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Biophys J, October 2000, p. 2155-2161, Vol. 79, No. 4
*Department of Biological Sciences, Boehringer Ingelheim (Canada)
Limited, Bio-Méga Research Division, Laval, Québec
H7S 2G5, Canada; and Laboratory of Physical Biology,
National Institutes of Health, Bethesda, Maryland 20892 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of ~210 kDa and the other of ~20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The ribonucleotide reductases (RR) are enzymes
that catalyze the conversion of ribonucleoside 5'-diphosphate to the
corresponding deoxyribonucleotide analog requisite for DNA replication.
Herpes simplex virus (HSV) encodes for its own RR enzyme, which is an essential enzyme for the replication of HSV in resting cells (Goldstein and Weller, 1988
; Jacobson et al., 1989). Like the mammalian and bacterial RRs, the HSV enzyme consists of two non-identical subunits (Lammers and Follman, 1983; Ingemarson and Lankinen, 1987). The small subunit (R2) is specified by a 1.2 kb mRNA encoding a 340-amino acid, 38,019 Da protein. The large subunit (R1) is specified by a 5.0 kb mRNA transcript that encodes a 1137-amino acid, 124,050 Da
protein (McGeoch et al., 1988). On denatured polyacrylamide gel
electrophoresis R2 migrates at the expected
Mr of 38,000 (Mann et al., 1991;
Lankinen et al., 1991; Lamarche et al., 1990). However, HSV R1 has been
reported to migrate at an Mr of
140,000, possibly because of the presence of a hydrophobic N-terminal
domain that retards the protein during electrophoresis (Furlong et al.,
1991; Massie et al., 1995).
Structurally, the R2 contains a tyrosyl radical and a µ-oxo bridged
binuclear ferric center that are crucial for the reduction process. R1
contains redox-active thiols that provide the hydrogen for nucleotide
reduction (Stubbe, 1990). Association of the R1 and R2 is essential for
catalytic activity. The C-terminus of R2 has been shown to be mobile in
solution (Laplante et al., 1994) and is critical for this process
(Filatov et al., 1992). These properties of HSV RR are common to other
iron-containing RR and are crucial to their catalytic activity.
Knowledge of the molecular mechanism of enzymatic reduction of
ribonucleotides is derived from elegant studies conducted with recombinant Escherichia coli ribonucleotide reductase
(Stubbe et al., 1983; Erikson and Sjoberg, 1989). The R1 protein binds the substrate and provides the hydrogens for nucleotide reduction. R2
provides a tyrosyl free radical which subsequently, via radical transfer, provides a protein radical on R1 in close enough proximity to
abstract the 3'-hydrogen atom of the substrate to yield the desired
deoxyribonucleotide (Stubbe, 1990; Bollinger et al., 1991). The
three-dimensional structure of the R1 and R2 proteins clearly supports
this model (Uhlin and Eklund, 1994; Nordlund et al., 1990; Sjoberg,
1994) and shows that the radical-carrying tyrosine is inaccessible to
solvent as it is buried in the interior of each protomer.
Although there exists a reasonable amount of information on the
molecular structure of HSV RR subunits, there is less information on
the nature of subunit interactions within the holoenzyme. However, it
is known from direct binding studies that the affinity of recombinant R2 for R1 is in the 100 nM range (Krogsrud et al., 1993) and that R1
and R2 each form stable homodimers (Lankinen et al., 1991; Connor et
al., 1993).
Radiation inactivation was chosen as the technique both to
independently evaluate existing theories on the subunit interactions for RR and to further explore their nature. This technique is based on
several general principles: gamma rays and high-energy electrons
interact randomly with proteins, dependent only on their mass. In each
interaction, large amounts of energy are deposited in protein
molecules, resulting in breakage of many covalent bonds. Every
polypeptide that is directly affected suffers severe and irreversible
structural damage and loses all biological activity. When irradiated in
the frozen state, damage to individual molecules has no effect
elsewhere. Only molecules that escape radiation damage retain both
structure and complete function. Because of the random nature of the
radiation interactions, the surviving biological functions decrease
exponentially with radiation dose at a rate directly proportional to
the mass of the active structure. In a complex mixture of polypeptides
and other molecules, radiation damage to other molecules that are not
involved in the measured activity has no effect on the measurement
(Kempner, 1988). Several individual experimental tests have confirmed
the basic principles of radiation target theory.
The method has been used successfully to study a wide variety of
biologically active proteins (Kempner, 1988). These revealed the mass
of all of the subunits required for the measured function
sometimes only one polypeptide of a complex. In addition to biological function, radiation target analysis can be applied to structure. The surviving monomers in irradiated proteins can be resolved by denaturing gel
electrophoresis and quantitated by staining or antibody reactions. Analysis of these data yields the mass of those structures destroyed by
a single radiation interaction independent of any biological function.
Both of these approaches have been applied here to the irradiated
holoenzyme RR and the individual subunits. The results give us new
insight into the structure and function of this critical enzyme.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials
Production of recombinant HSV-1 R2
Recombinant HSV-1 R2 protein was produced by induction of logarithmically growing BL21(DE3) pLysS E. coli that were transformed with the plasmid pETH2. The protein was purified to apparent homogeneity and reactivated by the aerobic method essentially as described previously (Mann et al., 1991).
Reactivated preparations of HSV-1 R2 had ~10-20% free radical
content, as determined by electron paramagnetic resonance, courtesy of
Dr. L. Thelander (University of Umea, Umea, Sweden). The iron content
of the reactivated R2 proteins was typically 60-85% when determined
as described previously (Krogsrud et al., 1993). These values for free
radical and iron content are in agreement with previous reports (Mann
et al., 1991; Filatov et al., 1992). Protein concentration for pure
HSV-1 R2 was determined from the absorbance difference at 280 nm and
310 nm using a molar extinction coefficient
(
280-310) of 52,000 M
1 as described
previously (Bradford, 1976; Bollinger et al., 1991). The concentration
of HSV-1 R2 was calculated by assuming a 38 kDa R2 monomer. Under the
standard assay conditions described below, the specific activity of the
HSV-1 R2 preparations used in this study varied from 50 to 100 units/mg
when tested in the presence of molar excess HSV R1. No activity was
observed in the absence of HSV R1 subunit.
Production of recombinant HSV-2 R1
Recombinant HSV-2 R1 was obtained from confluent 293 cells that had been infected with the recombinant adenovirus vector Ad5-RR1 as described elsewhere (Massie et al., 1995; Krogsrud et al., 1993;
Paradis et al., 1991). The HSV-2 R1 subunit was partially purified by
using the following procedure. Infected cell pellets were resuspended
in 1 volume of 50 mM HEPES, pH 8.0, 2 mM DTT containing the protease
inhibitors aprotinin and leupeptin at a concentration of 50 µg/ml
each, and the cell suspension was subjected to ultrasonic disruption
using a Branson Sonifier Model 350. The protein homogenate was
clarified by centrifugation for 10 min at 20,000 × g
and the soluble proteins were precipitated by adding to the supernatant
ammonium sulfate to a final concentration of 35% saturation. The
precipitated proteins were collected by centrifugation, dissolved in a
minimal volume of buffer A, and then dialyzed overnight against the
same buffer. The dialysate was clarified by centrifugation and stored
at 
80°C. Protein concentration of the HSV-2 R1 preparation was
measured by the method of Bradford (1976) using 
-globulin as a
standard. The specific activity of the enzyme preparations used in this
study ranged from 1 to 2 units/mg protein when measured in the presence
of excess HSV-1 R2 as described below. All HSV R1 preparations had no
activity in the absence of added HSV R2. The protein concentration of
the R1 preparation was 15 mg/ml with a HSV-2 R1 content of ~10%, as determined by SDS-polyacrylamide gel electrophoresis and densitometric scanning of Coomassie Brilliant Blue-stained gels.
Production of ribonucleotide reductase holoenzyme
For the production of ribonucleotide reductase holoenzyme, baby hamster kidney cells were infected with HSV-1 strain F at a multiplicity of infection of 5 for 12 h as described elsewhere (Cohen et al., 1985). The HSV-1 ribonucleotide reductase holoenzyme was
extracted from infected cell pellets and partially purified by
following the protocol described for the preparation of the R1 subunit.
The specific activity of the holoenzyme preparation was ~0.4 unit/mg
protein when assayed as described below with a total protein
concentration of 49.6 mg/ml.
Quantitation of RR subunits by SDS PAGE and Western blot
For immunoblot analysis, irradiated samples were run on 7.5% and 10% polyacrylamide gels for HSV R1 and R2, respectively. Twenty µg of partially purified R1 and 2 µg of purified R2 were loaded on the gel. In the case of the holoenzyme, 50 or 20 µg of partially purified enzyme were loaded for Western blots with the monoclonal antibodies 932 and 535, respectively (Ingemarson and Lankinen, 1987). For Western blotting, separated proteins were transferred to
nitrocellulose membranes, washed three times with 1% dry milk powder
in phosphate buffered saline, and probed with 10 µg/ml 932 antibody
or 0.1 µg/ml 535 antibody for 2 h, followed by a 2 h
incubation with 5 µCi sheep anti-mouse
[125I]IgG (Amersham, Québec,
Canada). Dried membranes were exposed to phosphorscreens and
quantitated on a Molecular Dynamics Phosphor Imager.
Radiation inactivation
Radiation inactivation was performed with 10 MeV electrons at135°C as described (Harmon et al., 1985). The irradiated samples were stored for no longer than 1 month before assay.
Ribonucleotide reductase activity assay
The catalytic activity of ribonucleotide reductase was determined from the rate of conversion of [14C]CDP to [14C]dCDP as described elsewhere (Krogsrud et al., 1993). One unit of reductase
activity is defined as that amount of enzyme which catalyzes the
formation of 1 nmol dCDP per min at 37°C. Briefly, the standard
reaction mixture, in a final volume of 60 µl, contained 50 mM HEPES,
pH 8.0, 4 mM NaF, 30 mM dithiothreitol, 54 mM CDP, 0.1 µCi
[14C]CDP (524 Ci/mol; NEN Dupont), 1 mM
bacitracin and enzyme. After 30 min incubation at 37°C, the reaction
was stopped by the addition of 20 µl of 40 mM HEPES, pH 8.0, 4 mM
MgCl2 containing 2.5 units of calf alkaline phosphatase
(Boehringer Mannheim), followed by incubation at 37°C for 15 min to
dephosphorylate all nucleotides. Deoxycytidine was separated from
cytidine using thin-layer chromatography and the radiolabel was
quantitated using the radioanalytical imaging system AMBIS (Ambis Inc.,
San Diego, CA). The specific activity (unit/mg) of R2 samples was
calculated from the linear portion of the activity versus R2
concentration (0.1-1 µg purified R2) curves in the presence of molar
excess HSV R1 (40 µg of partially purified R1 per assay). Likewise,
the specific activity of R1 samples was calculated from the linear
portion of the activity versus R1 concentration (10-250 µg partially
purified R1) curves in the presence of molar excess HSV R2 (5 µg
purified R2 per assay). The biological activities of HSV-1 R2 and R1
were also determined using the solid-phase competition binding assay
described below (Krogsrud et al., 1993).
Ribonucleotide reductase binding assay
Direct binding to R1 and competition for binding to R1 by R2 were evaluated using 125I-labeled 3-iodo-desamino-Tyr-(N-Me)Val-Ile-Asn(
-N, N-Et)Asp-Leu-OH (specific
activity 74 ± 7 GBq/µmol), essentially according to the methods
described earlier (Krogsrud et al., 1993).
Target size calculations
In the case of the enzyme assays and Western blot analysis where activity is represented by the amount of protein remaining in the gel band, the fraction of activity remaining following irradiation of samples was expressed as AD/A0, where AD represents the enzyme activity following a dose of radiation D and A0 represents the enzyme activity in the absence of irradiation. In the case of binding, activity was expressed as B/F, where B represents the amount of specific binding of radioligand and F represents the concentration of free radioligand. The fraction of binding activity remaining in irradiated samples was calculated by normalizing each value of B/F to that observed in non-irradiated controls, (B/F)0. In either case least-squares linear regression analysis was used to calculate the radiation inactivation constant from:
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(1) |
). The rate of radiation inactivation constant, k',
is corrected for its temperature dependence (Kempner and Haigler, 1982)
by a factor St, which is equal to 2.8 for irradiations performed at 135°C.
For the analyses presented here the simplest form of target analysis
was assumed, namely that the recognition site qualities of an enzyme or
binding site are either completely abolished or remain unaffected.
Thus, the loss of binding is due to a reduction in either
Vmax (maximal velocity of an enzyme
reaction) or Bmax (maximal binding
site capacity) with no changes in Km
(enzyme affinity constant) or Kd
(macromolecular dissociation constant), respectively. If this
assumption is valid, the target size can be calculated from:
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(2) |
). Several different-sized molecules are present that either independently bind to
the same ligand or (in the case of an enzyme) catalyze the same
reaction. If only two such species were present, the activity remaining
after exposure to a radiation dose D will be given by
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(3) |
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(4) |
and were obtained by using nonlinear regression analysis.
Statistics
Differences between groups were analyzed statistically by Student's t-tests.| |
RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Irradiation of holoenzyme
Samples of HSV-1 RR holoenzyme dissolved in 50 mM HEPES buffer, pH 7.5 containing 2 mM DTT in glass ampoules were frozen on dry ice and sealed with an oxygen-gas torch before irradiation and thawed before analysis. This procedure did not affect the activity of the holoenzyme when compared to samples that had not been processed for irradiation studies. After exposure of the holoenzyme to different doses of radiation, the remaining enzyme activity decreased as a single exponential function of radiation dose (Fig. 1), leading to an average target size of 315 kDa (Table 1), which is similar to the predicted molecular size of 324 kDa. Analysis of the target sizes of R1 and R2 following irradiation of the holoenzyme and quantitation by Western blot yielded single exponential decay curves and average target sizes of 281 kDa for R1 and 57 kDa for R2 (Fig. 1, Table 1).
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Irradiation of the free HSV R1 subunit
Partially purified recombinant R1 subunit was suspended in the same buffer as the holoenzyme. After exposure of the R1 subunit to different doses of radiation, the remaining functional R1 subunit was quantitated by enzyme assay following reconstitution with excess R2, Western blot, and tracer binding. All three techniques yielded single exponential decay curves and gave the following similar estimates: 153, 130, and 128 kDa, respectively (Fig. 2, Table 1).
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Irradiation of the free recombinant HSV R2 subunit
Purified recombinant R2 subunit was suspended in 20 mM bis-TRIS, pH 6.5 containing 2 mM DTT and 0.15 M NaCl. After exposure of R2 subunit to different doses of radiation, the remaining functional R2 subunit was also quantitated by enzyme assay following reconstitution with excess R1, Western blot, and competition for tracer binding. Surprisingly, by enzymatic assay the functional activity of R2 decreased as a single exponential with an estimated target size of 223 kDa (Fig. 3, Table 1) which is considerably larger than would be expected for either the monomer or the dimer. When Western blot analysis was used to quantitate R2, protein decreased as a single exponential with a target size of 19 kDa (Fig. 3, Table 1). Although this is smaller than would be expected for the R2 monomer, it indicates that only one monomer is damaged by each radiation event and there is no evidence for transfer of radiation energy to other monomers, as was seen in the holoenzyme.
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The amount of R2 capable of inhibiting tracer binding following radiation inactivation was quantitated from inhibition curves using R2 concentrations ranging from 1 to 10,000 nM. The destruction of R2 by irradiation resulted in a dose-dependent reduction of the inhibition of binding over all the concentrations of R2 used. In the absence of irradiation the IC50 for inhibition of tracer binding by R2 was 0.14 ± 0.03 µM. Two concentrations of R2 were chosen for measuring the amount of R2 remaining following irradiation, 0.1 µM and 1.0 µM, the amount of R2 remaining following each radiation dose being calculated from the inhibition curve by using the Hill equation. Upon radiation inactivation, both the high and low concentrations of R2 yielded biphasic exponential decay curves with increasing radiation dose (Fig. 3 shows only the results with the higher concentration of R2). The biphasic decay curve was resolved accounting for two target sizes. The target sizes and fraction of binding sites were 20 kDa, 0.15 and 210 kDa, 0.75, respectively (Table 1).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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HSV RR is an 
22
heterodimer of Mr 324,000. Isolated R1
and R2 subunits each exist as stable protein dimers in solution
(Lankinen et al., 1991; Conner et al., 1993). The functional RR enzyme
is believed to be a complex of the R1 and R2 because individual
subunits do not have any detectable enzymatic activity (Mann et al.,
1991; Lankinen et al., 1991; Lamarche et al., 1990; Furlong et al., 1991; Massie et al., 1995). Furthermore, truncated R2 derivatives lacking the essential C-terminal subunit interaction domain do not
display enzyme activity with R1 due to an inability to associate (Filatov et al., 1992; Krogsrud et al., 1993).
The interactions between R1 and R2 and monomers for each of these
subunits in the HSV RR holoenzyme were probed using radiation inactivation. Estimates of the functional target size (i.e., based on
enzymatic activity) of the holoenzyme suggested that a tetramer of two
R1 monomers and two R2 monomers was required for function. The target
size for the structure (as assessed by Western blot) of both the R1 and
R2 monomers in the irradiated holoenzyme was considerably larger than
the published values of 124 kDa and 38 kDa, respectively (McGeoch et
al., 1988) and corresponded to approximately twice their known
molecular weights. These results indicate that in the holoenzyme there
is transfer of radiation-deposited energy between monomers of R1 and
also between the monomers of R2. Energy transfer between subunit
monomers has been demonstrated previously (Kempner, 1995; Davis et al.,
1996, 1997), possibly occurring as a consequence of very close
juxtapositioning of monomers. The finding that the target size for each
monomer in the holoenzyme fit closely with the mass of a homodimer
implies, however, that energy transfer does not occur between R1 and
R2. Because of the transfer of radiation-deposited energy between
identical monomers in the holoenzyme, interpretation of the tetrameric
functional target size is obscured. If only one monomer of each species
were required for enzymatic activity, the expected target size would be
38 + 124 kDa; but because radiation damage appears in two monomers after only a single primary ionization, only the tetrameric target size
can be observed. Thus, from this experiment alone it cannot be
determined whether an dimer or an
22 structure is needed.
Irradiation of the free R1 gave target sizes of the monomer that were independent of the method used for quantitation: enzyme activity, binding of 125I-labeled peptide, or Western blot analysis. A target size by Western blot analysis equivalent to the monomer for irradiated free R1 indicates that energy transfer does not occur between purified monomers, even though this subunit exists in solution as a dimer. Thus, the minimum subunit for enzymatic and binding activity in R1 is the monomer.
The similar finding of a target size by Western blot analysis equivalent to the monomer for irradiated free R2 shows that radiation-deposited energy is not transferred between isolated R2 monomers either, even though it too exists as dimer. Because energy transfer did occur between monomers of R1 and between monomers of R2 during irradiation of the holoenzyme, this suggests that formation of the tetramer increases the interaction between two R1 monomers and also between two R2 monomers. The association of the subunits is probably accompanied by a change in conformation that results in a tighter interaction between the monomers of each subunit facilitating energy transfer. The tighter interaction between the monomers of each subunit may be important for stabilizing the holoenzyme complex, for optimal electron transfer from one subunit to the other, and for greater substrate binding at the active site.
The target size for enzyme activity in free R2 was consistently larger
than the expected molecular weight for either the monomer or the dimer.
RR is one of the few enzymes that are dependent on a native free
radical (Pedersen and Finazzi-Agro, 1993; Sun et al., 1993). Therefore,
the overestimation of the functional target size could be explained by
a radiation-induced destruction of the critical tyrosyl free radical in
R2, which was reported by Davydov et al. (1996). We analyzed their data
for the disappearance of tyrosyl radicals in R2 as a function of
radiation exposure at 77 K. Assuming no temperature correction (Kempner
et al., 1986) is required, their data yield a target size of 191 kDa.
This value is remarkably close to the activity target size for R2 (223 kDa) reported here. Confirmation of the disappearance of tyrosyl
radicals in our samples was obtained by electron paramagnetic resonance measurements on irradiated and control samples of R2. These
demonstrated a loss of tyrosyl free radical at lower doses of radiation
(data not shown; personal communication Dr. Lars Thelander, University of Umea, Umea, Sweden). This interpretation of the large target size
for enzyme activity upon irradiation of R2 is consistent with the known
structure and function of R2 in RR, but is in contrast with the
observed effects of radiation on other proteins.
Previously published data indicated that a primary ionization caused by radiation in a protein molecule resulted in major structural damage (covalent bond breaks) and consequent loss of biochemical activity. Molecules not suffering a direct hit were undamaged and fully functional. No indirect effects of radiation (damage to one molecule resulting from reaction with a radiation product formed elsewhere) were observed in frozen samples irradiated at very low temperature. In the present case, most tyrosyl free radicals disappeared from purified R2 molecules that had not suffered substantial structural damage (estimated by scission of the polymer backbone). In the holoenzyme this did not happen; the tyrosyl free radical on R2 may be shielded by R1.
Irradiation of R2 and quantitation by competition for tracer binding yielded a complex inactivation curve. This is indicative of destruction of independent molecular species with different masses. The data yielded target sizes of 20 kDa and 210 kDa, species that contributed 15% and 75% of the original binding, respectively. The smaller value is comparable to the mass of R2; the larger target size is close to the value for enzyme activity (and for the loss of the tyrosyl free radical) in irradiated R2. The radiation destruction of the tyrosyl free radical could result in an altered R2 with reduced affinity for inhibition of tracer binding to R1. This is supported by our observation that depletion of the tyrosyl free radical of the R2 by the radical scavenger hydroxyurea resulted in an approximately 10-fold loss of affinity for R1 (unpublished observation). Thus the tyrosyl free radical plays a role in the formation of an optimal conformation for binding of R2 to R1.
This is the first radiation inactivation study of an enzyme containing
a stable free radical. It appears that in this unusual molecule, the
loss of a crucial free radical occurs by an indirect action of
radiation, perhaps even in the frozen state. A role for free radicals
in low-temperature irradiations has been suggested previously (Symons
and Taiwo, 1987; Kempner and Miller, 1994; Kempner et al.,
1986), but in molecules that did not contain a stable free radical. The
well-defined protein structure of RR offers a new opportunity to
examine this phenomenon.
In summary, radiation inactivation of HSV RR has confirmed the tetrameric structure of the holoenzyme in solution. It was revealed that only one R1 monomer, when combined with R2, was necessary for enzymatic activity, and that the R1 and R2 dimers undergo a conformation change upon formation of the holoenzyme. The use of this unique technique has enabled us to independently confirm several earlier findings on the function of RR and to create a more consistent model relating the structure and function of RR. Results of irradiation of the various forms of this protein indicate that indirect radiation effects can abolish the stable free radical if it is accessible to the solvent.
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FOOTNOTES |
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Received for publication 12 May 2000 and in final form 7 July 2000.
Address reprint requests to Dr. Ellis S. Kempner, Bldg. 6, Room 140, NIH, Bethesda, MD 20892. Tel.: 301-496-6941; Fax: 301-402-0009; E-mail: kempnere{at}mail.nih.gov.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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centres to FeIII.
J. Chem. Soc., Faraday Trans. I.
83:3653-3661
Biophys J, October 2000, p. 2155-2161, Vol. 79, No. 4
© 2000 by the Biophysical Society 0006-3495/00/10/2155/07 $2.00
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), and R2 subunits (
)
detected by Western blot. Data are averages plus SD from three
independent experiments.
). Data are shown as averages plus SD from three independent
experiments.
