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Interaction of the hemin 2 and 4 substituents with apo horseradish peroxidase
BIOCHEMICAL
'dol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 698-7n3
February28,1978 INTERACTION
OF THE HEMIN 2 and 4 SUBSTITUENTS
WITH APO HORSERADISH PEROXIDASE Robert
K. DiNello
and David
Dolphin
Department of Chemistry University of British Columbia Vancouver, British Columbia V6T lW5 Canada
Received
October
17,1977
SUMMARY: Z-formyi!, 4-vinyl deuterohemin (1) and %vinyZ, I-fonnyZ deuterohemin(21 substituted horseradish peroxidases have been prepared from apoperoxidase and the respective hemins. The two hemins bind at different rates to the apoprotein and the resultant substituted peroxidases possess different vi&b Ze spectra and activities. These results indicate that the hemin 2 and 4 substituents interact with apoperotidase and are not exposed to the solvent. INTRODUCTION: tuents
The view has been expressed
do not
interact
with
the studies
of Ohlsson
phobic
ring
groups
action
should,
recent
experiments
in our
tuents
do interact
with
difficult hemin
substitution
differential
are
effects
electron
donating
Isomeric 4-formyl
hemins
deuterohemin
(2)
with
properties
of heme proteins interaction
0 I978
hemins Steric
properties with
since
the
brought protein
of the side
heme proteins
(3). substituted
of the hemin
deuterohemin
Therefore with
2 and 4 side
698
any differences hemins
chains
with
of
about
by
resulting chains.
(1 ) and 2-vinyl,
properties
these
is
polypeptide
properties
electronic
it
of
effects
or withdrawing
by Academic Press, Inc. in any form reserved.
of reproduction
the properties
properties
have identical
of
2 and 4 substi-
in heme protein
4-vinyl
hydro-
Such an inter-
are changes
0006~'r9lX/78/0804-0698$01,00/O Copyright All rights
hemin
and
chain.
effects.
chains
such as E-formyl,
associated
steric
side
the
2),
and results
in comparing
in heme protein
(1,
that
(2).
the
2 and 4 substituted
of hemin
Electronic
that
polypeptide
from electronic
changes
interaction
from the
energetically,
encountered
with
to indicate
unfavorable indicate
2 and 4 substi-
apoprotein
to solvent
the peroxidase
steric
peroxidase
are exposed
laboratory
invariably
to separate
chain.
be quite
reconstituted
the hemin
have been taken
of protohemin
are
heme proteins
horseradish
and Paul
however,
Difficulties
the
that
when not in the
must be due to the protein.
Such
Vol. 80,No.
BIOCHEMICAL
4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
32 0
'\\ H NT.N' 'N ' \' /
2
c%"
differences
have been demonstrated
the 2 and 4. substituents proteins
tlorseradish
from Sigma
and DiNello
8).
graphis
hemins is
et al
(1).
as peak positions
as far
and ferrotirs
monoxide
and intensities, spirographis
assay
hemin
is
is are
thus
apparent
differentially
differ
that
hemin.
similar
of the ferrous
by 8-10
wavelength.
Figures ferrous,
quite
in both
differing
than
peak posi-
nm with
those
As can be seen in
enzyme in the o-dianisidine
that
of the
of the protohemin
by their
of isospirographis
of the ferric,
considerably
the properties
affected
out as des-
and isospiro-
the spectra
hemin
higher to that
as
enzymes possess
enzyme at longer
equal
reconstituted
of spirographis spectra
wavelength
considerably
of
carried
of binding
concerned,
of the spirographis
enzyme and nearly It
enzymes
the long
1, the activity
peroxidase
study
carbon
that
the ferric are
were
by Hamilton
by the method
of spirographis time
the visible
spectra
as modified
hemin)
(11).
times
Although
(isospirographis
(6),
assays
The half
respectively,
deuterohemin
and the apoprotein
of binding
or twenty-six
B and C was obtained
from peroxidase
Peroxidase
to apoperoxidase.
130 minutes
of Clezy
(lo),
rates
enzymes.
hemin
4) where
of the
2-formyl-4-vinyl deuterohemin
Enzyme Manual
and ferrous-CO
Table
(3,
interior
of isozymes
was removed
by Yonetani
1 shows the
2, 3, and 4 show,
of the
in the
Missouri.
to the method
in the Worthington Figure
Louis,
Protohemin
by Tamura
RESULTS:
a mixture
and 2-vinyl-4-fotmyl
as modified
described
tions
and myoglobin
known to be buried
St.
according
(7,
(9),
cribed
Co.,
hemin)
synthesized
hemin
in hemoglobin
peroxidase,
Chemical
(spirographis
Teale
cv
(5).
METHODS:
were
are
H
of the interaction
isospirographis reconstituted
two hemins with
enzyme.
used in this
apo horseradish
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.00
N 6 0.60 0.40 0.20 0.0 0
50
100
Fig.
250
300
(MIN)
Rates of binding of apirographis hcmin (dashed line) and ieoepirographie hemin (solid line) to apo horoeradioh peroxidaoe. At t = 0, hemin (final concentration = 1.30 s IO-%I) and apoenzyme (final concentration = 1.16 x lo-4MMl in O.OlSM tris 1iC.l were combined. At the times indicated in the fiyure, oamples uere removed and chromatographed on DEAE cellulose to remove unbound hemin. The amount of bound hemin wa8 assayed by measuring the optical denoity at The OD 412 100X bound 412 nm, the Soret maxima of the substituted proteins. line refer8 to the spirographis hemin enzyme.
1.
Fig.
200
I50
TIME
2.
Visible
spectra
epirographis
peroxidase. formyl, polypeptide
of
(dotted
Although which
epirographis hemin (solid line) hemin horseradish peroxidases.
the 2 and 4 substituents
are quite chain.
Fe(III) %zel
small,
they
Such interaction
still argues
700
concerned
interact against
with
and Fe(III)
are
vinyl
ieo-
and
the peroxidase
the model
with
the vinyl
Vol. 80, No.&
Fig.
BIOCHEMICAL
1978
Spectra of FslIIll spirogrqhis hsmi.n (dashed line) horseradish
3.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hemin (solid peroxidases.
Table Activities
II
pKS values, chains exposed
(8,
sites
exhibit
small
side
Peroxidases
(n=lO)
3454 + 192
(n=lO)
Spirographis
3425 jr 174
(n=20)
Isospirographis
2704 + 168
(n=20)
Protohemin Reconstituted
advocated small
of hemin
12).
a vinyl
Studies
(1) side
relationship
thus
and Paul
chains
apoperoxidase
groups
in our
the rates
and the
withdrawing
power
to replace
enclosed
flexibility
with
between
seem prudent
+ S.D.
and Ohlsson
peroxidases
of the electron
conformational chains.
et al
2 and 4 hemin
substituted
It would with
by Tamura
of a regular
a measure
model
of Hemin Substituted
3904 + 117
the lack
compounds
1
Native
between
explains
isospirographis
Activity
exposed
interaction
and Fe(II)
Peroxidase
Protohemin
groups
line)
model
where
701
indicate
Steric also
of reaction
corresponding
of
porphyrin
of the porphyrin the vinyl
groups
the
binding
and can accommodate laboratory
(2).
that
vinyl
a variety only
side
of
when the
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
24
20
80
60 E 40
20
0
Fig.
Spectra graphis
4.
hemin
500
affected
chains
are made quite
(13).
This
substituents
where
even slight
has drastic
effects
on both
of the reconstituted ACKNOWLEDGEMENTS: scholar
teaching
the
This
Chemistry
fellowship.
Institutes
of Health
is
(soZid line) peroxidaees.
bulky
in marked
binding
binding
CO isospiro-
to apoperoxidase
to the hemin
of the propionic
to the peroxidase
6 and 7
acid
apoprotein
side
chains
and activity
(1, 8).
work was performed of Biological Department, The work
is
and Fe(II)
contrast
modification
heme protein
of the department
RKD thanks
Xcnm) 6c0
of Fe(IIll CO spirogr’czphis hemin hemin (dashed line) horseradish
2 and 4 side
adversely
0
400
while
RKD was a travelling
Chemistry, University
was supported
Harvard
Medical
of British by the
(AM-17989) and the National
Columbia
United
Research
School.
States Council
for
a
National of Canada.
REFERENCES:
1. 2. 3. 4. 5.
Tamura, M., Asakura, T., 268, 292-304. Ohlsson, P.I. and Paul, 293-305. Asakura, T.and Sono, M. Sono, M. and Asakura, T. Perutz, M.F., Muirhead,
219, 131-139.
and Yonetani, K.G.
T.
(1973) Biochim.
(1972) Biochim. Biophys.
Acta.,
Biophys.
Acta.,
315,
(1974) J. Biol. Chem., 249, 7087-7093. (1975) J. Biol. Chem., 250, 5227-5232. H., Cox, J.M., and Goaman, L.C.G. (1968) Nature,
702
Vol. 80, No. 4, 1978
6. 7. 8. 9. 10. 11. :::
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Clezy, P.S. and Fookes, C.J.R. (1974) Aust. J. Chem., 27, 371-382. Hamilton, A.D. (1976) Ph.D. Thesis, University of BritEh Columbia. DiNello,, R.K. (1977) Ph.D. Thesis, Harvard University. Teale, F.W.J. (1959) Biochim. Biophys. Acta., 35-, 543 (begins and ends on p. 543). Yonetanii, T. (1967) J. Biol. Chem., 242, 5008-5013. Worthington Enzyme Manual (1972) Wor%T?ngton Biochemical Co., Freehold, New Jersey, pp. 43-45. Makino, R. and Yamazaki, I. (1972) J. Biochem. (Tokyo), 72, 655-664. DiNello,, R.K. and Dolphin, D., manuscript in preparation.
703
'dol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 698-7n3
February28,1978 INTERACTION
OF THE HEMIN 2 and 4 SUBSTITUENTS
WITH APO HORSERADISH PEROXIDASE Robert
K. DiNello
and David
Dolphin
Department of Chemistry University of British Columbia Vancouver, British Columbia V6T lW5 Canada
Received
October
17,1977
SUMMARY: Z-formyi!, 4-vinyl deuterohemin (1) and %vinyZ, I-fonnyZ deuterohemin(21 substituted horseradish peroxidases have been prepared from apoperoxidase and the respective hemins. The two hemins bind at different rates to the apoprotein and the resultant substituted peroxidases possess different vi&b Ze spectra and activities. These results indicate that the hemin 2 and 4 substituents interact with apoperotidase and are not exposed to the solvent. INTRODUCTION: tuents
The view has been expressed
do not
interact
with
the studies
of Ohlsson
phobic
ring
groups
action
should,
recent
experiments
in our
tuents
do interact
with
difficult hemin
substitution
differential
are
effects
electron
donating
Isomeric 4-formyl
hemins
deuterohemin
(2)
with
properties
of heme proteins interaction
0 I978
hemins Steric
properties with
since
the
brought protein
of the side
heme proteins
(3). substituted
of the hemin
deuterohemin
Therefore with
2 and 4 side
698
any differences hemins
chains
with
of
about
by
resulting chains.
(1 ) and 2-vinyl,
properties
these
is
polypeptide
properties
electronic
it
of
effects
or withdrawing
by Academic Press, Inc. in any form reserved.
of reproduction
the properties
properties
have identical
of
2 and 4 substi-
in heme protein
4-vinyl
hydro-
Such an inter-
are changes
0006~'r9lX/78/0804-0698$01,00/O Copyright All rights
hemin
and
chain.
effects.
chains
such as E-formyl,
associated
steric
side
the
2),
and results
in comparing
in heme protein
(1,
that
(2).
the
2 and 4 substituted
of hemin
Electronic
that
polypeptide
from electronic
changes
interaction
from the
energetically,
encountered
with
to indicate
unfavorable indicate
2 and 4 substi-
apoprotein
to solvent
the peroxidase
steric
peroxidase
are exposed
laboratory
invariably
to separate
chain.
be quite
reconstituted
the hemin
have been taken
of protohemin
are
heme proteins
horseradish
and Paul
however,
Difficulties
the
that
when not in the
must be due to the protein.
Such
Vol. 80,No.
BIOCHEMICAL
4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
32 0
'\\ H NT.N' 'N ' \' /
2
c%"
differences
have been demonstrated
the 2 and 4. substituents proteins
tlorseradish
from Sigma
and DiNello
8).
graphis
hemins is
et al
(1).
as peak positions
as far
and ferrotirs
monoxide
and intensities, spirographis
assay
hemin
is
is are
thus
apparent
differentially
differ
that
hemin.
similar
of the ferrous
by 8-10
wavelength.
Figures ferrous,
quite
in both
differing
than
peak posi-
nm with
those
As can be seen in
enzyme in the o-dianisidine
that
of the
of the protohemin
by their
of isospirographis
of the ferric,
considerably
the properties
affected
out as des-
and isospiro-
the spectra
hemin
higher to that
as
enzymes possess
enzyme at longer
equal
reconstituted
of spirographis spectra
wavelength
considerably
of
carried
of binding
concerned,
of the spirographis
enzyme and nearly It
enzymes
the long
1, the activity
peroxidase
study
carbon
that
the ferric are
were
by Hamilton
by the method
of spirographis time
the visible
spectra
as modified
hemin)
(11).
times
Although
(isospirographis
(6),
assays
The half
respectively,
deuterohemin
and the apoprotein
of binding
or twenty-six
B and C was obtained
from peroxidase
Peroxidase
to apoperoxidase.
130 minutes
of Clezy
(lo),
rates
enzymes.
hemin
4) where
of the
2-formyl-4-vinyl deuterohemin
Enzyme Manual
and ferrous-CO
Table
(3,
interior
of isozymes
was removed
by Yonetani
1 shows the
2, 3, and 4 show,
of the
in the
Missouri.
to the method
in the Worthington Figure
Louis,
Protohemin
by Tamura
RESULTS:
a mixture
and 2-vinyl-4-fotmyl
as modified
described
tions
and myoglobin
known to be buried
St.
according
(7,
(9),
cribed
Co.,
hemin)
synthesized
hemin
in hemoglobin
peroxidase,
Chemical
(spirographis
Teale
cv
(5).
METHODS:
were
are
H
of the interaction
isospirographis reconstituted
two hemins with
enzyme.
used in this
apo horseradish
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.00
N 6 0.60 0.40 0.20 0.0 0
50
100
Fig.
250
300
(MIN)
Rates of binding of apirographis hcmin (dashed line) and ieoepirographie hemin (solid line) to apo horoeradioh peroxidaoe. At t = 0, hemin (final concentration = 1.30 s IO-%I) and apoenzyme (final concentration = 1.16 x lo-4MMl in O.OlSM tris 1iC.l were combined. At the times indicated in the fiyure, oamples uere removed and chromatographed on DEAE cellulose to remove unbound hemin. The amount of bound hemin wa8 assayed by measuring the optical denoity at The OD 412 100X bound 412 nm, the Soret maxima of the substituted proteins. line refer8 to the spirographis hemin enzyme.
1.
Fig.
200
I50
TIME
2.
Visible
spectra
epirographis
peroxidase. formyl, polypeptide
of
(dotted
Although which
epirographis hemin (solid line) hemin horseradish peroxidases.
the 2 and 4 substituents
are quite chain.
Fe(III) %zel
small,
they
Such interaction
still argues
700
concerned
interact against
with
and Fe(III)
are
vinyl
ieo-
and
the peroxidase
the model
with
the vinyl
Vol. 80, No.&
Fig.
BIOCHEMICAL
1978
Spectra of FslIIll spirogrqhis hsmi.n (dashed line) horseradish
3.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hemin (solid peroxidases.
Table Activities
II
pKS values, chains exposed
(8,
sites
exhibit
small
side
Peroxidases
(n=lO)
3454 + 192
(n=lO)
Spirographis
3425 jr 174
(n=20)
Isospirographis
2704 + 168
(n=20)
Protohemin Reconstituted
advocated small
of hemin
12).
a vinyl
Studies
(1) side
relationship
thus
and Paul
chains
apoperoxidase
groups
in our
the rates
and the
withdrawing
power
to replace
enclosed
flexibility
with
between
seem prudent
+ S.D.
and Ohlsson
peroxidases
of the electron
conformational chains.
et al
2 and 4 hemin
substituted
It would with
by Tamura
of a regular
a measure
model
of Hemin Substituted
3904 + 117
the lack
compounds
1
Native
between
explains
isospirographis
Activity
exposed
interaction
and Fe(II)
Peroxidase
Protohemin
groups
line)
model
where
701
indicate
Steric also
of reaction
corresponding
of
porphyrin
of the porphyrin the vinyl
groups
the
binding
and can accommodate laboratory
(2).
that
vinyl
a variety only
side
of
when the
BIOCHEMICAL
Vol. 80, No. 4, 1978
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
24
20
80
60 E 40
20
0
Fig.
Spectra graphis
4.
hemin
500
affected
chains
are made quite
(13).
This
substituents
where
even slight
has drastic
effects
on both
of the reconstituted ACKNOWLEDGEMENTS: scholar
teaching
the
This
Chemistry
fellowship.
Institutes
of Health
is
(soZid line) peroxidaees.
bulky
in marked
binding
binding
CO isospiro-
to apoperoxidase
to the hemin
of the propionic
to the peroxidase
6 and 7
acid
apoprotein
side
chains
and activity
(1, 8).
work was performed of Biological Department, The work
is
and Fe(II)
contrast
modification
heme protein
of the department
RKD thanks
Xcnm) 6c0
of Fe(IIll CO spirogr’czphis hemin hemin (dashed line) horseradish
2 and 4 side
adversely
0
400
while
RKD was a travelling
Chemistry, University
was supported
Harvard
Medical
of British by the
(AM-17989) and the National
Columbia
United
Research
School.
States Council
for
a
National of Canada.
REFERENCES:
1. 2. 3. 4. 5.
Tamura, M., Asakura, T., 268, 292-304. Ohlsson, P.I. and Paul, 293-305. Asakura, T.and Sono, M. Sono, M. and Asakura, T. Perutz, M.F., Muirhead,
219, 131-139.
and Yonetani, K.G.
T.
(1973) Biochim.
(1972) Biochim. Biophys.
Acta.,
Biophys.
Acta.,
315,
(1974) J. Biol. Chem., 249, 7087-7093. (1975) J. Biol. Chem., 250, 5227-5232. H., Cox, J.M., and Goaman, L.C.G. (1968) Nature,
702
Vol. 80, No. 4, 1978
6. 7. 8. 9. 10. 11. :::
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Clezy, P.S. and Fookes, C.J.R. (1974) Aust. J. Chem., 27, 371-382. Hamilton, A.D. (1976) Ph.D. Thesis, University of BritEh Columbia. DiNello,, R.K. (1977) Ph.D. Thesis, Harvard University. Teale, F.W.J. (1959) Biochim. Biophys. Acta., 35-, 543 (begins and ends on p. 543). Yonetanii, T. (1967) J. Biol. Chem., 242, 5008-5013. Worthington Enzyme Manual (1972) Wor%T?ngton Biochemical Co., Freehold, New Jersey, pp. 43-45. Makino, R. and Yamazaki, I. (1972) J. Biochem. (Tokyo), 72, 655-664. DiNello,, R.K. and Dolphin, D., manuscript in preparation.
703