Vol.
178,
July
15, 1991
No.
BIOCHEMICAL
1, 1991
AND
RESEARCH
COMMUNICATIONS Pages
OXYGEN DIFFUSION NEAR SITE OF HORSERADISH Victor
Vargas”‘,
‘Departamento
“Department
May 17,
Juan E. Brunet’
104-109
THE HEME BINDING PEROXIDASE and David
de Quimica, Facultad de Ciencias, Casilla 653 Santiago, Chile
‘Institute
Received
BIOPHYSICAL
M. Jameson’ Universidad
de Chile
de Quimica, Universidad CatMica de Valparaiso Casilla 4059 Valparaiso, Chile
of Biochemistry and Biophysics, University of Hawaii 1960 East-West Road, Honolulu, Hawaii 96822 1991
The quenching by molecular oxygen of the fluorescence of several probes complexed to apohorseradish peroxidase has been studied by intensity and timeresolved fluorescence methods. The probes utilized include 1-anilino-Snaphthalene sulfonic acid, 4,4’-bis (1-anilino-S-naphthalene sulfonic acid), and 2-ptoluidinylnaphthalene-6-sulfonic acid. These results are contrasted to those obtained using apohorseradish peroxidase complexed with protoporphyrin IX. The resistance of these complexes to denaturation by guanidine hydrochloride was also determined. The results demonstrate a dramatic increase in oxygen accessibility to the naphthalene probes compared to protoporphyrin IX, which can be correlated to the increased stability of the protein-protoporphyrin IX complex. B 1991Academic Press, Inc. The facile diffusion
of oxygen through
has been verified
by the methods
(1-8).
study of oxygen diffusion
A recent
complexed
with
protoporphyrin
quenching
constant
oxygen accounted
of fluorescence IX,
near 2 x lo+* M’
constants observed in any protein
of a number
of proteins
and phosphorescence
quenching
through
HRP(desFe),
apohorseradish determined
peroxidase a bimolecular
s-l whi ch is one of the lowest quenching
system (9). This poor accessibility
for the observations
1 To whom correspondence
the interior
of luminescence
to molecular
from the enzymatically
should be addressed.
AJ@RJZVIATIONS: 1,8-ANS, 1-anilino-8-naphathalene sulfonic acid; Bis-ANS, 4,4’bis (1-anilino-B-naphthalene sulfonic acid); 2,6-TNS, 2-p-toluidinylnaphthalene6-sulfonic acid; PPIX, protoporphyrin IX; apoHRP, apohorseradish peroxidase; HRP, horseradish peroxidase; HRP(desFE) apohorseradish peroxidaseprotoporphyrin IX adduct, GuHCl, guanidinium hydrochloride. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
104
Vol.
178,
No.
1, 1991
generated resistant Time
acetone
triplet
demonstrated
AND
state (10-11).
to denaturation
resolved
binding
BIOCHEMICAL
by GuHCl
fluorescence
compared
measurements
site (12). The low diffusional
in a rigid environment.
to denaturation measurements
by GuHCl. on several
AND
motion
in HRP(desFe)
in the heme
rate for oxygen to the heme pocket in HRP
fluorophore Here
(9). furthermore
of the fluorophore
of oxygen to fluorophores we report
is tightly
bound
on a relationship
bound in the heme pocket and as judged by their resistance
on oxygen quenching
complexes;
Bis-ANS,
moiety
to speculate
complexes
apo-HRP-probe
results using the probes 1,8-ANS, MATERIALS
on PPIX
It then seemed reasonable
of those apoHRP
COMMUNICATIONS
to other heme proteins
with the fact that the porphyrin
between the accessibility the stability
RESEARCH
It was also shown that HRP was very
the absence of any local
(desFe) is thus consistent
BIOPHYSICAL
2,6-TNS
and GuHCl
specifically
we compare
and PPIX.
METHODS
Preparation of apo-HRP Col&ANS, bis-ANS and 2,6-TNS from Molecular Probes, were used without further purification. Horseradish peroxidase type VI and GuHCl were from Sigma. The protein’s heme group was removed using Teale’s method of cold acid and butanone extraction (13), followed by exhaustive dialysis at 4°C against 0.1 M sodium phosphate buffer (pH 7.4). The concentration of apoHRP was determined spectrophotometrically using a molar absorption coefficient at 280 nm of 20,000 (14). The apoHRP conjugates with the naphthalene derivates were prepared with at least a 20fold molar excess of protein in phosphate buffer 0.1 M pH 7.4. The HRP(desFe) conjugate was prepared as previously described (12). The oxygen quenching experiments were carried out also as previously described (9), using a frequency of 30 MHz for l&ANS, 60 MHz for 2,6-TNS and 70 MHz for bis-ANS conjugates of HRP. Fluorescence Wved measurements: Lifetime measurements were carried out using either an ISS Greg 200 spectrofluorometer or a homebuilt multifrequency phase and modulation fluorometer based on the Gratton design as Excitation of the naphthalene probes was previously described (15-17). accomplished using the 364 nm line of an argon ion laser (Spectra Physics Model 2035-3.5S), and the emission was observed through a Schott KV399 cuton filter which passed wavelengths longer than 380 nm.
RESULTS Figure apoHRP
1 shows the displacement
by hemin
fluorescence. a 1:l molar 1:l conjugate
titration,
followed
of 1,8-ANS
with
identical
with an inflection
between the probe and the apoHRP. apoHPR
bound
by the decrease of the naphthalene
The two curves are virtually relation
and of 2,6-TNS
as described 105
by Mauk
The PPIX
and Girotti
to the probes’
point near also forms a
(18).
GuHCl
BIOCHEMICAL
Vol. 178, No. 1, 1991
01%
I I
I
HEMIN
I 2
MOLES/APO-HRP
AND BIOPHYSICAL
I 3
I
02
MOLES
RESEARCH COMMUNICATIONS
2
5
3 [Gu HCf;.
6
7
M
Pi_oure 1. Displacement of l&ANS (-0-) and 2,6-TNS (-•-) from apoHRP by hemin. The excitation wavelengths were 360 nm and 317 nm respectively and the fluorescence was monitored at 475 nm and 432 nm respectively. JQgur-&.Fluorescence decreasefor the 1,8-ANS (-a-), 2,6-TNS (-0-),bis-ANS (-A-) and PPIX (-0) conjugates of apoHRP as a function of GuHCl concentration. The fluorescence was measuredfrom the integrated area of the spectra. The excitation wavelength was 364 nm for the naphthalene probes and 514 nm for the HRP(desFe).
unfolding
of the HRP conjugates
and PPIX protein
show a decrease
as the GuHCl Stem-Volmer
fluorescence in Figures constants
fluorescence
plots of the intensity
3-a through
from
the
yield in buffer.
quenching
by oxygen of the
probes and PPIX
From the slopes of the intensity
discrete exponential
contributions
and lifetime
I were obtained.
probes
upon displacement
bound to the naphthalene
3-d.
shown in Table
fractional
in their
increases due to their low fluorescence
of the apoHRP
a two component
are shown in Figure 2. The naphthalene
are shown
quenching, obtained
from
decay analysis and weighted according
to the
to the intensity,
The average lifetimes,
the Ksv
are shown also in Table
I.
DISCUSSION Details
of the dynamics of oxygen diffusion
obscure, but are generally protein
matrix.
believed
Most of the measurements
and phosphorescence
have been carried
In those studies, the tryptophan the protein,
i.e., interior
been accessible
protein
to nanosecond
tryptophan
residues have been located
remain
fluctuations
on oxygen quenching
out on intrinsic
versus exterior,
interiors
of fluorescence residues (l-7).
at various
but all of the tryptophans
in the
regions in
studied
have
to oxygen.
In this report, binding
to be related
through
we have studied
site, and endeavored
one particular
to elucidate 106
differences
region
of HRP,
in the protein
the heme matrix
in
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
2.6
1.8
0.04
0.08
0.12 [OXYGEN],
0.04 M
0.08
0.12
w. Intensity (-El-), phase lifetime (-a-) and modulation lifetime (-0-) quenching of the emission of 1,8-ANS (a); bis-ANS (b); 2,6-TNS (c) and PPIX (d) conjugates of apoHRP. For the naphthalene probes,the exciting wavelength was 364 nm and the emission was observed through a KV399 cuton filter. The conditions for the HRP(desFe) experiments are the same as described in the text.
response to the presence of different accessibility in Table
to the heme binding
I, the naphthalene
to oxygen than is PPIX. PPIX
is very tightly
surrounding resistance though
protein to GuHCl
probes.
probes,
in general, is consistent
bound (KdclO”‘M)
rigidity
The naphthalene
TABLE STERN QUENCHING
Probe PPIX 1,8-ANS 2,6-TNS bis-ANS
VOLMER CONSTANTS OF HRP
-zr>(nsec)
As shown
more accessible
with the interpretation
and presumably
This conferred
denaturation.
are significantly
(Kd 1U5 - 10-6) (19), and hence the protein
LIFETIMES,
that oxygen
site depends upon the probe utilized.
This finding
matrix.
Our results demonstrate
that the
confers a rigidity
also is manifested
in that site’s
probes are bound less tightly matrix
comprising
I
CONSTANTS AND BIOMOLECULAR FOR OXYGEN QUENCHING CONJUGATES
Ksv (M”)
16.9
3.2 10.2
17.7 9.5
15.5
8.1
14.2
107
to the
1.9 5.7 16.4 17.4
x x x x
1o+s lot8 lo+’ lo+8
the heme
Vol.
178,
No.
binding
BIOCHEMICAL
1, 1991
site should
denaturation
be expected
AND
to be less rigid
to previous
conferred
observations
by hemin
relevance
photoinactivation
and 1,8-ANS
by ultraviolet
with hemin
the relative
by hemin.
efficiencies
or 1,8-ANS.
1,8-ANS
complex
facile diffusion decreased
susceptible
Given
to
This protection
to the PPIX of 1,8-ANS
against
of the inactivated was much less
to be not simply due to
between the amino of increased
complex,
it was
some protection
was judged
acid residues and
oxygen diffusion
in the
we could now suggest that more
matrix
relative
could also play a role in the
to hemin
we have shown that the accessibility
site of HRP
varies depending
occupying
that site. This observation
in protein
interiors
dynamic
Specifically,
in oxygen
requiring
processes.
In summary, heme binding
(20).
assay), but that this protection
our observations
role
to HRP
in
to photoinactivations
to HRP, afforded
of oxygen in the HRP protein
protective
protection
complexed
of energy transfer
relative
photoinactivation
overall
and more
light (assessed by recombination
and enzymatic
than that conferred hemin
COMMUNICATIONS
of our data on oxygen diffusion
on the relative
observed that 1,8-ANS, when complexed protein
RESEARCH
by GuHCl.
We may also note the possible HRP
BIOPHYSICAL
of molecular
upon the nature
suggests that the diffusion
depends not just on the protein
aspects of the particular
oxygen to the
of the molecule of small molecules
matrix per se, but rather on the
protein-probe
system being studied.
Acknowledgments We are grateful to Carolina Jullian for initial work in this area and Genny Leinenweber for technical assistance. This work was supported by National Science Foundation Grants INT-8916623 (JB and DMJ) and DMB 9005195 (DMJ), and Direcci6n General de Investigaci6n of the Universidad Cat6lica de Valparaiso. The Greg 200 spectrofluorometer was obtained through the Chilean Interuniversity grant 8012/86 sponsored by FONDECYT-Chile. The support of the Univeristy Research Council of the University of Hawaii is also acknowledged. DMJ is an Established Investigator of the American Heart Association.
REFERENCES 1. 2. Z: 5. 6.
Lakowicz, J.R. and Weber, G. (1973) Biochemistry Lakowicz, J.R. and Weber, G. (1973) Biochemistry Eftink, M. and Jameson, D.M. (1982) Biochemistry Calhoun, D.B., Vanderkooi, J.M., Woodrow, G.V. S.W. (1983) Biochemistry 22, 1526-1532. Strambini, G.B. (1987) Biophys. J. 52, 23-28. Strambini, G.B. and Caballieri, E. (1990) Photochem. 643-648. 108
12, 4161-4170. 12, 4171-4179. 21, 4443-4449. and Englander, Photobiol.
51,
Vol.
178,
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Vanderkooi, J.M., Calhoun, D.B. and Englander, S.W. (1987) Science 236, 568. Jameson, D.M., Gratton, E., Weber, G. and Alpert, B. (1984) Biophys. J. 45, 789-794. Brunet, J.E., Jullian, C. and Jameson, D.M. (1990) Photochem. Photobiol. 5 1, 487-489. Cilento, G. (1980) Act. Chem. Res. 13, 225230. Cilento, G. (1982) In Chemical and Biological Generation of Excited States (W. Adam and G. Cilento, ed.) p. 277-307. Academic Press, New York. Jullian, C., Brunet, J.E., Thomas, V. and Jameson, D.M. (1989) Biochim. Biophys. Acta 997, 206-210. Teale, F.W.J. (1959) Biochim. Biophys. Acta 35, 543. Tamura, M., Asakura, T. and Yonetani, T. (1972) Biochim. Biophys. Acta 2678, 292-304. Gratton, E. and Limekeman, M. (1983) Biophys. J. 44, 315-324. Spencer, R.D. and Weber, G. (1969) Ann. NY Acad. Sci. 158,361-375. Jameson, D.M., Gratton, E. and Hall, R.D. (1984) App. Spec. Rev. 20, 55-106. Mauk, M.R. and Girotti, A.W. (1974) Biochemistry 13, 1757-1763. Rosen, C.G. (1970) FEBS Letters 6, 158-160. Rosen, C.G. and Nilsson, R. (1971) Biochim. Biophys. Acta 236, l-7.
109
178,
July
15, 1991
No.
BIOCHEMICAL
1, 1991
AND
RESEARCH
COMMUNICATIONS Pages
OXYGEN DIFFUSION NEAR SITE OF HORSERADISH Victor
Vargas”‘,
‘Departamento
“Department
May 17,
Juan E. Brunet’
104-109
THE HEME BINDING PEROXIDASE and David
de Quimica, Facultad de Ciencias, Casilla 653 Santiago, Chile
‘Institute
Received
BIOPHYSICAL
M. Jameson’ Universidad
de Chile
de Quimica, Universidad CatMica de Valparaiso Casilla 4059 Valparaiso, Chile
of Biochemistry and Biophysics, University of Hawaii 1960 East-West Road, Honolulu, Hawaii 96822 1991
The quenching by molecular oxygen of the fluorescence of several probes complexed to apohorseradish peroxidase has been studied by intensity and timeresolved fluorescence methods. The probes utilized include 1-anilino-Snaphthalene sulfonic acid, 4,4’-bis (1-anilino-S-naphthalene sulfonic acid), and 2-ptoluidinylnaphthalene-6-sulfonic acid. These results are contrasted to those obtained using apohorseradish peroxidase complexed with protoporphyrin IX. The resistance of these complexes to denaturation by guanidine hydrochloride was also determined. The results demonstrate a dramatic increase in oxygen accessibility to the naphthalene probes compared to protoporphyrin IX, which can be correlated to the increased stability of the protein-protoporphyrin IX complex. B 1991Academic Press, Inc. The facile diffusion
of oxygen through
has been verified
by the methods
(1-8).
study of oxygen diffusion
A recent
complexed
with
protoporphyrin
quenching
constant
oxygen accounted
of fluorescence IX,
near 2 x lo+* M’
constants observed in any protein
of a number
of proteins
and phosphorescence
quenching
through
HRP(desFe),
apohorseradish determined
peroxidase a bimolecular
s-l whi ch is one of the lowest quenching
system (9). This poor accessibility
for the observations
1 To whom correspondence
the interior
of luminescence
to molecular
from the enzymatically
should be addressed.
AJ@RJZVIATIONS: 1,8-ANS, 1-anilino-8-naphathalene sulfonic acid; Bis-ANS, 4,4’bis (1-anilino-B-naphthalene sulfonic acid); 2,6-TNS, 2-p-toluidinylnaphthalene6-sulfonic acid; PPIX, protoporphyrin IX; apoHRP, apohorseradish peroxidase; HRP, horseradish peroxidase; HRP(desFE) apohorseradish peroxidaseprotoporphyrin IX adduct, GuHCl, guanidinium hydrochloride. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
104
Vol.
178,
No.
1, 1991
generated resistant Time
acetone
triplet
demonstrated
AND
state (10-11).
to denaturation
resolved
binding
BIOCHEMICAL
by GuHCl
fluorescence
compared
measurements
site (12). The low diffusional
in a rigid environment.
to denaturation measurements
by GuHCl. on several
AND
motion
in HRP(desFe)
in the heme
rate for oxygen to the heme pocket in HRP
fluorophore Here
(9). furthermore
of the fluorophore
of oxygen to fluorophores we report
is tightly
bound
on a relationship
bound in the heme pocket and as judged by their resistance
on oxygen quenching
complexes;
Bis-ANS,
moiety
to speculate
complexes
apo-HRP-probe
results using the probes 1,8-ANS, MATERIALS
on PPIX
It then seemed reasonable
of those apoHRP
COMMUNICATIONS
to other heme proteins
with the fact that the porphyrin
between the accessibility the stability
RESEARCH
It was also shown that HRP was very
the absence of any local
(desFe) is thus consistent
BIOPHYSICAL
2,6-TNS
and GuHCl
specifically
we compare
and PPIX.
METHODS
Preparation of apo-HRP Col&ANS, bis-ANS and 2,6-TNS from Molecular Probes, were used without further purification. Horseradish peroxidase type VI and GuHCl were from Sigma. The protein’s heme group was removed using Teale’s method of cold acid and butanone extraction (13), followed by exhaustive dialysis at 4°C against 0.1 M sodium phosphate buffer (pH 7.4). The concentration of apoHRP was determined spectrophotometrically using a molar absorption coefficient at 280 nm of 20,000 (14). The apoHRP conjugates with the naphthalene derivates were prepared with at least a 20fold molar excess of protein in phosphate buffer 0.1 M pH 7.4. The HRP(desFe) conjugate was prepared as previously described (12). The oxygen quenching experiments were carried out also as previously described (9), using a frequency of 30 MHz for l&ANS, 60 MHz for 2,6-TNS and 70 MHz for bis-ANS conjugates of HRP. Fluorescence Wved measurements: Lifetime measurements were carried out using either an ISS Greg 200 spectrofluorometer or a homebuilt multifrequency phase and modulation fluorometer based on the Gratton design as Excitation of the naphthalene probes was previously described (15-17). accomplished using the 364 nm line of an argon ion laser (Spectra Physics Model 2035-3.5S), and the emission was observed through a Schott KV399 cuton filter which passed wavelengths longer than 380 nm.
RESULTS Figure apoHRP
1 shows the displacement
by hemin
fluorescence. a 1:l molar 1:l conjugate
titration,
followed
of 1,8-ANS
with
identical
with an inflection
between the probe and the apoHRP. apoHPR
bound
by the decrease of the naphthalene
The two curves are virtually relation
and of 2,6-TNS
as described 105
by Mauk
The PPIX
and Girotti
to the probes’
point near also forms a
(18).
GuHCl
BIOCHEMICAL
Vol. 178, No. 1, 1991
01%
I I
I
HEMIN
I 2
MOLES/APO-HRP
AND BIOPHYSICAL
I 3
I
02
MOLES
RESEARCH COMMUNICATIONS
2
5
3 [Gu HCf;.
6
7
M
Pi_oure 1. Displacement of l&ANS (-0-) and 2,6-TNS (-•-) from apoHRP by hemin. The excitation wavelengths were 360 nm and 317 nm respectively and the fluorescence was monitored at 475 nm and 432 nm respectively. JQgur-&.Fluorescence decreasefor the 1,8-ANS (-a-), 2,6-TNS (-0-),bis-ANS (-A-) and PPIX (-0) conjugates of apoHRP as a function of GuHCl concentration. The fluorescence was measuredfrom the integrated area of the spectra. The excitation wavelength was 364 nm for the naphthalene probes and 514 nm for the HRP(desFe).
unfolding
of the HRP conjugates
and PPIX protein
show a decrease
as the GuHCl Stem-Volmer
fluorescence in Figures constants
fluorescence
plots of the intensity
3-a through
from
the
yield in buffer.
quenching
by oxygen of the
probes and PPIX
From the slopes of the intensity
discrete exponential
contributions
and lifetime
I were obtained.
probes
upon displacement
bound to the naphthalene
3-d.
shown in Table
fractional
in their
increases due to their low fluorescence
of the apoHRP
a two component
are shown in Figure 2. The naphthalene
are shown
quenching, obtained
from
decay analysis and weighted according
to the
to the intensity,
The average lifetimes,
the Ksv
are shown also in Table
I.
DISCUSSION Details
of the dynamics of oxygen diffusion
obscure, but are generally protein
matrix.
believed
Most of the measurements
and phosphorescence
have been carried
In those studies, the tryptophan the protein,
i.e., interior
been accessible
protein
to nanosecond
tryptophan
residues have been located
remain
fluctuations
on oxygen quenching
out on intrinsic
versus exterior,
interiors
of fluorescence residues (l-7).
at various
but all of the tryptophans
in the
regions in
studied
have
to oxygen.
In this report, binding
to be related
through
we have studied
site, and endeavored
one particular
to elucidate 106
differences
region
of HRP,
in the protein
the heme matrix
in
Vol.
178, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
2.6
1.8
0.04
0.08
0.12 [OXYGEN],
0.04 M
0.08
0.12
w. Intensity (-El-), phase lifetime (-a-) and modulation lifetime (-0-) quenching of the emission of 1,8-ANS (a); bis-ANS (b); 2,6-TNS (c) and PPIX (d) conjugates of apoHRP. For the naphthalene probes,the exciting wavelength was 364 nm and the emission was observed through a KV399 cuton filter. The conditions for the HRP(desFe) experiments are the same as described in the text.
response to the presence of different accessibility in Table
to the heme binding
I, the naphthalene
to oxygen than is PPIX. PPIX
is very tightly
surrounding resistance though
protein to GuHCl
probes.
probes,
in general, is consistent
bound (KdclO”‘M)
rigidity
The naphthalene
TABLE STERN QUENCHING
Probe PPIX 1,8-ANS 2,6-TNS bis-ANS
VOLMER CONSTANTS OF HRP
-zr>(nsec)
As shown
more accessible
with the interpretation
and presumably
This conferred
denaturation.
are significantly
(Kd 1U5 - 10-6) (19), and hence the protein
LIFETIMES,
that oxygen
site depends upon the probe utilized.
This finding
matrix.
Our results demonstrate
that the
confers a rigidity
also is manifested
in that site’s
probes are bound less tightly matrix
comprising
I
CONSTANTS AND BIOMOLECULAR FOR OXYGEN QUENCHING CONJUGATES
Ksv (M”)
16.9
3.2 10.2
17.7 9.5
15.5
8.1
14.2
107
to the
1.9 5.7 16.4 17.4
x x x x
1o+s lot8 lo+’ lo+8
the heme
Vol.
178,
No.
binding
BIOCHEMICAL
1, 1991
site should
denaturation
be expected
AND
to be less rigid
to previous
conferred
observations
by hemin
relevance
photoinactivation
and 1,8-ANS
by ultraviolet
with hemin
the relative
by hemin.
efficiencies
or 1,8-ANS.
1,8-ANS
complex
facile diffusion decreased
susceptible
Given
to
This protection
to the PPIX of 1,8-ANS
against
of the inactivated was much less
to be not simply due to
between the amino of increased
complex,
it was
some protection
was judged
acid residues and
oxygen diffusion
in the
we could now suggest that more
matrix
relative
could also play a role in the
to hemin
we have shown that the accessibility
site of HRP
varies depending
occupying
that site. This observation
in protein
interiors
dynamic
Specifically,
in oxygen
requiring
processes.
In summary, heme binding
(20).
assay), but that this protection
our observations
role
to HRP
in
to photoinactivations
to HRP, afforded
of oxygen in the HRP protein
protective
protection
complexed
of energy transfer
relative
photoinactivation
overall
and more
light (assessed by recombination
and enzymatic
than that conferred hemin
COMMUNICATIONS
of our data on oxygen diffusion
on the relative
observed that 1,8-ANS, when complexed protein
RESEARCH
by GuHCl.
We may also note the possible HRP
BIOPHYSICAL
of molecular
upon the nature
suggests that the diffusion
depends not just on the protein
aspects of the particular
oxygen to the
of the molecule of small molecules
matrix per se, but rather on the
protein-probe
system being studied.
Acknowledgments We are grateful to Carolina Jullian for initial work in this area and Genny Leinenweber for technical assistance. This work was supported by National Science Foundation Grants INT-8916623 (JB and DMJ) and DMB 9005195 (DMJ), and Direcci6n General de Investigaci6n of the Universidad Cat6lica de Valparaiso. The Greg 200 spectrofluorometer was obtained through the Chilean Interuniversity grant 8012/86 sponsored by FONDECYT-Chile. The support of the Univeristy Research Council of the University of Hawaii is also acknowledged. DMJ is an Established Investigator of the American Heart Association.
REFERENCES 1. 2. Z: 5. 6.
Lakowicz, J.R. and Weber, G. (1973) Biochemistry Lakowicz, J.R. and Weber, G. (1973) Biochemistry Eftink, M. and Jameson, D.M. (1982) Biochemistry Calhoun, D.B., Vanderkooi, J.M., Woodrow, G.V. S.W. (1983) Biochemistry 22, 1526-1532. Strambini, G.B. (1987) Biophys. J. 52, 23-28. Strambini, G.B. and Caballieri, E. (1990) Photochem. 643-648. 108
12, 4161-4170. 12, 4171-4179. 21, 4443-4449. and Englander, Photobiol.
51,
Vol.
178,
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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