concentrations
hydrophobic
many macromolecules are involved..In time of singlet deleterious
action
kable resistance be brought
superoxide
oxygen will
dismutase.
Throughout this
oxida-
bilayers
of
presented
in
increased
and the
oxygen species may proceed
are scavenging enzymes available. dismutaae is of a remar-
An attractive
conclusion
be accepted by the protein
The enzyme itself
will
might
portion
of
be in an excited
given away to adjacent
H20 mole-
process both the metal and the protein
play a major role.
ture is essential
during
The 22 kcal of -'AgO2 above the triplet
and the energy finally
conformation
However, a
these systems the half-life-
superoxide
versus 'ng02.
forward.
ground state
cules.
unless there
has been shown that
regions
be substantially
of these excited
uninhibited It
state
oxygen will
dismutase.
macromolecular lipid
membranes or the above cited
be rapidly
up to 55 M
has to be considered
processes where lipids,
rather
oxygen decon-
in aqueous systems. Both oxygen species will
compared to the nM amounts of superoxide
should
we do not really
dismutase nor a "singlet
scavenged by H20 which is present
tive
and membranes or in
to fulfill
all
An appropriate
tertiary
the requirements
struc-
of singlet
Vol. 66, No. 2, 1975
BIOCHEMICAL
'A 0 4 2
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
'A 0 92
Figure 2:
'A 0 92
Proposed decay of a X02 complex
oxygen decontamination. Looking at the reaction component it
between oxygen and a reducing
can be envisaged that
all
sorts
of excited
species may be generated depending on the nature macromolecular
portion
decay are dictated this
(Fig.
2).
The different
by the respective
system superoxide
is just
gen could be formed. The conclusion dismutase prevents may react latter
level.
systems, however, this
ligand.
action
superoxide
In
case where singlet
of singlet
studies
dismutation
oxy-
superoxide oxygen.
oxygen directly.
must be answered by separate
The exclusive
pathways of oxygen
can be drawn that
with X02 complexes or singlet
question
cular
the deleterious
of the surrounding
macromolecular
one special
oxygen
It
This on a mole-
in biological
cannot be regarded as the predominant role
of
copper protein.
ACKNOWLEDGEMENTS This study was aided by DFG grants on metal proteins to U.W. W.P. is a recipient of a predoctoral fellowship of the Land Baden-Wiirttemberg. M.Y. is a recipient of a German Academic Exchange Service (DAAD) fellowship. Special thanks go to Miss R. Prinz and G. Strobe1 for valuable help and discussions. REFERENCES 1)
(1973)
Weser,U.
IJ
Structure
& Bonding (Spr inger
I-65
776
Verlag,
Berlin
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
(1974) Advances in Enzymology (Meister, A. ed.) 2) Fridovich,I. 29 35-97 and Weser,U. (1972) 3) Joester,K.E.,Jung,G.,Weber,U. FEBS Letters 3, 25-28 and 4) Brigeliue,R.,Sp~ttl,R.,Bors,W.,Lengfelder,E.,Saran,M. FEBS Letters 47, 72-75 Weser,U. (1974) 5) Brigelius,R.,Hartmann,H.J.,Bors,W.,Lengfelder,E.,Saran,M. and Weser,U. (1975) Z. Physiol. Chem. a, 739-745 Biochim. Biophys. Acta 327, 6) Paschen,W. and Weser,U. (1973)
217-222
Paschen,W. and Weser,U. (1975) Z. Physiol. Chem. s, 727-737 Richter,C.,Wendel,A.,Weser,U. and Azzi,A. (1975) FEBS Letters 2, 300-303 (1974) Biochemistry 1 , 3811-3815 9) Hodgson,E.K. and Fridovich,I. and Wexler,S. (1964) J. Am. Chem. Sot. d IO) Foote,C.S. 3879-3880 and Wexler,S. (1964) J. Am. Chem. Sot. 3: 3880-3881 11) Foote,C.S. (1937) Biochem. Z. 291, 271-284 12) Kautsky,H. and Kearn6,D.R. (1972) J. Am. Chem. Sot. '& 13) Merke1,P.B. 7244-7253
14) 15) 16) 17) 18) 19) 20)
Matheson,I.B.C. and Lee,J. (1970) Chem. Phys. Lett. 4, 475-476 Adams,D.R. and Wilkinson,F. (1972) J. Chem. Sot. Farad. Trans. 68, 586-593 Weser,U.,Prinz,R.,Schallies,A.,Fretzdorff,A.,Krauss,P.,Voelter,W. Z. Physiol. Chem. m, 1821-1831 and Voetsch,W. (1972) and Weser,U. (1975) Z. Physiol. Chem. z, 767-776 Prinz,R. Le Berre,A. and Ratsimbazafy,R. (1963) Sot. Chim. 2, 229-230 Pederson,T.C. and Aust,S.D. (1973) Biochem. Biophys. Res. Comm. 2,
1071-1078
Forman,H.J.,Evans,H.J.,Hill,R.L. Biochemistry l2, 823-827
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