From the Department of Biochemistry, University of Bergen, Årstadveien 19, 5009 Bergen, Norway
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ABSTRACT |
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The high-spin (S = 5/2) Fe(III)
ion at the active site of recombinant human phenylalanine hydroxylase
(PAH) has a paramagnetic effect on the longitudinal relaxation rate of
water protons. This effect is proportional to the concentration of
enzyme, with a paramagnetic molar-relaxivity value at 400 MHz and
25 °C of 1.3 (± 0.03) × 103 s Mammalian phenylalanine hydroxylase
(PAH,1 EC 1.14.16.1)
catalyzes the hydroxylation of L-Phe to
L-Tyr, which is the rate-limiting step in the catabolic
pathway for L-Phe, taking place mainly in the liver (1, 2).
Like the other mammalian aromatic amino acid hydroxylases, tyrosine
hydroxylase, and tryptophan hydroxylase, PAH is a mononuclear nonheme
iron containing enzyme which requires (6R)-L-erythro-tetrahydrobiopterin
(BH4) and dioxygen as additional substrates (3). The iron
is ferric in the enzyme as isolated and in the catalytic cycle it is
reduced to Fe(II) by BH4 (2, 4, 5). Deficiency of human PAH
activity causes phenylketonuria, which represents the most prevalent
inborn error of amino acid metabolism (6).
Our group has recently described the expression and the molecular and
kinetic characterization of recombinant human PAH (7-10). The
catalytic and physicochemical properties of this enzyme are essentially
the same as those reported for the human liver enzyme (7, 11). The
catalytic domain of the human PAH has been crystallized and the
three-dimensional structure has recently been solved (12, 13). This
crystal structure provides a frame in the understanding of the effect
of mutations in PAH causing phenylketonuria. Moreover, large efforts
are being made by several groups in this field to solve the mechanism
for catalysis and regulation in light of the three-dimensional
structure. The largest body of information about PAH has been
previously obtained on the enzyme isolated from rat liver (2, 3). The
rat and human PAH share a 96% sequence homology (14) and they have
many similar molecular and kinetic properties (2, 3, 7, 8, 10, 15):
(a) they have comparable Km-values for
L-Phe and tetrahydropterin cofactors; (b) they
consist of dimers and tetramers in equilibrium; (c) they are
activated by a number of processes, e.g. incubation with
lysolecithin, high pH, deletion of the N-terminal domain,
phosphorylation at Ser-16 and by the substrate L-Phe, the
two latter mechanisms being of physiological importance; (d)
the activation by L-Phe, which binds with positive
cooperativity, results in conformational changes involving the tertiary
and the quaternary structure, shifting the dimer By using 1H NMR paramagnetic relaxation of the water
protons, we have shown earlier that the Fe(III) ion at the active site of resting rat PAH contains coordinated water and that following the
binding of both L-Phe and L-noradrenaline at
least one water molecule is displaced from coordination (18). In this
study we have measured the paramagnetic effect of the ferric site in recombinant human PAH on the water protons in the absence and the
presence of the substrate L-Phe. The differences
encountered between the accessibility of the solvent to the iron in the
rat and the human enzymes are interpreted based on the different degree of activity in the resting enzymes.
Materials--
L-Phenylalanine and
bathophenanthroline disulfonic acid were from Sigma and
2H2O (99.8%) from Aldrich. Expression of
recombinant human PAH in Escherichia coli (TB1) as fusion
protein with maltose-binding protein, purification of the fusion
proteins by affinity chromatography on amylose resin followed by
high-performance size exclusion chromatography, cleavage by factor Xa
or enterokinase, and further purification of the hydroxylase was
performed as described (7). Rat PAH was isolated from rat liver by the
method (procedure II D) of Shiman et al. (19). The enzyme
activity was measured by determination of L-Tyr formed by
high pressure liquid chromatography and fluorimetric detection as
described (7). The concentration of purified PAH (both rat and human)
was estimated using an absorption coefficient at 280 nm of 1.0 cm Metal Analyses--
The metal content of the enzyme preparations
was determined by a Perkin-Elmer model 402 atomic absorption
spectrophotometer equipped with a graphite furnace (type HGA-76B from
Perkin-Elmer).
Preparation of Iron-free Apoenzymes--
The iron was extracted
from the isolated rat and human holoenzymes by a modification of the
method of Gottschall et al. (20) using 1 mM
bathophenanthroline, 1 mM L-Phe, 0.5 mM BH4, and 1 mM dithiothreitol.
The formation of the Fe(II)-bathophenanthroline complex was followed by
measuring the increase in absorbance at 535 nm. The concentration of
the released iron was calculated from the molar extinction coefficient
for the Fe(II)-bathophenanthroline complex ( NMR Measurements--
Longitudinal relaxation rates of the
residual water signal (HDO) were measured on enzyme samples prepared by
3-4 cycles of 20-fold concentration and dilution in
2H2O, containing 20 mM potassium
phosphate of pH 7.2 (pH value determined with an Ingold electrode and
representing the uncorrected value in 2H2O) and
0.2 M KCl, using Centricon 30 microconcentrators (Amicon). The NMR spectra were recorded on a Bruker DMX-400 and in experiments with variable field, measurements at 250 and 600 MHz were made on
Bruker AM-250 and Bruker DRX-600 spectrometers, respectively. The
longitudinal relaxation times (T1OBS) were
measured at the indicated probe temperatures by using a standard
inversion-recovery sequence, with acquisition parameters including 16K
data points, four transients per time increment and recycle delay
(>5 × T1). In the titration experiments,
the enzyme solution was allowed to equilibrate at the indicated
concentrations of L-Phe in the NMR tubes for 5 min at room
temperature, before measurements. Titrations were performed by adding
microliter amounts of concentrated solutions of L-Phe.
Final dilution of the samples was Theoretical Details--
The longitudinal paramagnetic
relaxation time of the bound water proton (T1M) is
described by the dipolar term of the Solomon-Bloembergen equation (22,
23), i.e.
The observed longitudinal relaxation rate of water protons in a protein
solution (T1OBS The Enzyme Preparations--
The specific activities of the
isolated recombinant human and rat liver PAH preparations with 1 mM L-Phe and 500 µM
BH4 in the presence and the absence of prior incubation (5 min, 25 °C) with 1 mM L-Phe are shown in
Table I. Although the activity of the rat
liver holoenzyme was not stimulated by addition of ferrous ammonium
sulfate (100 µM) in the assay mixture, the activity of the recombinant human PAH increased by about 10% in the presence of
ferrous ions, indicating the presence of some iron-free apoenzyme in
the preparations. Thus, as determined by atomic absorption spectroscopy
the preparations of rat and human PAH used in this study contained
0.95 ± 0.03 and 0.48 ± 0.05 atom of iron/mol subunit, respectively. By measuring the formation of the
Fe(II)-bathophenanthroline complex at 535 nm, it was determined that a
maximal amount of about 0.5 atom of iron/mol subunit was extracted from
both the rat and human enzymes after a 30-45-min incubation period
with the chelator at reducing conditions (see "Experimental
Procedures"). After iron extraction, the resulting proteins were
devoid of catalytic activity and were referred to as apoenzymes (Table
I). However, as shown by atomic absorption spectroscopy measurements,
although no remaining iron was present in the apoenzyme forms of the
recombinant human PAH, the apoenzyme of the rat liver enzyme contained
0.4-0.45 atom/subunit. This population of inactive iron in rat PAH has previously been found not to be reduced by the tetrahydropterin cofactor and not to participate in catalysis (4, 5, 26). After
reconstitution of the holoenzyme from the apoenzyme by incubation with
0.1 mM ferrous ammonium sulfate, full activity was
recovered.
Effect of Recombinant Human Phenylalanine Hydroxylase on the Water
Proton Relaxation
Rates--
T1OBS
Although the paramagnetic molar relaxivity
(T1P Temperature and Field Dependence of Water Proton Relaxation
Rates--
The effect of temperature (16-40 °C) on the
paramagnetic molar relaxivity of water protons is shown in Fig.
3. The Arrhenius activation energy
(Ea) for the paramagnetic contribution to the
relaxation (T1P
The paramagnetic contribution to the relaxation time
(T1P) for solutions of human PAH was measured at
295 K and three different Larmor frequencies, i.e. 250, 400, and 600 MHz, and found to be frequency-dependent both in
the presence and the absence of 5 mM L-Phe
(data not shown), also consistent with a fast exchange condition of
water protons in the paramagnetic environment (25). A linear fit of the
T1P·Cs values at the
three frequencies was used to calculate the effective dipolar
correlation time ( Estimation of the Fe(III)-Water Proton Internuclear Distances in
Recombinant Human Phenylalanine Hydroxylase and the Enzyme-Substrate
Complex--
For nonexchange-limited processes, the paramagnetic
relaxation rate of the water proton resonance is dependent on the
distance between the exchangeable water molecules and the ferric iron. Distances can be estimated from the paramagnetic molar relaxivity values (Table II) using the value of
The estimated water protons-Fe(III) distances increased by 13% on
incubation of the enzyme with L-Phe, regardless of the
value of q, indicating a displacement of, at least, one of
the coordinated water molecules, as earlier found for rat PAH in the
presence of L-Phe (18) and for other systems in which bound
water is known to be displaced upon binding of ligands (27, 31, 32). However, to date there is no crystal structure of complexes of the
enzyme with L-Phe, and a possible displacement of water on binding of the substrate has not been proved. The binding of
L-Phe to either the rat or the human PAH does not affect
the spin state of the Fe(III)
(26)2 and the decrease in
paramagnetic molar relaxivity upon binding cannot be because of
high-spin to low-spin transition of the iron.
Most groups working with PAH interpret the activation of PAH by
L-Phe as the result of its cooperative binding at an
allosteric site, which is physically different to the binding site of
L-Phe at the active site (1, 2). We have, however,
interpreted the activation as the result of the cooperative binding of
L-Phe at the active site (18, 33) because:
L-noradrenaline, a ligand binding at the active site of the
enzyme by coordination to the iron, binds with positive cooperativity
to rat PAH and induces conformational changes similar to
L-Phe (18, 26, 33). Thus, displacement of water from the
active site Fe could be because of the rearrangement of the
coordination geometry of the metal upon substrate binding and enzyme
activation. Although the local effects related to water displacement
seem to be similar for rat and human PAH, larger conformational effects
seem to be induced on the rat enzyme by activation with
L-Phe increasing the limited accessibility of the active
site, a limitation that is not observed in the human enzyme.
In conclusion, the results presented here support that the observed
limitation to the exchange of the coordinated water in PAH isolated
from the rat liver is not detected for the human enzyme. This may be
related to the state of activation, which is higher in human than in
rat PAH (1). These results have important implications for
understanding the structural and regulatory differences between the
hydroxylases from both sources, as well as the phenylalanine
homeostasis in man (1). Moreover, we have further shown that the
binding of L-Phe and activation of both human and rat
enzyme is accompanied by the displacement of at least one water
molecule from coordination to the iron.
1
M
1. The value of the Arrhenius activation
energy (Ea) for the relaxation rate was
14.4 ± 1.1 kJ/mol for the resting enzyme, indicating a fast exchange of
water protons in the paramagnetic environment. The frequency dependence
of the relaxation rate also supported this hypothesis. Thus, the
recombinant human PAH appears to have a more solvent-accessible
catalytic iron than the rat enzyme, in which the water coordinated to
the metal is slowly exchanging with the solvent. These findings may be
related to the level of basal activity before activation for these
enzymes, which is higher for human than for rat PAH. In the presence of saturating (5 mM) concentrations of the substrate
L-Phe, the paramagnetic molar relaxivity for human PAH
decreased to 0.72 (± 0.05) × 103 s
1
M
1 with no significant change in the
Ea. Effective correlation times (
C)
of 1.8 (± 0.3) × 10
10 and 1.25 (± 0.2) × 10
10 s
1 were calculated for the enzyme and
the enzyme-substrate complex, respectively, and most likely represent
the electron spin relaxation rate (
S) for Fe(III) in
each case. Together with the paramagnetic molar-relaxivity values, the
C values were used to estimate Fe(III)-water distances.
It seems that at least one of the three water molecules coordinated to
the iron in the resting rat and human enzymes is displaced from
coordination on the binding of L-Phe at the active site.
INTRODUCTION
Top
Abstract
Introduction
References
tetramer
equilibrium toward the tetrameric form. However, there is a clear
difference between the human and the rat PAH with respect to the extent
of the response to preincubation with L-Phe and to other
modes of activation. Thus, the native and recombinant rat PAH are
activated 8- to 30-fold by L-Phe (1, 16), whereas the
recombinant human PAH is only activated about 2-3-fold (8, 10). Even
lower values of activation of the human enzyme have been reported by
Kowlessur et al. (17). The degree of activation by
phosphorylation is also lower for human than for rat PAH (10). Thus,
although the activity of the maximally activated enzyme is about 2-fold
lower for the human than for the rat PAH (1, 8, 16), the level of basal
activity in the absence of activating treatments is higher for the
recombinant human than for the rat enzyme, which seems to be in
agreement with the properties of PAH from human liver (16, 17).
EXPERIMENTAL PROCEDURES
1 for 1 mg/ml (7, 19).
535 = 22,000 M
1 s
1) (21). The enzyme was
separated from the chelating agent and other low molecular weight
compounds by gel filtration on a G-25 Sephadex column (1.5 × 20-cm) equilibrated in 20 mM Na-Hepes, 0.2 M
NaCl, pH 7.0, and 1 mM L-Phe, included to
stabilize the apoenzyme. The catalytically inactive apoenzyme was
concentrated in Centriplus 30 concentrators (Amicon, MA).
2.5%.
where (
(Eq. 1)
I is the nuclear gyromagnetic ratio,
g is the electronic g factor (isotropic splitting
factor),
is the Bohr magneton, S is the electronic spin
at the ground state of the paramagnetic ion (24), r is the
metal-proton internuclear distance,
I and
S are the nuclear and electron Larmor precession
frequencies, respectively, and
C the effective dipolar
correlation time, which describes the molecular events which modulate
the electron-nuclear dipolar coupling and can be calculated from the
frequency dependence of the longitudinal paramagnetic relaxation (25).
1) is equal to
T1P
1 + T1D
1, where
T1P
1 is the longitudinal
relaxation rate of water protons due to the paramagnetic ion and
T1D
1 is the diamagnetic
contribution of the protein due to the effects of protein
residues-solvent interactions.
T1D
1 was estimated using sodium
dithionite-treated enzyme, in which the iron is fully reduced (26), or
using apoenzyme forms without iron.
T1P
1 values for water protons,
normalized to a subunit concentration (Cs) of 1 M are expressed as paramagnetic molar relaxivity
(T1P
1·Cs
1),
which is related to T1M by the following
expression (27):
where q is the number of water ligands that are
coordinated to the paramagnetic ion (i.e. in the first
coordination sphere) and TO.S.
(Eq. 2)
1 is
the outer sphere contribution to the relaxation rate.
TO.S.
1 is usually small in
paramagnetic systems with coordinated water. The effect of varying
temperature and frequency on T1P
1
was used to determine the predominant contributions
(T1M,
M, or
TO.S.) to the observed relaxation rate (25).
Further theoretical considerations relevant for this paper have been
described (18).
RESULTS AND DISCUSSION
Activity for the apo- and holoenzyme forms of the rat and
recombinant human PAH
1 values were
measured at 400 MHz on the bulk residual water signal (HDO) (4.8 ppm)
at 295 K in deuterated samples of recombinant human PAH at various
concentrations (up to 110 µM enzyme subunit), in the
absence and presence of 5 mM L-Phe (Fig. 1). In this concentration range we found
no significant diamagnetic contribution of the protein to the
relaxation rate, measured either with the iron-free human apoenzyme or
with the dithionite reduced enzyme (Fig. 1). The large effect of the
enzyme as isolated on T1OBS
1
indicates that the high-spin Fe(III) (S = 5/2) site in
the recombinant human PAH is accessible to exchangeable water
molecules, as was previously found for the rat liver enzyme (18).
Accordingly, the x-ray structure of the catalytic domain of human PAH
has shown that the ferric iron is six-coordinated to His-285, His-290,
Glu-330 and to three water molecules, referred to as Wat (1), Wat (2), and Wat (3) (12). As shown by magnetic circular dichroism and x-ray
absorption spectroscopy, the iron sites for both the resting ferric
(inactive) and ferrous (active) forms in the rat enzyme also seem to be
six-coordinate distorted octahedral and substrate binding results in
geometric and electronic structural changes at the iron center
(28).
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Fig. 1.
Longitudinal relaxation rate of the residual
water signal in solutions of recombinant human PAH. Measurements
were made at 295 K and 400 MHz in enzyme solutions (pH 7.2) in the
absence ( ) and the presence of 5 mM L-Phe
(
), and after reduction with 1 mM sodium dithionite
(
). The effect of the enzyme concentration on the relaxation rate of
the water protons can be expressed by the equation:
T1OBS
1 = (1.31(±
0.03)·103·Cs + 0.07)
s
1 and that of the enzyme-substrate complex by
T1OBS
1 = (0.72(±
0.02)·103·Cs + 0.07)
s
1; Cs = subunit concentration
(M).
1·Cs
1)
at 298 K is higher for the rat than for the human enzymes, the values become similar when they are normalized to molar concentration of iron
(T1P
1·CFe
1)
(Table II). The paramagnetic molar
relaxivity of human PAH was found to decrease about 2-fold when 5 mM L-Phe was added (Table II), indicating
either occlusion or displacement of the coordinated water molecules.
The effect of L-Phe binding to the human enzyme on
1/T1P was studied in more detail as a function
of ligand concentration (Fig. 2). The
titration curve was found to be hyperbolic, unlike the curves obtained
on titration of rat liver PAH either with L-Phe or
L-noradrenaline, which are three-phasic and nonhyperbolic (18). The three-phasic curves have been interpreted as being the result
of the change from a system in which water is coordinated to Fe(III) at
the active site and slowly exchanging with the bulk water, to a system
in which water is fast exchanging at a site close to the iron, but not
coordinated (18). In the case of the recombinant human PAH the
hyperbolic titration curve (Fig. 2) seems to indicate the absence of
exchange limitations in the enzyme as isolated, as well as in the
L-Phe-enzyme complex.
Paramagnetic molar relaxivity at 298 K and 400 MHz normalized to
subunit concentration (T1P1·CS
1),
paramagnetic molar relaxivity normalized to iron concentration
(T1P
1·CFe
1), Arrhenius activation
energy (Ea) for the paramagnetic contribution to the
relaxation, effective correlation times (
C), and estimated
water-iron distances (r) for the rat and the recombinant human PAH in
the absence and the presence of L-Phe
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Fig. 2.
Effect of L-Phe concentration on
the paramagnetic longitudinal relaxation of the residual water proton
resonance in solutions of recombinant human PAH. The enzyme
samples (100 µM subunit) were allowed to equilibrate with
the indicated concentrations of substrate for 5 min. Measurements were
made at 400 MHz and 295 K, pH 7.2.
1) was only
slightly affected by the binding of L-Phe and showed just a
small decrease from
14.4 ± 1.1 kJ/mol in the resting enzyme to
17.9 ± 0.4 kJ/mol in the presence of 5 mM
L-Phe (enzyme-substrate complex) (Table II). Typical
Ea for T1P
1 in
slow exchange processes are
9 kJ/mol because, in general, the
exchange-lifetime,
M, has positive temperature
coefficients (25, 29). Our results thus indicate that both in the
resting human enzyme and in its complex with L-Phe, the
relaxation of water protons is not exchange-limited. This is different
from the situation in the resting nonactivated rat liver PAH, in which Ea was found to be 11.3 ± 0.8 kJ/mol (18)
consistent with exchange limitations for the water molecules
coordinated to the iron, indicating that the protein may impose
hindrances to the free exchange of the coordinated water. This
limitation was abolished in the L-Phe-activated enzyme
(Ea =
1.5 ± 0.2 kJ/mol) (Table II).
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Fig. 3.
Temperature dependence of the paramagnetic
molar relaxivity. Measurements were made at 400 MHz in solutions
(pH 7.2) of human PAH (73 µM subunit) in the absence
( ) and the presence of 5 mM L-Phe (
). The
slopes of linear regression lines yield activation energies
(Ea) of
14.4 and
17.9 kJ/mol,
respectively.
C, Eq. 1) under the assumption that it
is constant in this frequency range (25, 30) (Table II). The
C values thus obtained for both the human enzyme (1.8 (±0.3) × 10
10 s) and its enzyme-substrate complex (1.25 (±0.2) × 10
10 s) seem to be dominated by
S, the electron-spin relaxation time for the high-spin
Fe(III) center (18, 25, 27). For the rat PAH a frequency dependence of
the relaxation rates was only observed for the enzyme-substrate
complex, yielding a
C value of 4.2 (±0.5)·10
10 s (18). These
C values
should be considered as an approximation, because they were calculated
from measurements at high field (
100 MHz), where dispersion of
magnetization may occur (24).
C (see Refs. 24 and
25, and Equation 1). Assuming that the paramagnetic contribution to the
relaxation rate of the bulk water is mainly because of the exchange of
one of the water molecules coordinated to the paramagnetic ion
(q = 1, Eq. 2), T1M is
calculated to be 6.6 × 10
6 s and the estimated
distance (r) between the iron and the water protons
(averaged distance for the two protons) is 3.0 ± 0.3 Å (Eq. 1).
Assuming that two (q = 2) or three (q = 3) of the water molecules coordinated to the Fe(III) (12) contribute
equally to the relaxation of the bulk water r increases to
3.4 ± 0.4 Å and 3.7 ± 0.4 Å, respectively. Thus, our data
fit best with the enhancement of solvent bulk water proton relaxation
rates being due to one or two of the coordinated water molecules
transferring the paramagnetic effects to the bulk water through
exchange. These water molecules are most likely Wat (1) and Wat (2),
the most mobile of the three coordinated water molecules to the Fe(III) in the crystal structure of the catalytic domain (12). Although Wat (1)
has a temperature factor that is slightly higher than that of Wat (2)
(48.7 Å2 versus 35.8 Å2), it is
hydrogen bonded (2.8 Å) to the hydroxyl group in the phenolic ring of
Tyr-325, whereas Wat (2) is not stabilized by any additional
interaction with the protein. Moreover, Wat (3) is hydrogen bonded (2.7 Å) to Glu-286 and has a low temperature factor (18.7 A2)
and probably contributes little to the transfer of paramagnetic effect
to the bulk water.
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ACKNOWLEDGEMENTS |
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We are very grateful to Professor Torgeir Flatmark and Dr. Anne Døskeland for valuable discussions. We thank Randi Svebak and Ali J. Sepulveda Muñoz for expert technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Research Council of Norway.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Science Institute, University of Iceland, Dunhaga
3, 107 Reykjavik, Iceland.
§ To whom correspondence should be addressed. Tel.: 47-55586427; Fax: 47-55586400; E-mail: aurora.martinez{at}pki.uib.no.
2 K. K. Andersson, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
PAH, phenylalanine
hydroxylase;
Cs, subunit concentration;
CFe, iron concentration;
Ea, activation energy;
BH4, (6R)-L-erythro-tetrahydrobiopterin;
T1D1, diamagnetic contribution to
the longitudinal relaxation rate;
T1M
1, longitudinal paramagnetic
relaxation rate of the bound water protons;
T1P
1, paramagnetic contribution to
the longitudinal relaxation rate;
T1OBS
1, observed water proton
longitudinal relaxation rate;
TO.S.
1, outer-sphere contribution
to the relaxation rate;
C, dipolar correlation time;
M, exchange lifetime;
S, electron spin
relaxation rate;
I and
S, proton and
electron Larmor frequencies, respectively.
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REFERENCES |
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