Betaineprotectsurea-induceddenaturationof myosin
subfragment-1
Susana Ortiz-Costa
1,2
, Martha M. Sorenson
2
and Mauro Sola-Penna
1
1 Laborato
´
rio de Enzimologia e Controle do Metabolismo, Departamento de Fa
´
rmacos, Universidade Federal do Rio de Janeiro, Brazil
2 Instituto de Bioquı
´
mica Me
´
dica, Universidade Federal do Rio de Janeiro, Brazil
Osmolytes are osmotically active solutes that can be
grouped into four major classes: polyols (sugar and
sugar derivatives), amino acids (and their derivatives),
methyl ammonium compounds and urea [1,2]. Often,
they are further classified as ‘compatible’ or ‘counter-
acting’ on the basis of their ability to affect the func-
tional activity of proteins. Compatible osmolytes
increase protein stability against denaturation with
little or no effect on their function [1–20], whereas
counteracting osmolytes have, in addition, the ability
to offset the deleterious effects of denaturants on the
catalytic activity [1,3,6–8,11–13,17–25]. The counteract-
ing solute effect can be viewed as a situation in which
the tendency of a denaturant to unfold the protein
structure is counterbalanced by a tendency of the sol-
ute to minimize the protein surface area in contact
with water [18–20,25].
Myosin (EC 3.6.4.1) belongs to the restricted family
of motor proteins that transform the chemical energy
of ATP into mechanical work. The motor function of
myosin is located in its two globular heads, which can
be separated by proteolysis as subfragment-1 (S1),
equipped with separate sites for actin binding and
hydrolysis of ATP [26]. Crystallographic determination
of the S1 structure and electron microscopic studies
have contributed a great deal to our understanding of
the structure and mapping of the functional subdo-
mains [27,28]. Despite the extensive knowledge about
the structure, our understanding of the folding path-
way is limited, and experiments on protein denatur-
ation–renaturation may help us to understand how a
multidomain oligomeric protein, such as the myosin
head, folds, and how its subunits and domains acquire
stability.
The portion ofmyosin studied in this work is the
myosin head or myosin S1 obtained after chymotryptic
proteolysis. It consists of two nonidentical essential
light chains with a molecular mass of 17–23 kDa and
Keywords
methylamine; muscle; myosin; protection;
urea
Correspondence
M. Sola-Penna, Laborato
´
rio de Enzimologia
e Controle do Metabolismo (LabECoM),
Departamento de Fa
´
rmacos, Faculdade de
Farma
´
cia, Universidade Federal do Rio de
Janeiro, Ilha do Funda˜o, Rio de Janeiro –
RJ, 21941-590, Brazil
Fax ⁄ Tel: +55 21 2260 9192 ext 251
E-mail: maurosp@ufrj.br
(Received 30 January 2008, revised 19
March 2008, accepted 30 April 2008)
doi:10.1111/j.1742-4658.2008.06487.x
We have demonstrated previously that urea inhibits the activity and alters
the tertiary structure of skeletal muscle myosin in a biphasic manner. This
was attributed to differential effects on its globular and filamentous por-
tion. The inhibition of catalytic activity was counteracted by methylamines.
With the aim of comprehending the effects of urea on the catalytic (globu-
lar) portion of myosin, this study examines the effects of urea and the
countereffects ofbetaine on the catalytic activity and structure of myosin
subfragment-1. It is shown that urea inactivates subfragment-1 in parallel
with its ability to induce exposure of the enzyme hydrophobic domains, as
assessed using intrinsic and extrinsic fluorescence. Both effects are counter-
acted by betaine, which alone does not significantly affect subfragment-1.
Urea also enhances the accessibility of thiol groups, promotes aggregation
and decreases the a-helix content of S1, effects that are also counteracted
by betaine. We conclude that urea-induced inactivation of the enzyme is
caused by partial unfolding of the myosin catalytic domain.
Abbreviations
bis-ANS, 4,4¢-bis(1-anilinonaphthalene 8-sulfonate); Nbs
2,
5,5¢-dithiobis(2-nitrobenzoic acid); nd-PAGE, nondenaturing PAGE; S1, subfragment-1
of skeletal muscle myosin II; TMAO, trimethylamine-N-oxide.
3388 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS
one heavy chain (approximately 95 kDa). In a previ-
ous study [29], we investigated the counteracting effects
of methylamines against urea-induced changes in skele-
tal muscle myosin. We showed that myosin loses its
ATPase activity when the assay is performed in the
presence of 2 m urea. However, when the assay is per-
formed in the presence of 2 m urea combined with 1 m
methylamine, the myosin ATPase activity is preserved.
Nevertheless, these modifications in ATPase activity by
urea were not directly correlated with the modifica-
tions in myosin structure detected by intrinsic fluores-
cence spectroscopy [29]. In the present work, we
studied the ability ofbetaine (an amino acid derivative
of methylamine) to counteract the effects of urea on
the function and structure of S1. Our purpose was to
study, in more detail, the protective effect of betaine
on the function of skeletal muscle myosin, and, for
this, we used the motor domain (S1). We investigated
the effects of urea and betaine on K
+
EDTA ATPase
activity, intrinsic fluorescence emission, binding of the
hydrophobic fluorescent probe 4,4¢-bis(1-anilinonaph-
thalene 8-sulfonate) (bis-ANS), aggregation, exposure
of thiol groups and far-UV CD of S1. The addition of
urea revealed that slight changes in the structure of S1
could account for the loss of its functional activity. In
addition, betaine was able to protect against these
small changes in structure, thereby preserving the
enzyme’s catalytic activity.
Results and Discussion
The effects of urea and betaine on S1 ATPase activity
were evaluated (Fig. 1). Urea inhibits S1 ATPase activ-
ity in a dose-dependent manner (Fig. 1A, open circles),
presenting an I
0.5
value of 1.32 ± 0.04 m and a coop-
erativity index (n) of 5.3 ± 0.9, calculated as described
in Materials and methods. When the same experiment
is performed in the presence of 1 m betaine, the inhibi-
tory effects of urea are attenuated (Fig. 1A, filled cir-
cles): I
0.5
increases to 2.11 ± 0.04 m (P < 0.05,
Student’s t-test) without a significant change in the
cooperativity index (n) (6.7 ± 1.0; P > 0.05, Student’s
t-test). These results are similar to those obtained for
the effects of urea and betaine on native myosin [29].
Thus, although urea promotes significant changes in
the myosin rod [29], the observed effects on ATPase
activity are exclusively the result of alterations in the
globular head domain.
The effects of 2 m urea on S1 ATPase activity are
counteracted by increasing concentrations of betaine
(Fig. 1B, filled squares). Betaine alone promotes a
slight decrease in activity, so that the remaining activ-
ity is 93.6 ± 0.9% and 68.7 ± 0.5% of the original
value in the presence of 1 and 2 m betaine, respectively
(Fig. 1B, open squares). There is no significant differ-
ence in the S1 ATPase activity when the enzyme is
incubated with 1–2 m betaine in the absence or pres-
ence of 2 m urea, indicating that the denaturant has no
effect on the enzyme catalytic activity in the presence
of these concentrations of betaine, consistent with the
known counteracting effects of methylamines on
urea-induced inactivation and denaturationof other
proteins using a 2 : 1 concentration ratio of urea to
methylamines [1].
In order to understand the effects of urea on S1
ATPase activity at the structural level, we measured
the intrinsic fluorescence emission of S1 in the presence
of increasing concentrations of urea (Fig. 2). The
intrinsic fluorescence of S1 is altered in the presence of
increasing concentrations of urea (Fig. 2A). Urea pro-
motes a dose-dependent decrease in fluorescence inten-
sity which can be evaluated by means of the spectral
Fig. 1. Effects of urea and betaine on S1 ATPase activity. S1 ATPase activity was assessed as described in Materials and methods in the
presence of the concentrations of urea and betaine indicated for each panel. Plotted values are the mean ± SE of at least four independent
experiments performed in duplicate. Full lines are the results of nonlinear regression as described in Materials and methods.
S. Ortiz-Costa et al. Betaineprotectssubfragment-1 against urea
FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3389
area (Fig. 2B, open circles). The I
0.5
value calculated
for this phenomenon is 1.34 ± 0.09 m, which is not
statistically different from the I
0.5
value calculated for
the urea-induced inhibition of S1 ATPase (P > 0.05,
Student’s t-test), suggesting that the effects of urea on
the enzyme activity are a result of alterations in its
structure. However, the presence of 1 m betaine does
not counteract the effects of urea on S1 fluorescence
(Fig. 2B, filled circles): the calculated value of I
0.5
is
1.45 ± 0.11 m, not statistically different from the
value obtained in the absence ofbetaine (P > 0.05,
Student’s t-test). The center of mass of the intrinsic
fluorescence spectrum is not affected by urea up to
4 m, being shifted to the red region of the spectrum in
the presence of 8 m urea (Fig. 2B, inset). This red shift
is also not counteracted by 1 m betaine (data not
shown).
The center of mass of intrinsic fluorescence spectra
has been used to evaluate protein unfolding or dissoci-
ation [9,16,17,30–33]. However, in many cases, changes
in this parameter do not correlate with changes in
enzyme catalytic activity, which decreases under condi-
tions in which no changes are observed in the center of
mass [7–12,16]. Nevertheless, the attenuation of the flu-
orescence intensity is generally correlated with the
exposure of protein tryptophan residues to a more
polar environment, thus affording a means of evaluat-
ing perturbations in tertiary structure [34]. Our data
clearly show that the effects of urea on S1 ATPase
activity occur in parallel with its effects on the intrinsic
fluorescence emission. Despite the coincident I
0.5
values
for the effects of urea on both the ATPase activity and
intrinsic fluorescence emission, the fact that betaine
does not counteract the decrease in fluorescence emis-
sion does not support the hypothesis that urea inhibi-
tory effects are only a result of the exposure of
tryptophan residues.
As a means of evaluating other tertiary structural
changes related to the effects of urea on S1 ATPase
activity and its counteraction by betaine, we assessed
the binding of bis-ANS. This fluorescent dye binds to
distinct hydrophobic regions of S1, including its cata-
lytic ATP binding site [35], increasing the fluorescence
AB
Fig. 2. Area and center of mass of intrinsic fluorescence spectra of S1 in the presence of urea and betaine. Experiments were performed
as described in Materials and methods. (A) Intrinsic fluorescence spectra of S1 in the absence (control) and presence of the concentrations
of urea indicated in the figure. This is a representative experiment of a series of four independent experiments. (B) Calculated spectral area
of S1 in the presence of the concentrations of urea indicated on the abscissa, in the absence (filled circles) and presence (open circles) of
1
M betaine. Experiments shown in (A) and (B) are representative of a series of four experiments presenting similar results. Inset: center
of mass of S1 intrinsic fluorescence spectra obtained in the presence of the concentrations of urea indicated on the abscissa. Plotted values
are the mean ± SE of four independent experiments.
Fig. 3. bis-ANS binding to S1 in the presence of urea and betaine.
Experiments were performed as indicated in Materials and meth-
ods in the presence of the concentrations of urea indicated on the
abscissa with and without 1
M betaine. Plotted values are the
mean ± SE of four independent experiments. Inset: representative
spectrum of bis-ANS alone (dotted line) or in the presence of S1
(full line).
Betaine protectssubfragment-1 against urea S. Ortiz-Costa et al.
3390 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS
emission of the dye (Fig. 3, inset: dotted line, without
S1; full line, with S1). Urea up to 2 m induces a dose-
dependent increase in bis-ANS fluorescence, which is
not observed in the presence of 1 m betaine (Fig. 3).
This result indicates that urea is promoting the expo-
sure of S1 hydrophobic domains, allowing bis-ANS to
bind to the protein. This phenomenon is prevented by
the presence of 1 m betaine in the medium, which pro-
tects the S1 tertiary structure against this unfolding
promoted by urea. Higher concentrations of urea
(3–8 m) induce a progressive decrease in the bis-ANS
fluorescence (Fig. 3), indicating that these urea concen-
trations are disrupting S1 hydrophobic domains. The
presence of 1 m betaine does not counteract this effect,
and the fluorescence signal decreases in the presence
and absence of the methylamine (Fig. 3).
The exposure of protein hydrophobic groups to a
polar environment often favors the formation of pro-
tein aggregates. In order to assess this possibility, S1
was analyzed by electrophoresis by nondenaturing
PAGE (nd-PAGE) in the absence and presence of urea
and betaine. In the absence of additives, nd-PAGE
reveals a single band at the top of the 6% gel (Fig. 4,
lane 1). In the presence of increasing concentrations of
urea (0.5–8 m), this band is progressively replaced by a
band at the top of the 3.6% gel, indicating the forma-
tion of aggregates that do not enter the 3.6% gel
(Fig. 4, lanes 2–7). Betaine, which alone does not sig-
nificantly alter the nd-PAGE pattern of S1 (Fig. 4,
lanes 13–15), at a 2 : 1 concentration ratio of urea to
betaine, partially protects S1 from the urea-induced
formation of aggregates (Fig. 4, lanes 9, 11 and 12).
Comparing lane 5 (in the presence of 2 m urea) and
lane 9 (in the presence of 2 m urea and 1 m betaine), it
can be seen that the S1 aggregate formed at the top of
the 3.6% gel in lane 5 is not present in lane 9. Con-
versely, the loss of native S1 at the top of the 6% gel
in lane 5 does not occur in lane 9. Similar results are
obtained with 4 m urea in the absence (lane 6) and
presence (lane 12) of 2 m betaine, and with 8 m urea in
the absence (lane 7) and presence (lane 11) of 4 m
betaine. It is important to note that, in the presence of
1or4m urea, both with 1 m betaine, the electropho-
retic patterns are no different from the results obtained
in the presence of 1 or 4 m urea alone, indicating the
need for a 2 : 1 concentration ratio of urea to betaine.
Taken together, these results indicate that betaine pre-
vents S1 from the unfolding and aggregation induced
by urea.
In order to better understand the kinetics of urea-
induced S1 aggregation, we performed light scattering
measurements of S1 as a function of incubation time in
the presence of 1 m betaine, 2 m urea or both together
in the medium. Corroborating the results presented in
Fig. 4, light scattering experiments revealed that S1
aggregates in the presence of 2 m urea (Fig. 5, open
triangles), an effect that is counteracted by the simul-
taneous presence of 1 m betaine (Fig. 5, filled triangles).
A small but significant increase in light scattering can be
observed when S1 is incubated in the presence of 1 m
betaine alone (Fig. 5, open circles). From a comparison
of lanes 1 (control) and 13 (1 m betaine) in Fig. 4, it can
be seen that a weak band is visualized in the 3.6% gel,
which was neglected when analyzing this result alone.
Nevertheless, the results presented in Fig. 5 suggest that
betaine induces, to a much lesser extent when compared
Fig. 4. nd-PAGE of S1 in the presence of urea and betaine. Experi-
ments were performed as described in Materials and methods in
the presence of the urea and betaine concentrations indicated
in the figure. This is a representative experiment of a series of
three.
Fig. 5. Light scattering of S1 in the presence of urea and betaine.
Experiments were performed as indicated in Materials and meth-
ods in the presence of the concentrations of urea indicated in the
figure and in the absence (filled circles, open triangles) or presence
(open circles, filled triangles) of 1
M betaine. Plotted values are the
mean ± SE of four independent experiments.
S. Ortiz-Costa et al. Betaineprotectssubfragment-1 against urea
FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3391
with urea, the aggregation of S1. This aggregation is
probably not caused by the same mechanism as that of
urea as, under the same conditions, S1 is still active in
the presence of betaine, but not in the presence of urea
alone (see Fig. 1). Moreover, it is possible that the beta-
ine-induced aggregation of S1 helps protect the enzyme
against the deleterious effects of urea, preventing the lat-
ter osmolyte from interacting with some domains of S1.
It has been shown that guanidinium hydrochloride-
induced unfolding of S1 increases the accessibility of
the reactive thiol groups of the enzyme prior to its
complete unfolding [36]. In order to determine whether
betaine counteracts the initial steps ofurea-induced S1
unfolding, we assessed the effects of urea on the acces-
sibility of S1 thiol groups to 5,5¢-dithiobis(2-nitroben-
zoic acid) (Nbs
2
) (Fig. 6). Urea increases the
accessibility of S1 thiol groups, in a dose-dependent
manner, from 2 molÆmol
)1
in the absence of urea to
4 molÆmol
)1
in the presence of 2 m urea (Fig. 6, open
circles). These results are similar to those obtained
with guanidinium chloride [36], and are consistent with
the concept that the effects of urea on S1 catalytic
activity are a result of the initial stages of unfolding of
the enzyme. Moreover, 1 m betaine totally prevents
this unfolding effect of 2 m urea (Fig. 6, filled circles),
consistent with its protection of S1 activity at this con-
centration (Fig. 1A). So far, our findings reveal that
urea inactivates S1 ATPase as a result of the initial
changes in the enzyme tertiary structure promoted by
this compound. Furthermore, both structural and
catalytic changes are counteracted by betaine at a 2 : 1
concentration ratio of urea to betaine.
The effects of urea and methylamines on the acces-
sibility of thiol groups have been evaluated by
Yancey and Somero [37] using another enzyme:
bovine liver glutamate dehydrogenase. These authors
found similar results, with urea increasing the accessi-
bility of thiol groups and trimethylamine-N-oxide
(TMAO) counteracting this effect. Nevertheless, they
showed that TMAO alone diminished the accessibility
of thiol groups in the enzyme, an effect that was not
observed using betaine and S1 (see Fig. 6). However,
in our previous work, we have found that TMAO is
more effective than betaine in protecting myosin
activity against the deleterious effects of urea [29].
Although we did not observe any difference between
the effects ofbetaine and TMAO on the myosin fluo-
rescence spectra, Go
¨
ller and Galinsky [38], studying
the rabbit muscle lactate dehydrogenase, demon-
strated that TMAO was more effective than betaine
in protecting the enzyme against denaturation, as
measured by intrinsic fluorescence. These results sug-
gest that, as stated for the protection conferred by
carbohydrates on many enzymes [8], the degree of
protection provided by distinct methylamines depends
on the nature of the enzyme, and must be evaluated
on a case-by-case basis.
In order to investigate whether urea and betaine
affect the secondary structure of S1, we recorded the
far-UV CD spectra from S1 in the presence of urea
and betaine. Figure 7A shows that increasing concen-
trations of urea progressively decrease the a-helix con-
tent of S1, as observed at 222 nm. Betaine alone does
not significantly alter the S1 CD spectrum, although it
counteracts the effect of urea observed through this
technique (Fig. 7B). About one-half of the total effect
of urea on the ellipticity at 222 nm occurs when urea
is increased to 2 m (Fig. 7A, inset) and, at this concen-
tration of urea, 1 m betaine (purple triangle) blocks
the effect. These results indicate that folding intermedi-
ates may be formed during urea titration into the solu-
tion containing S1, and that these intermediates may
not be produced in the presence of betaine. Thus, beta-
ine counteracts the effects of urea by preventing the
formation of folding intermediates which favor the
denaturation of the enzyme.
Together, our results indicate that urea-induced S1
inactivation and its counteraction by betaine are
caused by changes in the S1 structure at secondary
and tertiary levels. Although many reports have not
revealed a direct correlation of distinct enzyme inacti-
vation and unfolding [7–12,36,39–41], the present work
shows that, for myosin S1, this correlation is quite
precise, at least for secondary structure and early
unfolding steps. Moreover, it is clear from the current
Fig. 6. Nbs
2
titration of S1 in the presence of urea and betaine.
Experiments were performed as indicated in Materials and meth-
ods in the presence of the concentrations of urea indicated on the
abscissa and in the absence (open circles) or presence (filled
circles) of 1
M betaine. Plotted values are the mean ± SE of four
independent experiments.
Betaine protectssubfragment-1 against urea S. Ortiz-Costa et al.
3392 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS
results that intrinsic fluorescence measurements may
not be an accurate technique to evaluate the initial loss
of S1 tertiary structure, which is better assessed by
other methods, such as the accessibility of thiol
groups, extrinsic fluorescence and nondenaturing gel
electrophoresis. Thus, we conclude that the protection
granted by betaine against S1 inactivation by urea is a
result of the prevention of secondary structure loss
and the initial unfolding process.
Materials and methods
Materials
Urea, betaine, ATP (Grade I), a-chymotrypsin (type I-S),
Nbs
2
, phenylmethanesulphonyl fluoride, Hepes and the
dialysis membranes were obtained from Sigma (St Louis,
MO, USA). The DE-52 anion-exchange resin was from
Whatman (Clifton, NJ, USA). All other reagents used were
of analytical grade.
[
32
P]Pi was obtained from Instituto de Pesquisas Energe
´
t-
icas e Nucleares (Sa
˜
o Paulo, Brazil). [c-
32
P]ATP was
purchased from New England Nuclear (Boston, MA, USA)
or synthesized as described in [42].
Protein purification
Skeletal muscle myosin was prepared from chicken breast
according to [43]. The purity (> 99%) was checked by
SDS-PAGE [44]. Purified myosin was stored at )20 °Cina
solution consisting of 300 mm KCl, 50 mm imidazole
(pH 7.0), 1 mm dithiothreitol and 50% (v ⁄ v) glycerol.
Before use, the protein was diluted 12-fold in ice-cold
water, centrifuged and resuspended in 600 mm KCl and
50 mm imidazole (pH 7.0).
S1 was prepared from purified myosin by digestion with
chymotrypsin for 10 min at room temperature [45]. Phen-
ylmethanesulphonyl fluoride was added to stop the reaction
and the suspension was centrifuged to remove the rod
region. The supernatant was loaded onto a Whatman
DE-52 column (3.5 cm · 20 cm) equilibrated with 50 mm
imidazole buffer (pH 7.0) and 1 mm dithiothreitol, and
eluted with a linear NaCl gradient (0–200 mm NaCl ⁄ 50 mm
imidazole, pH 7.0). Fractions containing the cleaved heavy
chain ($ 95 kDa), together with light chain 1 or light
chain 3, were pooled and concentrated nearly 15-fold by
ultrafiltration under nitrogen across an Amicon YM 30
membrane (Millipore, Billerica, MA, USA). After dialysis
against 50 mm KCl, 10 mm imidazole (pH 7.0), 20 mm
potassium phosphate buffer (pH 7.0), 0.2 mm EDTA and
1mm dithiothreitol, the protein was diluted to 50% using
glycerol and stored under liquid nitrogen in small aliquots.
Before use, S1 was dialyzed against 50 mm KCl containing
50 mm imidazole or 50 mm Hepes (pH 7.0) for measurement
of ATPase activity or fluorescence, respectively, and was
centrifuged for 25 min at 350 000 g to eliminate aggregates.
The myosin concentration was estimated by the biuret
assay [46]. S1 was determined spectrophotometrically,
assuming an absorption coefficient of A
1%
280
= 7.5 cm
)1
.
ATPase activity
The assay medium used to measure the K
+
EDTA ATPase
activity of S1 contained 0.05 mgÆmL
)1
protein, 3 mm ATP,
600 mm KCl, 50 mm imidazole (pH 7.0) and 5 mm EDTA.
The hydrolysis of ATP was measured at 30 °C after
10 min of incubation with osmolytes (urea or betaine). The
effect of increasing urea concentration was measured in the
absence or presence of 1 m betaine. The reactions were
started by the addition of [c-
32
P]ATP and quenched with
activated charcoal suspended in 0.1 m HCl. The reaction
rate was measured by taking samples between 2 and 8 min
on the linear portion of the progress curve. After centrifu-
gation, an aliquot containing [
32
P]Pi was applied to filter
paper, dried and counted for radioactivity in a liquid
A
B
Fig. 7. CD spectra of S1 in the presence of urea and betaine.
Experiments were performed as indicated in Materials and meth-
ods in the presence of the concentrations of urea or betaine indi-
cated in the figure. Inset: ellipticity measured at 222 nm in the
absence (black circle) or presence of 0.5 (red circle), 1 (green cir-
cle), 2 (yellow circle), 4 (blue circle) or 8
M (magenta circle) urea,
1
M betaine (cyan triangle) or both 2 M urea and 1 M betaine (purple
triangle). This is a representative experiment of a series.
S. Ortiz-Costa et al. Betaineprotectssubfragment-1 against urea
FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3393
scintillation counter. The velocity of hydrolysis was calcu-
lated by linear regression. Appropriate blanks were carried
out in duplicate as described previously [29].
Kinetic parameters for the inhibition of S1 ATPase activ-
ity by urea were calculated by fitting the experimental data
to obtain the parameters of the following equation:
R
a
¼
100 Â I
n
0:5
I
n
0:5
þ½urea
n
ð1Þ
where R
a
is the residual relative ATPase activity at a given
concentration of urea ([urea]), I
0.5
is the [urea] value that
promotes 50% of maximal inhibition and n is the cooper-
ativity index for this phenomenon.
Fluorescence experiments
The intrinsic fluorescence measurements were carried out
using an F-4500 spectrofluorimeter (Hitachi, Tokyo, Japan).
The excitation wavelength was 280 nm, and emission spec-
tra were recorded between 300 and 400 nm. Assays were
performed at 30 °C in a medium containing 0.05 mgÆmL
)1
S1, 600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA
in the absence or presence of increasing concentrations of
each osmolyte (urea or betaine) or increasing urea concen-
trations combined with 1 m betaine. The osmolytes (solid
powder) were added to the same sample in increasing quan-
tities, followed by a 10 min incubation period in which no
further changes were observed, as described previously [29].
The fluorescence intensity and osmolyte concentrations were
corrected for changes in sample volume after addition of the
chemical agent.
The spectral area and spectral center of mass of the fluo-
rescence spectra were calculated using sigmaplot 10.0
(Systat, San Jose, CA, USA), employing the following
equations:
Area ¼
X
k  I
k
ð2Þ
and
Centre of mass ¼
X
ðk  I
k
Þ=
X
I
k
ð3Þ
where S is the sum over all wavelengths (k) 300–400 nm
and I
k
is the fluorescence intensity at a given k [29–32,34].
In order to evaluate the binding of bis-ANS to S1,
2.5 lm of the dye was added to the same buffer as used for
the intrinsic fluorescence measurements with 0.5 lm protein
(0.05 mgÆmL
)1
). The excitation was set at 360 nm and the
fluorescence emission was measured in the range 400–
600 nm; measurements were performed 10 min after all
additions.
Light scattering experiments
Light scattering measurements were performed in a Jasco
spectrofluorimeter (Jasco Corporation, Tokyo, Japan) in the
same medium as described above for intrinsic fluorescence,
in the absence and presence of 2 m urea, 1 m betaine or
both. Appropriate reference spectra were subtracted from
the data to correct for background interference, which was
always less than 5% of the total signal. The incident light
wavelength was set at 510 nm, and scattering was recorded
at 90° between 500 and 520 nm. The spectral area was cal-
culated using Eqn (2), and plotted values represent the area
of the light scattering experiment at each incubation time
divided by the area obtained before the additions.
Reactivity of thiol groups
The reactivity of thiol groups in S1 was measured by moni-
toring, at 405 nm, the time course of reaction of the protein
(2.5 lm) with Nbs
2
(90-fold excess over S1) in the presence
of 600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA
at 30 °C. The reaction was followed for 20 min, at 6 s
intervals, using a Thermomax Microplate Reader (Molecu-
lar Devices, Menlo Park, CA, USA). For urea denaturation
studies and counteracting effects, stock solutions of 10 m
urea and 5 m betaine, prepared in the same buffer as
described above, were varied to give the appropriate final
osmolyte concentrations.
CD spectra
CD spectra were recorded on a Jasco J-715 (Jasco Corpora-
tion). The results were expressed as the mean residue molar
ellipticity [h] (degÆcm
2
Ædmol
)1
) and calculated using the
following equation:
½h¼ð½h
obs
 MRWÞ=ð10lcÞð4Þ
where [h]
obs
is the observed ellipticity (mdeg), MRW is the
mean residue molecular mass (molecular mass of the poly-
peptide divided by the number of amino acid residues), c is
the peptide concentration (mgÆmL
)1
) and l is the optical
path length (0.1 cm). CD spectra were the average of three
scans obtained by collecting data at 0.2 nm intervals from
260 to 200 nm. Stock protein solutions were prepared in
600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA.
Stock solutions of 10 m urea and 5 m betaine were pre-
pared in the same buffer. The ratios of buffer and osmolyte
solutions added were varied to give the appropriate final
osmolyte concentrations, and protein stock solution was
added to give a final concentration of 0.05 mgÆmL
)1
(0.5 lm).
Nondenaturing electrophoresis
nd-PAGE was performed in a Mini-Protean 3 system (Bio-
Rad Laboratories, Hercules, CA, USA) according to [47].
Stacking (3.6% polyacrylamide) and running (6% poly-
acrylamide) gels were prepared according to [47]. S1 samples
Betaine protectssubfragment-1 against urea S. Ortiz-Costa et al.
3394 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS
were pre-incubated for 10 min in the absence (control) or
presence of distinct concentrations of urea and ⁄ or betaine,
as indicated; 15 lg of protein was submitted to electro-
phoresis for 1.5 h at constant voltage (200 V). After the
runs, samples were stained using Coomassie blue dye, as
described previously [48].
Acknowledgements
This work was supported by grants from the Conselho
Nacional de Desenvolvimento Cientı
´
fico e Tecnolo
´
gico
(CNPq), Fundac¸ a
˜
o Carlos Chagas Filho de Amparo a
Pesquisa do Estado do Rio de Janeiro (FAPERJ),
Fundac¸ a
˜
o Ary Frausino ⁄ Fundac¸ a
˜
o Educacional
Chales Darwin (FAF⁄ FECD Programa de Oncobiolo-
gia) and Programa de Nu´ cleos de Exceleˆ ncia (PRO-
NEX).
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Betaine protectssubfragment-1 against urea S. Ortiz-Costa et al.
3396 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS
. Betaine protects urea-induced denaturation of myosin
subfragment-1
Susana Ortiz-Costa
1,2
, Martha M aim of comprehending the effects of urea on the catalytic (globu-
lar) portion of myosin, this study examines the effects of urea and the
countereffects of