BIOCHEMICAL
Vol. 74, No. 3, 1977
SENSITIZED
PHOTOOXIDATION
OF
Yeou-Jan Department
October
Kang” of
Salt
Received
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LOW SPIN
HORSERADISH
and
D.
John
Biology, Lake City,
PEROXIDASE
Spikes
University Utah 84112
of
Utah
18,1976
Summary: Horseradish peroxidase differs from most enzymes in that it is almost completely resistant to photodynamic action due to the paramagnetic ferric ion Chelation of horseradish peroxidase at the in the prosthetic group, heme. sixth coordination position of the iron with a cyanide or hydroxyl group converts it to a low spin diamagnetic state. Upon illumination with visible light with eosin Y, flavin mononucleotide or methylene blue as sensitizer, the low spin enzyme lost both peroxidative and oxidative activities with the same Several amino acid residues, quantum yields. including one histidine and one tyrosine were destroyed in the low spin enzyme after 60 min of illumination with eosin Y as sensitizer.
Peroxidases such
as
They
are
heat also
are
(l-3))
was
are
shown
not
endowed
horse
In
communication
this
dynamic
hydroxyl
as
derivatives
changing
we of
heme groups
to the
in
which
HRP*”
iron to
an
spin
yield
were
(high
spin
examination the state)
diamagnetic sensitive
sixth
heme to
light
the
in
(low
a high
to
it
a low
spin
state
to
light
(II).
sensitivity
to
position with
spin
presence
photoof
cyanide
EOY,
Such FMN or
low MEB
sensitizers.
College,
*Present Address: Taipei, Taiwan,
Department Republic
of of
Biochemistry, China.
National
Yang-Ming
a”Abbreviations used: HRP, horserad i sh peroxi dase; HRP-OH, hydroxidecomplexed horseradish peroxidase; HRP-CN, cyanide-complexed horseradish peroxidase; apoHRP, horseradish apoperoxidase; EOY, eosin Y; FMN, flavin mononucleotide; MEB, methylene blue.
Copyright All rights
the
or
state). of
spin
However,
sensitivity the
(2).
(dye-sensitized iron
iron
coordinated
in
radiation
coordination
iron
agents
(11-12).
heme
of
is
heme
light
marked
report
ultraviolet
the
visible
high
inactivating
inactivation
have
sperm-whaleniyoglobinwith
treatment
paramagnetic
spin
and
hemoproteins
of
and
photodynamic
sensitive
that
a variety
(2,4,5) to
Most
directly
recently
to
radiation
resistant
(6-10).
and
resistant
ionizing
unusually
photooxidation) state
unusually
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
1160
Medical
BIOCHEMICAL
Vol. 74, No. 3, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.6
HRP-OH
,I 321
I
3
400 Wavelength
Fig. 1. The near-ultraviolet HRP in 0.5 fi sodium phosphate in NaCN and 0.5 M in NazHP04
I
480
I 560
(nm)
and visible absorption spectra of 12.6 buffer, pH 8; of HRP-CN in a solution and of HRP-OH in 0.85 l NaOH, pH 12.4.
MATERIALS
AND
ufi 20
mM -
METHODS
Horseradish peroxidase (EC 1.11.1.7, Sigma Chemical Co., Type II, lot 20R.Z. 1.37) was further purified on CM-and DEAE-cellulose columns (13) and the isoenzyme C fraction with R.Z. 3.2 was isolated as described elsewhere HRP-CN was prepared by dissolving the enzyme (1 mg/ml) in a solution (IO). 20 mM in NaCN and 0.5 fi in Na2HP04. HRP-OH was prepared by dissolving the protein (1 mg/ml) in 0.85 L NaOH (pH 12.4). Reaction mixtures for photooxidation contained 1.0 ml peroxidase solution, 0.5 ml dye solution (I x 10b4M EOY or MEB, or 1 x 10s3M FMN) and 0.5 ml of 0.5 M pH 8 sodium phosphate buffer (for the HRP-CN experiments) or 0.5 ml of 0.85 K NaOH (for the HRP-OH experiments). The reaction mixtures were illuminated in air with stirring at 25OC in a plexiglass cell. Illumination was provided by a 500 watt projector; Baird-Atomic multilayer interference filters were used to give narrow wavelength bands corresponding to the absorption peak of the sensitizer used (437 nm for FMN, 517 nm for EOY, 675 nm for MEB). At intervals during illumination, samples were removed for enzyme assay. Light measurements and calculation of the quantum yields of inactivation (defined as the number of enzyme molecules inactivated per number of photons absorbed, extrapolated to zero time of illumination) were performed as described earlier
c-2820,
(14) * 100 ul samples of the HRP-CN system were For activity measurements, removed and mixed with 1.0 ml of 28 mM silver nitrate. The resulting precipitate was removed by centrifugation and filtration, after which 100 ~1 aliquots of the mixture were used for the enzyme assay. For the HRP-OH system, 100 ~1 samples of the reaction mixture were mixed with 1.0 ml of 0.5 -M sodium phos-
1161
Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I -
z-
I -
- 0. l250
300 WAVELENGTH
Fig. 2. NazHP04 equimolar in 0.85 distilled
350 (nm)
The ultraviolet difference spectra of 32 PM HRP-CN in 0.5 l (front cell of Cary Model 14M spectrophotometer) against an solution of HRP in 0.5 y Na2HP04 (rear cell) and 32 I.@ HRP-OH M NaOH (front cell) against an equimolar solution of HRP in water (rear cell).
phate buffer, pH 6.0; 50 ~1 aliquots of this solution were then used for the The peroxidative activity of the HRP preparations was assayed with assay. o-dianisidine coupled with hydrogen peroxide as substrate (15) while the oxidative activity was measured with indoleacetic acid as substrate by a slight modification of the method of Siegel and Galston (16). The photometric assays and spectral measurements were performed with a Cary Model 14M spectrophotometer. Amino acid analyses were carried out with a Beckman Model 12OC amino acid analyzer on enzyme samples hydrolyzed in acid or in base as described before (10). RESULTS Spectral violet band
and shifts
properties visible from
of
HRP-CN
absorption 403
nm for
AND and
spectra the
native
DISCUSSION
HRP-OH of
HRP, enzyme
1162
-
Fig.
1 shows
HRP-CN
and
to
nm and
416
the
HRP-OH. 423
near The nm for
ultraSoret HRP-OH
Vol.
74,
and
No.
HRP-CN,
spin
respectively;
ferriheme
around two
357
replaced
bands
a shoulder)
the
change
of
The
ultraviolet are
given
peak
may
resulting
in
325 HRP-OH
result
from
an
absence
of
renders
these
an spin
dynamic
and
watt
tral
regions
the
rapidly
pressure
contrast treatment are
presented
to
illumination
is
first
(8-10).
arc
the
appearance
of
the
cause
or
visible
280 nm; in
from
a glass-filtered
in
02,
low
spin
In
contrast, to
observed
high
CO or heme
cyanide
acting the
as low
No photo-
after
General
a very
the
light.
even
residues
sensitizers
with
the
complex acid
light
insensitive
was
the
amino added
heme
a new
significance
approximately
(II).
has
alterations
the
to
with
538 nm and
at
versus
aromatic
the
are
HRP-OH
of
residues
which
and
absence
of
were
and
the
6 hr
of
Electric
output
in
the
A-H6 spec-
hemes. of
in
which
1.
HRP-CN
oxidative
order
Table
study
and
(unfolding)
of
light
peaks Further
spectral
at
sensitizer
mercury
Quantum in
insensitive
to
the
The
HRP-OH of
presence
enzyme is
yields
and
activities
in
HRP,
to
change
are
6 cm
and
on
peak
the
a high
Small
HRP-OH
show
a new
in
acid
in
HRP-CN
as
some
from
formed.
latter
the
HRP-OH
our
also
nm
known
sensitive
inactivation
native
is
for
of
by
peroxidative
inactivation
complexes
Ligation
amino
absorbed
lost
Both
dyes.
in
a distance
Photodynamic Both
used
of
high
for
photodynamic
HRP-OH
at
spectra
shift
640 nm disappear
These
exhibits
and
very
destruction
1000
state.
nothing
sensitizing
proteins
illumination
spin
myoglobin
endogeneous
HRP-CN
HRP-CN.
572
COMMUNICATIONS
(17).
were
nm and
a conformational
HRP-CN
ferric
added
efficient
in
2.
nm;
HRP-OH 498
the
protein
nm and
environment
of
whale
at
545
also
altered
Illumination and
in
Fig.
The
peak.
Horse
in
and
HRP
difference
approximately
this
this
to
RESEARCH
for
ferriheme
HRP-CN
at
BIOPHYSICAL
characteristic
spin
native
as
due
at
of
bands
also
HRP
a low
absorption
568 nm (appears
peak
to
AND
is
366 nm for
nm and
by
native
this
protein
absorption
are
BIOCHEMICAL
3, 1977
values
added
HRP-CN EOY,
and
FMN This
completely
insensitive
the
photodynamic
for
the
1163
loss
sensitizers HRP-OH
or
concentration.
almost for
of
with
MEB. is
inactivation of
peroxidative
-
are The
in to
sharp photodynamic of and
HRP-CN oxida-
Vol.
74,
No.
3, 1977
Table
BIOCHEMICAL
I.
Quantum Yields the Peroxidative
Sensitizer
x
lo-3
1.75
x
1o-3
Oxidative Activity
1.79
x
1o-3
1.53
x
lo-3
1.75
x
10
2.
Quantum Yields the Peroxidative
being action
mixture.
efficiency show and
and
an
lo-3
1.55
x
10
5.20
x
1O-3
0.96
x
IO-~
1.54
x
1o-3
identical
efficiency
may
up
to
with decreases
1.2
same
yields
shows pH
for x
differ
the
10s3,
HRP-OH.
the
the
typically
increasing
actually
for
This
efficient.
not
much
with
photodynamic
0.35
x
differdestruction
10m3
and
1.2
x
10m3
(18).
with
however,
do
yields
are
data lost
and
quantum
respectively
are
increasing
efficiency
x
similar
EOY with
-3
0.95
conditions
MEB,
results, most
MEB
1O-3
the
similar
again,
the
FMN
of HRP-OH
x
essentially
2 gives
HRP-CN
Photodynamic inactivation Oxidative Activities of
5.21
comparison,
under
activities,
the and
-3
ive
For
FMN
for
EOY
are
Table
the
MEB
1.52
dyes.
EOY,
FMN
of HRP-CN
IO-~
activities
with
Photodynamic Inactivation Oxidative Activities of
x
Oxidat Activity
apoHRP
COMMUNICATIONS
1.80
Peroxidative Activity
of
the and
RESEARCH
Peroxidative Activity
Sensitizer
ent
for
BIOPHYSICAL
EOY
Table
tive
AND
result
peroxidat
ive
yields.
In
quantum
depend
on
from
very
higher
1164
high
the
the
a progressive
increasing at
The
high
values;
values
oxidat
pH
(12.4) in
FMN but with
and
this
to
dye
with
of
the
EOY re-
sensitizing MEB
tends FMN
ive
contrast
sensitizing
increase
pH,
and
(7,14).
initially to
level
off
Vol. 74, No. 3, 1977
The
marked
BIOCHEMICAL
increase
in
magnetic
species
in
photodynamic
systems.
iron)
is
an
photodynamic
the
photooxidizable
since
ion
of
protective
is
excited
would
arises
have
shown
that
suggests some
unfolding
in
Fig.
2. Amino
As
results per
in
could
the
not
residue
that
heme
This
might
be
to
substrates in
ions as
of expected
protect
involved
act
the
showed
no
photodynamic
60
min
sensitive
spin
major
are
to
the
one
by
heme
conformational residues
from the
the
native
in
1165
the
native
the
of
(Fig.
the
2) There
may spectrum
low the
presence one
cyanide enzyme.
spin of
HRP
-
EOY
tyrosine
photodynamic difference
in
this
difference
and
ultraviolet
of
enzyme.
to
acids
to
exposure
complex.
in
of
we
spectrum
ultraviolet
lack
which
sensitive
answer
this
residue
changes buried
the
HRP-CN
histidine
since
in
It
example,
the
the
HRP-CN
iron.
ways
photodynamically-treated of
results
in
amino
unfolding
in
from
ultraviolet
aromatic
shown
other
becomes
buried
the
heme
For
unambiguous in
of
the
in
results
were
change
as
of
altered
this
illumination of
state
treatment.
which
of
sensitivity
hydrochloride
significant
probably
low
indicate
expose
para-
certain
metal
spin
a completely
HRP-OH
destruction
the
residues
no
in
photodynamic
destruction
3,
This by
of
for
shown
not
HRP
agent
states
do
spin
earlier
photodynamic
guanidine
give
the
the
presumably
absence
is
Table
molecule.
function does
in
and
complexes
corresponding
of
acid
shown
the
there
be
the
in
acid
region
that
been
excited
change
to
cannot
although the
the
(18-19);
studies
in
have
diamagnetic
whether
sensitivity
amino
question, HRP-CN
that
treatment
present
(18-19).
low behavior
shown
HRP
quenching
(24)
to
HRP unfolded
photooxidizable The
ions
contrast,
from
course, the
photodynamic
as
simply
of
increase
the
protective in
photosensitizers
results
possible,
In
about
We have
metal by
known
of
(10,23).
question
HRP-OH
is
residues
(20-23).
agents The
acid
treatment
sequence
quenching
what
paramagnetic
photodynamic
react
and
of
with
efficient
amino
a variety
against
well
RESEARCH COMMUNICATIONS
sensitivity
above
spin
in
photodynamic
described
(high
fits
the
AND BIOPHYSICAL
residue protective
spectrum
(Fig.
derivative Since
which at
least
2)
BIOCHEMICAL
Vol. 74, No. 3, 1977
3. Amino
Table
Acid Analyses Photodynamic
Photosensitive Amino Acid
one
histidine
near
the
by
2.7
1.8
Tryptophan
I.1
0.9
0.7
Tyrosine
4.9
4.0
0.6
histidine
are
in
these
HRP,
residues
is
present
might
also
some
the
3).
to
the
same
to
from
an
tyrosine
be
(25-26)
the
only
are
present
residues
the
proteins
This
the
high are
work
Administration
the
result
from
exposure in
are
the
of the
destroyed
to
was
by
supported Contract
No.
the
has
tyrosine
of
the
been
solvent.
amino protein amino
reported
This
at
Energy
Theorel Rosen,
I, C.
H. G.
and and
Maehly, Nilsson,
A. C. (1950) Acta R. (1971) Biochim.
tyrosine
residues pH
values
Research
and
E(ll-I)-875.
1166
Chem. Stand. Biophys.
4, Acta
HRP-OH.
residues
REFERENCES I. 2.
which
could in
tyrosine
higher
(27), 2)
photooxidized
since
U.S.
of
280 nm (Fig.
of
rapidly
one
photosensitive
at
system,
more
under
more
photooxidation
photooxidized
the
a change
residues
HRP-OH
of
than
destruction
HRP-OH
exposed
tyrosine
more
all
absorption
increased pH of
almost extensive
increased
of
conditions,
more
may
residues number
that
and
change
shows
possible
other
to
This
HRP-CN
leading
increased
ACKNOWLEDGMENT. Development
compared
more
result
residue
expected
residue
(Table
study
for
tyrosine
under
A conformational
that
one
be
methionine
HRP-OH
residues.
is
might
photooxidized one
as of
suggests
and
.4
HRP-CN.
destroyed
conformation
in
(lO,l8,25)
residue,
residues
It
HRP-OH
3.2
residue
of
Molecule
Methionine
HRP-OH
account
per
I
heme
the
Residues HRP-CN
2.2
When
and
of
3.1
of
acid
60 Min
of Low Spin HRP After Treatment with EOY
Number Control
Dark
RESEARCH COMMUNICATIONS
Histidine
treatment
acid
AND BIOPHYSICAL
422-434.
236,
l-7.
(28).
BIOCHEMICAL
Vol. 74, No. 3, 1977
3.
Diehl,
4. 5. 6. 7.
Checcucci, Crippa, Nakatani, Spikes, edited Academic Hopkins, Kang, Kang, Folin,
8. 9. 10. 11.
J.
F.
(1972)
Deut.
Ges.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chem.
Apparatew.
70,
Monogr.
265-277.
A. and Crippa, P. R. (1966) Studia Biophys. I, 343-346. P. R. and Vecli, A. (1969) Studia Biophys. 13, 65-68. M. (1960) J. Biochem. (Tokyo) 48, 640-644. J. D. and Livingston, R. (1969) in Advances in Radiation Biology by Augenstein, L., Mason, R. and Zelle, M., Vol. 3, pp. 29-113, Press, New York. T. R. and Spikes, J. D. (1969) Radiation Res. 37, 253-260. Y. J. and Spikes, J. D. (1973) Biophysical J. 13, 273a. Y. J. and Spikes, J. D. (1976) Arch. Biochem. Biophys. 172, 565-573. M., Gennari, G. and Jori, G. (1974) Photochem. Photobiol. 20, 357-
370. 12. 13.
Spikes, Shannon,
14.
2172. Glad,
15.
Worthington Freehold, 16. Siegel, 17. Brill, 18. Kang, 19. Kang, American 20. Tomita, 21 . Tomi ta, 22. Sone, 23. Jori, 749-766. 24. Linschitz, 25. Shih, Chem. 26. Phelps, 27. Epstein, 28. Spikes, 149-162.
J.
B.
L.
D. M.,
W.
and
(1975) Kay, Spikes,
Annal. E. and J.
N. Y. Acad. Lew, J. Y. D.
(1966)
Sci. (1966)
244,
496-508.
J.
Radiation
Biol. Res.
Chem. 27,
241,
2166-
237-249.
Biochemical Corporation, Descriptive Manual No. II, (1972), New Jersey. B. Z. and Galston, A. W. (1967) Science 157, 1557-1559. A. S. and Williams, R. J. P. (1961) Biochem. J. 78, 246-253. Y. J. (1973) Ph.D. dissertation, University of Utah. Y. J. and Spikes, J. D. (1974) Program 2nd Annual Meeting of Society for Photobiology, p. 152, Vancouver, British Columbia. G. (1966) Nature 212, 898-901. G. (1967) Experientia 23, 25-26. K. (1962) Bitamin 26, 13-17. G., Gennari, G. and Scoffone, E. (1971) Biochim. Biophys. Acta H. and Pekkarinen, L. (1960) J. Amer. J. H. C., Shannon, L. M., Kay, E. and Lew, 246, 4546-4551. C., Forlani, L. and Antonini, E. (1971) N. and Schejter, S. (1972) FEBS Letters J. D. and MacKnight, M. L. (1970) Annal.
1167
Chem. J. Y.
Sot. (1971)
Biochem. J. 25, 46-48. N. Y. Acad.
82, J.
124, Sci.
236,
2411-2416. Biol.
605-614. 171,
Vol. 74, No. 3, 1977
SENSITIZED
PHOTOOXIDATION
OF
Yeou-Jan Department
October
Kang” of
Salt
Received
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
LOW SPIN
HORSERADISH
and
D.
John
Biology, Lake City,
PEROXIDASE
Spikes
University Utah 84112
of
Utah
18,1976
Summary: Horseradish peroxidase differs from most enzymes in that it is almost completely resistant to photodynamic action due to the paramagnetic ferric ion Chelation of horseradish peroxidase at the in the prosthetic group, heme. sixth coordination position of the iron with a cyanide or hydroxyl group converts it to a low spin diamagnetic state. Upon illumination with visible light with eosin Y, flavin mononucleotide or methylene blue as sensitizer, the low spin enzyme lost both peroxidative and oxidative activities with the same Several amino acid residues, quantum yields. including one histidine and one tyrosine were destroyed in the low spin enzyme after 60 min of illumination with eosin Y as sensitizer.
Peroxidases such
as
They
are
heat also
are
(l-3))
was
are
shown
not
endowed
horse
In
communication
this
dynamic
hydroxyl
as
derivatives
changing
we of
heme groups
to the
in
which
HRP*”
iron to
an
spin
yield
were
(high
spin
examination the state)
diamagnetic sensitive
sixth
heme to
light
the
in
(low
a high
to
it
a low
spin
state
to
light
(II).
sensitivity
to
position with
spin
presence
photoof
cyanide
EOY,
Such FMN or
low MEB
sensitizers.
College,
*Present Address: Taipei, Taiwan,
Department Republic
of of
Biochemistry, China.
National
Yang-Ming
a”Abbreviations used: HRP, horserad i sh peroxi dase; HRP-OH, hydroxidecomplexed horseradish peroxidase; HRP-CN, cyanide-complexed horseradish peroxidase; apoHRP, horseradish apoperoxidase; EOY, eosin Y; FMN, flavin mononucleotide; MEB, methylene blue.
Copyright All rights
the
or
state). of
spin
However,
sensitivity the
(2).
(dye-sensitized iron
iron
coordinated
in
radiation
coordination
iron
agents
(11-12).
heme
of
is
heme
light
marked
report
ultraviolet
the
visible
high
inactivating
inactivation
have
sperm-whaleniyoglobinwith
treatment
paramagnetic
spin
and
hemoproteins
of
and
photodynamic
sensitive
that
a variety
(2,4,5) to
Most
directly
recently
to
radiation
resistant
(6-10).
and
resistant
ionizing
unusually
photooxidation) state
unusually
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
1160
Medical
BIOCHEMICAL
Vol. 74, No. 3, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.6
HRP-OH
,I 321
I
3
400 Wavelength
Fig. 1. The near-ultraviolet HRP in 0.5 fi sodium phosphate in NaCN and 0.5 M in NazHP04
I
480
I 560
(nm)
and visible absorption spectra of 12.6 buffer, pH 8; of HRP-CN in a solution and of HRP-OH in 0.85 l NaOH, pH 12.4.
MATERIALS
AND
ufi 20
mM -
METHODS
Horseradish peroxidase (EC 1.11.1.7, Sigma Chemical Co., Type II, lot 20R.Z. 1.37) was further purified on CM-and DEAE-cellulose columns (13) and the isoenzyme C fraction with R.Z. 3.2 was isolated as described elsewhere HRP-CN was prepared by dissolving the enzyme (1 mg/ml) in a solution (IO). 20 mM in NaCN and 0.5 fi in Na2HP04. HRP-OH was prepared by dissolving the protein (1 mg/ml) in 0.85 L NaOH (pH 12.4). Reaction mixtures for photooxidation contained 1.0 ml peroxidase solution, 0.5 ml dye solution (I x 10b4M EOY or MEB, or 1 x 10s3M FMN) and 0.5 ml of 0.5 M pH 8 sodium phosphate buffer (for the HRP-CN experiments) or 0.5 ml of 0.85 K NaOH (for the HRP-OH experiments). The reaction mixtures were illuminated in air with stirring at 25OC in a plexiglass cell. Illumination was provided by a 500 watt projector; Baird-Atomic multilayer interference filters were used to give narrow wavelength bands corresponding to the absorption peak of the sensitizer used (437 nm for FMN, 517 nm for EOY, 675 nm for MEB). At intervals during illumination, samples were removed for enzyme assay. Light measurements and calculation of the quantum yields of inactivation (defined as the number of enzyme molecules inactivated per number of photons absorbed, extrapolated to zero time of illumination) were performed as described earlier
c-2820,
(14) * 100 ul samples of the HRP-CN system were For activity measurements, removed and mixed with 1.0 ml of 28 mM silver nitrate. The resulting precipitate was removed by centrifugation and filtration, after which 100 ~1 aliquots of the mixture were used for the enzyme assay. For the HRP-OH system, 100 ~1 samples of the reaction mixture were mixed with 1.0 ml of 0.5 -M sodium phos-
1161
Vol. 74, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I -
z-
I -
- 0. l250
300 WAVELENGTH
Fig. 2. NazHP04 equimolar in 0.85 distilled
350 (nm)
The ultraviolet difference spectra of 32 PM HRP-CN in 0.5 l (front cell of Cary Model 14M spectrophotometer) against an solution of HRP in 0.5 y Na2HP04 (rear cell) and 32 I.@ HRP-OH M NaOH (front cell) against an equimolar solution of HRP in water (rear cell).
phate buffer, pH 6.0; 50 ~1 aliquots of this solution were then used for the The peroxidative activity of the HRP preparations was assayed with assay. o-dianisidine coupled with hydrogen peroxide as substrate (15) while the oxidative activity was measured with indoleacetic acid as substrate by a slight modification of the method of Siegel and Galston (16). The photometric assays and spectral measurements were performed with a Cary Model 14M spectrophotometer. Amino acid analyses were carried out with a Beckman Model 12OC amino acid analyzer on enzyme samples hydrolyzed in acid or in base as described before (10). RESULTS Spectral violet band
and shifts
properties visible from
of
HRP-CN
absorption 403
nm for
AND and
spectra the
native
DISCUSSION
HRP-OH of
HRP, enzyme
1162
-
Fig.
1 shows
HRP-CN
and
to
nm and
416
the
HRP-OH. 423
near The nm for
ultraSoret HRP-OH
Vol.
74,
and
No.
HRP-CN,
spin
respectively;
ferriheme
around two
357
replaced
bands
a shoulder)
the
change
of
The
ultraviolet are
given
peak
may
resulting
in
325 HRP-OH
result
from
an
absence
of
renders
these
an spin
dynamic
and
watt
tral
regions
the
rapidly
pressure
contrast treatment are
presented
to
illumination
is
first
(8-10).
arc
the
appearance
of
the
cause
or
visible
280 nm; in
from
a glass-filtered
in
02,
low
spin
In
contrast, to
observed
high
CO or heme
cyanide
acting the
as low
No photo-
after
General
a very
the
light.
even
residues
sensitizers
with
the
complex acid
light
insensitive
was
the
amino added
heme
a new
significance
approximately
(II).
has
alterations
the
to
with
538 nm and
at
versus
aromatic
the
are
HRP-OH
of
residues
which
and
absence
of
were
and
the
6 hr
of
Electric
output
in
the
A-H6 spec-
hemes. of
in
which
1.
HRP-CN
oxidative
order
Table
study
and
(unfolding)
of
light
peaks Further
spectral
at
sensitizer
mercury
Quantum in
insensitive
to
the
The
HRP-OH of
presence
enzyme is
yields
and
activities
in
HRP,
to
change
are
6 cm
and
on
peak
the
a high
Small
HRP-OH
show
a new
in
acid
in
HRP-CN
as
some
from
formed.
latter
the
HRP-OH
our
also
nm
known
sensitive
inactivation
native
is
for
of
by
peroxidative
inactivation
complexes
Ligation
amino
absorbed
lost
Both
dyes.
in
a distance
Photodynamic Both
used
of
high
for
photodynamic
HRP-OH
at
spectra
shift
640 nm disappear
These
exhibits
and
very
destruction
1000
state.
nothing
sensitizing
proteins
illumination
spin
myoglobin
endogeneous
HRP-CN
HRP-CN.
572
COMMUNICATIONS
(17).
were
nm and
a conformational
HRP-CN
ferric
added
efficient
in
2.
nm;
HRP-OH 498
the
protein
nm and
environment
of
whale
at
545
also
altered
Illumination and
in
Fig.
The
peak.
Horse
in
and
HRP
difference
approximately
this
this
to
RESEARCH
for
ferriheme
HRP-CN
at
BIOPHYSICAL
characteristic
spin
native
as
due
at
of
bands
also
HRP
a low
absorption
568 nm (appears
peak
to
AND
is
366 nm for
nm and
by
native
this
protein
absorption
are
BIOCHEMICAL
3, 1977
values
added
HRP-CN EOY,
and
FMN This
completely
insensitive
the
photodynamic
for
the
1163
loss
sensitizers HRP-OH
or
concentration.
almost for
of
with
MEB. is
inactivation of
peroxidative
-
are The
in to
sharp photodynamic of and
HRP-CN oxida-
Vol.
74,
No.
3, 1977
Table
BIOCHEMICAL
I.
Quantum Yields the Peroxidative
Sensitizer
x
lo-3
1.75
x
1o-3
Oxidative Activity
1.79
x
1o-3
1.53
x
lo-3
1.75
x
10
2.
Quantum Yields the Peroxidative
being action
mixture.
efficiency show and
and
an
lo-3
1.55
x
10
5.20
x
1O-3
0.96
x
IO-~
1.54
x
1o-3
identical
efficiency
may
up
to
with decreases
1.2
same
yields
shows pH
for x
differ
the
10s3,
HRP-OH.
the
the
typically
increasing
actually
for
This
efficient.
not
much
with
photodynamic
0.35
x
differdestruction
10m3
and
1.2
x
10m3
(18).
with
however,
do
yields
are
data lost
and
quantum
respectively
are
increasing
efficiency
x
similar
EOY with
-3
0.95
conditions
MEB,
results, most
MEB
1O-3
the
similar
again,
the
FMN
of HRP-OH
x
essentially
2 gives
HRP-CN
Photodynamic inactivation Oxidative Activities of
5.21
comparison,
under
activities,
the and
-3
ive
For
FMN
for
EOY
are
Table
the
MEB
1.52
dyes.
EOY,
FMN
of HRP-CN
IO-~
activities
with
Photodynamic Inactivation Oxidative Activities of
x
Oxidat Activity
apoHRP
COMMUNICATIONS
1.80
Peroxidative Activity
of
the and
RESEARCH
Peroxidative Activity
Sensitizer
ent
for
BIOPHYSICAL
EOY
Table
tive
AND
result
peroxidat
ive
yields.
In
quantum
depend
on
from
very
higher
1164
high
the
the
a progressive
increasing at
The
high
values;
values
oxidat
pH
(12.4) in
FMN but with
and
this
to
dye
with
of
the
EOY re-
sensitizing MEB
tends FMN
ive
contrast
sensitizing
increase
pH,
and
(7,14).
initially to
level
off
Vol. 74, No. 3, 1977
The
marked
BIOCHEMICAL
increase
in
magnetic
species
in
photodynamic
systems.
iron)
is
an
photodynamic
the
photooxidizable
since
ion
of
protective
is
excited
would
arises
have
shown
that
suggests some
unfolding
in
Fig.
2. Amino
As
results per
in
could
the
not
residue
that
heme
This
might
be
to
substrates in
ions as
of expected
protect
involved
act
the
showed
no
photodynamic
60
min
sensitive
spin
major
are
to
the
one
by
heme
conformational residues
from the
the
native
in
1165
the
native
the
of
(Fig.
the
2) There
may spectrum
low the
presence one
cyanide enzyme.
spin of
HRP
-
EOY
tyrosine
photodynamic difference
in
this
difference
and
ultraviolet
of
enzyme.
to
acids
to
exposure
complex.
in
of
we
spectrum
ultraviolet
lack
which
sensitive
answer
this
residue
changes buried
the
HRP-CN
histidine
since
in
It
example,
the
the
HRP-CN
iron.
ways
photodynamically-treated of
results
in
amino
unfolding
in
from
ultraviolet
aromatic
shown
other
becomes
buried
the
heme
For
unambiguous in
of
the
in
results
were
change
as
of
altered
this
illumination of
state
treatment.
which
of
sensitivity
hydrochloride
significant
probably
low
indicate
expose
para-
certain
metal
spin
a completely
HRP-OH
destruction
the
residues
no
in
photodynamic
destruction
3,
This by
of
for
shown
not
HRP
agent
states
do
spin
earlier
photodynamic
guanidine
give
the
the
presumably
absence
is
Table
molecule.
function does
in
and
complexes
corresponding
of
acid
shown
the
there
be
the
in
acid
region
that
been
excited
change
to
cannot
although the
the
(18-19);
studies
in
have
diamagnetic
whether
sensitivity
amino
question, HRP-CN
that
treatment
present
(18-19).
low behavior
shown
HRP
quenching
(24)
to
HRP unfolded
photooxidizable The
ions
contrast,
from
course, the
photodynamic
as
simply
of
increase
the
protective in
photosensitizers
results
possible,
In
about
We have
metal by
known
of
(10,23).
question
HRP-OH
is
residues
(20-23).
agents The
acid
treatment
sequence
quenching
what
paramagnetic
photodynamic
react
and
of
with
efficient
amino
a variety
against
well
RESEARCH COMMUNICATIONS
sensitivity
above
spin
in
photodynamic
described
(high
fits
the
AND BIOPHYSICAL
residue protective
spectrum
(Fig.
derivative Since
which at
least
2)
BIOCHEMICAL
Vol. 74, No. 3, 1977
3. Amino
Table
Acid Analyses Photodynamic
Photosensitive Amino Acid
one
histidine
near
the
by
2.7
1.8
Tryptophan
I.1
0.9
0.7
Tyrosine
4.9
4.0
0.6
histidine
are
in
these
HRP,
residues
is
present
might
also
some
the
3).
to
the
same
to
from
an
tyrosine
be
(25-26)
the
only
are
present
residues
the
proteins
This
the
high are
work
Administration
the
result
from
exposure in
are
the
of the
destroyed
to
was
by
supported Contract
No.
the
has
tyrosine
of
the
been
solvent.
amino protein amino
reported
This
at
Energy
Theorel Rosen,
I, C.
H. G.
and and
Maehly, Nilsson,
A. C. (1950) Acta R. (1971) Biochim.
tyrosine
residues pH
values
Research
and
E(ll-I)-875.
1166
Chem. Stand. Biophys.
4, Acta
HRP-OH.
residues
REFERENCES I. 2.
which
could in
tyrosine
higher
(27), 2)
photooxidized
since
U.S.
of
280 nm (Fig.
of
rapidly
one
photosensitive
at
system,
more
under
more
photooxidation
photooxidized
the
a change
residues
HRP-OH
of
than
destruction
HRP-OH
exposed
tyrosine
more
all
absorption
increased pH of
almost extensive
increased
of
conditions,
more
may
residues number
that
and
change
shows
possible
other
to
This
HRP-CN
leading
increased
ACKNOWLEDGMENT. Development
compared
more
result
residue
expected
residue
(Table
study
for
tyrosine
under
A conformational
that
one
be
methionine
HRP-OH
residues.
is
might
photooxidized one
as of
suggests
and
.4
HRP-CN.
destroyed
conformation
in
(lO,l8,25)
residue,
residues
It
HRP-OH
3.2
residue
of
Molecule
Methionine
HRP-OH
account
per
I
heme
the
Residues HRP-CN
2.2
When
and
of
3.1
of
acid
60 Min
of Low Spin HRP After Treatment with EOY
Number Control
Dark
RESEARCH COMMUNICATIONS
Histidine
treatment
acid
AND BIOPHYSICAL
422-434.
236,
l-7.
(28).
BIOCHEMICAL
Vol. 74, No. 3, 1977
3.
Diehl,
4. 5. 6. 7.
Checcucci, Crippa, Nakatani, Spikes, edited Academic Hopkins, Kang, Kang, Folin,
8. 9. 10. 11.
J.
F.
(1972)
Deut.
Ges.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chem.
Apparatew.
70,
Monogr.
265-277.
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Biochemical Corporation, Descriptive Manual No. II, (1972), New Jersey. B. Z. and Galston, A. W. (1967) Science 157, 1557-1559. A. S. and Williams, R. J. P. (1961) Biochem. J. 78, 246-253. Y. J. (1973) Ph.D. dissertation, University of Utah. Y. J. and Spikes, J. D. (1974) Program 2nd Annual Meeting of Society for Photobiology, p. 152, Vancouver, British Columbia. G. (1966) Nature 212, 898-901. G. (1967) Experientia 23, 25-26. K. (1962) Bitamin 26, 13-17. G., Gennari, G. and Scoffone, E. (1971) Biochim. Biophys. Acta H. and Pekkarinen, L. (1960) J. Amer. J. H. C., Shannon, L. M., Kay, E. and Lew, 246, 4546-4551. C., Forlani, L. and Antonini, E. (1971) N. and Schejter, S. (1972) FEBS Letters J. D. and MacKnight, M. L. (1970) Annal.
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