A comparison of the urea-induced unfolding of apo¯avodoxin
and ¯avodoxin from
Desulfovibrio vulgaris
Brian O
Â
Nuallain* and Stephen G. Mayhew
Department of Biochemistry, University College Dublin, Bel®eld, Dublin, Ireland
1
The kinetics and thermodynamics of the urea-induced
unfolding of ¯avodoxin and apo¯avodoxin from Desulfo-
vibrio vulgaris were investigated by measuring changes in
¯avin and protein ¯uorescence. The reaction of urea with
¯avodoxin is up to 5000 times slower than the reaction with
the apoprotein (0.67 s
)1
in 3
M
urea in 25 m
M
sodium
phosphate at 25 °C), and it results in the dissociation of
FMN. The rate of unfolding of apo¯avodoxin depends on
the urea concentration, while the reaction with the holo-
protein is independent of urea. The rates decrease in high salt
with the greater eect occurring with apoprotein. The ¯uo-
rescence changes ®t two-state models for unfolding, but they
do not exclude the possibility of intermediates. Calculation
suggests that 21% and 30% of the amino-acid side chains
become exposed to solvent during unfolding of ¯avodoxin
and apo¯avodoxin, respectively. The equilibrium unfolding
curves move to greater concentrations of urea with increase
vibrio desulfuricans and Clostridium beijerinckii,anda
second group that has about 20 amino acids inserted in
one strand of the b sheet, and that includes proteins from
Azotobacter vinelandii and Anabaena PCC 7119. The
¯avodoxin fold is shared by a range of un related proteins
(nine superfamilies) with different functions [3].
The FMN of ¯avodoxins can be reversibly removed with
acid. The resulting apoproteins are stable, and it has been
proposed that they are useful models to investigate the
folding/unfolding reactions of a/b proteins [4±8]. However,
their ability to bind ¯avin is a property that has yet to be
explored in the context of protein folding, and it is like ly that
they will also prove to be useful a s models for folding of a/b
proteins that require a tightly bound organic cofactor for
activity. An early study showed that guanidine HCl disso-
ciates ¯avodoxin from Clostridium pasteurianum into
apo¯avodoxin and FMN [9]. M ore recently, this denaturant
has b een used to study unfolding of the apoproteins of
¯avodoxins from A. vinelandii [6±8] and D. desulfurican s
[10], and similar studies have been carried out with
apo¯avodoxin from Anabaena PCC 7119 but using urea
as the denaturant [4,5]. In the ®rst two cases, evidence was
obtained for a stable intermediate in the equilibrium
between the folded and unfolded states. In contrast, urea
causes apo¯avodoxin from Anabaena to unfold directly
without stabilizing an intermediate. The study with apo-
¯avodoxin from D. desulfuricans was the only one to
investigate t he effect of bound FMN on the unfolding
equilibrium. It w as concluded that FMN has no effect on
the stability of the protein, and that FMN remains tightly
protein and apoprotein. A mechanism is proposed for
the unfolding/folding reactions of the t wo forms o f the
protein in urea. This ¯avodoxin resembles ¯avodoxin from
D. desulfuricans both in amino-acid sequence (148 amino
acids) and in three-dimensional structure [11,12]. H owever,
we ®nd that the effects of denaturant on the ¯avodoxin and
apo¯avodoxin from D. vulgaris are s urprisingly d ifferent
from those reported for the protein from D. desulfuricans
[10].
MATERIALS AND METHODS
Preparation and estimation of ¯avodoxin
and apo¯avodoxin
Flavodoxin from D. vulgaris was obtained as the recombi-
nant protein that was puri®ed from extracts of E. coli [13].
Apo¯avodoxin was prepared by acid precipitation [14]. The
protein precipitate was dissolved in 25 m
M
sodium phos-
phate and 0.3 m
M
EDTA, pH 7.0 (buffer A), and dialysed
against the same buffer. The concentrations of holoprotein
and apoprotein were determined using absorption coef®-
cients at 458 nm (10 700
M
)1
ácm
)1
) and at 280 nm
(22 400
cence at 525 nm with excitation at 445 nm (see below).
Determination of unfolding rate constants
Studies on the rate of protein unfolding were carried out by
diluting a stock solution of ¯avodoxin or apo¯avodoxin in
buffer A at least 100-fold to obtain 1 l
M
protein in urea
(0.1±10
M
). The relatively rapid unfolding of apo¯avodoxin
in urea and 25 m
M
sodium phosphate buffer pH 7 (buffer B)
was measured by using a stopped-¯ow spectro¯uorimeter
that consisted of a Rapid Kinetics Spectrometer Accessory
(Applied Photophysics Ltd; RX-1000) interfaced to the
optical system of a Baird Nova ¯uorimeter, a home-made
Fig. 1. Structure o f ¯avodoxin from
D. vulgaris showing the relative positions of
the FMN, tryptophan side chains and tyrosine
side chains. TheFMNisshowninyellow,
tryptophan side chains in magenta, and
tyrosine side chains in green. The ®gure was
produced with
RASMOL
.
Ó FEBS 2002 Flavodoxin unfolding (Eur. J. Biochem. 269) 213
signal ampli®er, an oscilloscope (Hameg Instruments 203-7)
and a digital storage adaptor (Thurlby±Thandor DSA524).
Progress curves for the unfolding of ¯avodoxin under all
was followed by a further very slow change in the protein
and ¯avin ¯uorescence from the holoprotein, and of the
protein ¯uorescence in experiments with the apoprotein, at
rates that were insigni®cant compared with the initial
reaction. The rates of the slow reactions were not affected by
the concentration of u rea (0.6±5
M
) or by c hanging the
phosphate buffer concentration in the range 25±250 m
M
,
indicating that these reactions are not associated with urea-
dependent unfolding. Therefore, the reactions were disre-
garded and the ¯uorescence at the end of the relatively rapid
phase of ¯uorescence change was taken as a measure of the
equilibrium between folded and unfolded protein.
The positions of the unfolded/fo lded equilibria of ¯avo-
doxin and apo¯avodoxin at different concentrations of urea
were also measured in refolding experiments. The protein
(50 l
M
) was unfolded in buffer B and 6
M
urea as described
above. It was then diluted 50-fold into buffer B and urea
(0±6
M
). Refolding of the diluted protein was monitored
from the changes in ¯avin and/or protein ¯uorescence.
Analysis of the equilibrium between folded
DG
D
ÀRT ln
U
F
and
U
F
S
F
À S
S À S
U
2
where S is the observed ¯uorescence signal; S
F
and S
U
are
the ¯uorescence signals f or the f olded and unfolded protein,
respectively, and F and U are the proportions of the folded
and unfolded states; R is the gas constant; and T is the
temperature in K. T he urea-unfolding curves for ¯avodoxin
and apo¯avodoxin were analysed using an equation derived
by Santoro & Bolen (Eqn 3) [18] that incorporates Eqns (1)
and (2).
S
5
300 340 380 420
Fluorescence (arbitrary units)
Wavelength (nm)
1
2
3
4
Fig. 2. Fluorescence emission spectra of folded and unfolded ¯avodoxin
and apo¯avodoxin. (1) Folded apo¯avodoxin; (2) folded ¯avod oxin; (3)
unfolded ¯avodoxin; (4) unfolded a po¯avodo xin. The solutions con-
tained at 25 °C: 1 l
M
protein; 25 m
M
sodium phosphate, pH 7.0; and
for ( 3) and (4) 6
M
urea. T he spectra (3) and (4) were re corded after all
¯uorescence changes were complete. Fluorescence excitation was at
280 nm.
214 B. O
Â
Nuallain and S. G. Mayhew (Eur. J. Biochem. 269) Ó FEBS 2002
Eqn ( 3) gives empirically derived m and DG
W
values without
having to determine t he value for DG
D
at each concentration
a buried amino-acid side chain that occurs on unfolding of
the protein when the concentration o f denaturant is in®nite;
K
den
is an empirical constant that represents the concentra-
tion of denaturant at which half DG
s,m
is achieved. T he
values used for DG
s,m
(5.024 kJ mol
)1
)andK
den
(25.25
M
)
in urea were obtained from [19]. These values represent the
behaviour of an ÔaverageÕ protein, determined from the
solvent-excluded side chains of 55 proteins in the Protein
Data Bank, and using solvation e nergies of model com-
pounds in guanidine HCl and in urea [20±23].
Assuming that salt ions bind preferentially to the folded
state, the number of salt ions (NaCl) that are released from
apo¯avodoxin when the protein unfolds can be obtained by
®tting the unfolding curves to Eqn (5) [24].
Dln K
app
Da
10 min. A value for the dissociation rate constant (k
o
)was
determined from the progress curve. The dissociation of the
holoprotein can be described by Eqns (6) and (7).
FMN-apoprotein
ÀÀB
AÀÀ
k
off
k
on
FMN apoprotein 6
K
d
k
off
k
on
FMNapoprotein
FMN À apoprotein
x
2
e
a À x
e
7
e
À x
8
The values for x
e
, x, a,andt were obtained f rom each
progress curve. A p lot of the right hand side of Eqn (8) vs.
time gives a straight line whose slope is k
o
. Values for k
o
were determined from the average slope of the plots for
three measurements.
Values for the dissociation constant for the holoprotein
(K
d
) were calculated f rom the end point of the p rogress
curves using E qn (8). It was assumed that the concentrations
of apoprotein and free FMN in the equilibrium were the
same. As the experiments were carried out in a low
concentration of u rea, it was necessary to corre ct f or a
small proportion of unfolded apoprotein. This was calcu-
lated from the appropriate unfolding curves ®tted to Eqn
(3). Values for k
on
were then calculated by substituting the
values calculated for k
o
and K
d
phosphate concentration is increased from 25 m
M
to
250 m
M
, the rate constant decreases % 60-fold. Further-
more, when chloride ion was u sed to raise the ionic s trength
to the same value as that of 250 m
M
phosphate, t he decrease
in the rate constant for apo¯avodoxin was somewhat less,
indicating that the rate dep ends in addition on the nature of
the salt (Table 1). The rate constant increases exponentially
with increasing urea. Tanford [27] proposed that the rate
constant for unfolding of a protein in urea, k
u
, is related to
the rate constant in the absence of urea, k
w
,andtotheurea
concentration by Eqn (9).
ln k
u
lnk
w
m
u
urea9
Ó FEBS 2002 Flavodoxin unfolding (Eur. J. Biochem. 269) 215
where m
which the ®nal 20±30% of the progress curve was monit ored
suggest that the reaction in the presence of equimolar FMN
follows second-order kinetics (16.4 2.1 ´ 10
5
M
)1
ás
)1
).
The rate constan t was f ound to be similar to that observed
with apo¯avodoxin that had not been through the unfolding
procedure (14.1 3.1 ´ 10
5
M
)1
ás
)1
).
Plots of the extent of ¯uorescence change at equilibrium
vs. the concentration of urea also suggest that in the case of
this apo¯avodoxin a simple two-state transition occurs
between the folded and unfold ed protein (Fig. 5). Dilution
of the completely unfolded apoprotein with urea of different
concentrations showed that the ¯uorescence at equilibrium
mirrored that observed during unfolding with urea, and that
the unfolding is reversible (Fig. 5). The conformational
stability in buffer B, determined by ®tting the urea-
unfolding curve for 1 l
M
apo¯avodoxin, is 9.99 0.4
small increases with increasing phosphate, the main effect
being a shift of the transition midpoint. Similar but slightly
smaller increases in the stability of apo¯avodoxin are
observed when the ionic strength is increased with NaCl
(Fig. 5 ; Table 2). By assuming that the increase in stab ility
of apo¯avodoxin with salt is due to the preferential binding
of salt to the f olded p rotein, it can be calculated that
approximately two ions are released when the apoprotein
unfolds in urea (Fig. 5, inset). The values of m, DG
w
and the
concentration of urea to g ive half-unfolded apoprotein were
found to be independent of the protein concentration
(0.25±23 l
M
; Table 3).
Equilibrium unfolding curves for apo¯avodoxin were
also obtained using guanidine HCl as denaturant in buffer
B. The midpoint of the unfolding curve o ccurs at a lower
concentration of denaturant (1.0
M
guanidine HCl vs. 1.35
M
with urea) and the s lope is steeper (20.7 1.4 kJámolá
M
)1
).
The calculated conformational stability in guanidine HCl
(21.15 1.45 kJámol
)1
25 m
M
phosphate, 500 m
M
NaCl and 6
M
urea; m,250m
M
phosphate
and 6
M
urea. The inset shows the corresponding logarithmic plots.
Table 1. Eects of salt o n t he rates of urea-unfolding of ¯avodoxin and apo¯avodoxin. Value s for the ®rst-order rate co nstants f or the u nfolding o f th e
protein (k
u
)inureaatpH7.0and25°C were determined as described in Figs 3 and 6. Th e errors are the standard deviations.
Buer
[urea]
(
M
)
10
4
´ k
u
(s
)1
)
Flavodoxin Apo¯avodoxin
25 m
with the apoprotein, the rate constant is independent of the
urea concentration [k
u
in 6
M
urea 1.42 0.02 ´
10
)4
s
)1
(3); k
u
in 10
M
urea 1.48 0.05 ´ 10
)4
s
)1
(3)], and similar to the rate constant determined for
dissociation of the holoprotein in the absence of urea
(k
o
1.81 ´ 10
)4
s
)1
). This suggests that the dissociation of
the holoprotein complex to apo¯avodoxin and FMN is the
rate-determining step during unfolding of this ¯avodoxin.
Salt inhibits the rate of unfolding of ¯avodoxin but the
app
) for the unfolding of apo-
¯avodoxin calculated at 2.94
M
(j) and 3.43
M
(d)ureavs.the
logarithm of the mean activity of NaCl (ln a
).
Table 2. Eects of salt on the energetic parameters for the conformational stabilities of ¯avodoxin and apo¯avodoxin. The parameters were
determined at 25 °C from urea-unfolding curves for 1 l
M
protein in the buer at pH 7.0 as indicated.
Buer
m
(kJámol
)1
á
M
)1
)
Urea
1/2
(
M
)
DG
w
(kJámol
-4
-3.6
04812
ln
k
u
(s
-1
)
Urea (M)
Fig. 4. The eect of urea concentration on the rate constant of unfolding
of apo¯avodoxin. The logarithm of the observed ® rst-order rate con-
stant (k
u
) for the unfolding of apo¯avodoxin at 25 °C in 250 m
M
sodium ph osphate, pH 7.0, con taining 5.2±10
M
urea, is plo tted
against the c oncentration of urea. The values for k
u
are the averages of
three kinetic traces. The error bars show the standard deviations.
Ó FEBS 2002 Flavodoxin unfolding (Eur. J. Biochem. 269) 217
(< 1
M
urea in buffer B) the FMN ¯ uore scence increases b y
up to threefold (equivalent to % 3% of the FMN ¯uores-
cence of fully dissociated ¯avodoxin), and comparable
changes occur in the protein ¯uorescence. Several explana-
must re¯ect a decrease in the value for the association
Table 3. E ects of protein concentration on the energetic parameters f or the conformational stabilities of ¯avodoxin and apo¯avodoxin. The energetic
parameters were determined by ®ttin g unfolding curves as in Figs 5 and 7 using Eqn (3). Data were obtained at 25 °Cin25 m
M
sodium phosphate
and 0±7.1
M
urea.
Protein
[protein]
7(M)
m
(kJámol
)1
á
M
)1
)
urea
1/2
(
M
)
DG
w
(kJámol
)1
)
Flavodoxin
0.25 ) 4.79 0.4 2.44 11.68 1.1
Protein (open symbols) and ¯avin (closed symbols) ¯uorescence was
measured at 380 nm a nd 525 n m with excitation at 280 nm and
445 nm, respectively. m,n,25m
M
phosphate and 6
M
urea; j,h,
25m
M
phosphate, 500 m
M
NaCl a nd 6
M
urea; d, 250 m
M
phosphate
and 8
M
urea. The inset shows the corresponding logarithmic plots.
218 B. O
Â
Nuallain and S. G. Mayhew (Eur. J. Biochem. 269) Ó FEBS 2002
rate constant (k
on
; Table 4). It i s c oncluded t hat t he increase
in ¯avin ¯uorescence at low urea concen trations results
mainly from a direct effect of urea on the holoprotein that
weakens the FMN±protein interactions and shifts the
equilibrium in Eqn ( 6) to the right. In addition, apo¯avo-
doxin in equilibrium Eqn (6) starts to become unfolded at
period used to unfold apoprotein (20 min). This conclusion
is supported by the observation that when unfolded
holoprotein is exposed to urea for a longer period than is
required to completely unfold the protein even less folded
protein is formed when t he urea is subsequently diluted out
(6% recovery of folded holoprotein after 48 h unfolding). A
control experiment showed that when apoprotein is incu-
bated in urea for up to 5 days before diluting out the
denaturant, the extent of refolding also progressively
decreases (data not shown). The urea-unfolding and refold -
ing experiments were carried out in the a bsence of EDTA, a
chelating agent that is used to protect thiol groups from
heavy metal-catalysed oxidation. The inclusion of 2 m
M
EDTA in the incubations did not improve the reversibility of
the reactions after long-term treatment with urea.
The c onformational stability determined f or the holo-
protein of ¯avodoxin in buffer B (17.6 1.0 kJámol
)1
,
Table 3) is almost twice that of the apoprotein. The greater
stability of the holoprotein results from an increase in the
transition midpoint which is a pproximately doubled. The ®t
of the urea-unfolding curve for ¯avodoxin with Eqn (4)
suggests that the number o f amino-acid side chains that
become exposed when the holoprotein unfolds (30.6 1.7)
is % 9 less than for the apoprotein.
In contrast to the a poprotein for which the transition
midpoint was found to be independent of the protein con-
centration, the transition midpoint for the holoprotein
increase the ionic strength (Table 2, Fig. 7).
DISCUSSION
The experiments described in this paper show that
D. vulgaris apo¯avodoxin is reversibly unfolded by urea
in a reaction whose equilibrium midpoint depends on the
ionic composition of the solution. The change in protein
¯uorescence as a function of the concentration of urea is
consistent with a two-state model of unfolding, as are the
kinetics of the reaction. However, two observations suggest
that the a poprotein of D. vulgaris ¯avodoxin is not
completely unfolded in urea. First, the maximum ¯uores-
cence emission occurs at 351 nm, and is therefore blue-
shifted compared with the protein emission from unfolded
apo¯avodoxins from A. vinelandii [28] and Anabaena [4]
which occurs at 354± 355 nm. T he 3±4 nm difference
suggests that the side chains of the aromatic amino acids
in the urea-treated D. vulgaris protein may not all be as
exposed to solvent as those in the unfolded apo¯avodoxins
from the other two organisms [29]. Second, it is calculated
that only 30% of the amino-acid side chains become
exposed to solvent a fter urea t reatment, a value that is
similar to t he value that can b e calculated for the urea-
unfolding of apo¯avodoxin from Anabaena (using the data
of Fig. 6 in [4]).
The observation that the ®nal p rotein ¯uo rescence of
urea-treated ¯avodoxin is the same as that of apoprotein
treated with the same concentration of urea suggests that
when the urea concentration is suf®ciently high both forms
of the protein unfold to the s ame extent. The observa tion
that the ¯uorescence due to FMN in urea-unfolded
´ k
o
(s
)1
)
10
10
´ K
d
(
M
)
0.0 8.32 1.81 2.18
0.2 5.07 2.31 4.55
0.4 4.33 1.58 3.66
0.6 3.92 1.79 4.56
0.8 3.40 2.56 7.53
1.0 2.83 2.13 7.54
Ó FEBS 2002 Flavodoxin unfolding (Eur. J. Biochem. 269) 219
¯avodoxin is the same as that of protein-free FMN under
the same conditions indicates that the FMN is fully
dissociated from the protein. The kinetics of the two
unfolding reactions involve a single exponential, similar to
those observed with other small single-domain proteins [29],
and because the rate constants for the increase in FMN
¯uorescence and protein ¯uorescence are so similar, we
conclude that the rate-limiting s tep in unfolding of the
holoprotein is the slow dissociation of FMN from the
apoprotein. The only evidence so far that urea perturbs
the holoprotein at urea concentrations less than that
tion of a folding intermediate, as well as additional effects
such as preferential binding of salt ions to the folded protein
or an effect on the properties of the solvent. The use of a
single spectroscopic techniqu e to monitor unfolding, as used
in the present study, cannot exclude the formation of stable
intermediates in the reaction, nor does it allow the
conclusion that the protein at the end point of the transition
is devoid of all secondary and tertiary structure. Measure-
ments of the unfolding equilibrium by additional techniques
such as far UV circular dichroism might reveal different
unfolding equilibria, as recently observed in the guani-
dine HCl unfolding of apo¯avodoxins from A. vinelandii
[6±8] and D. desulfurica ns [10]. It is known that other
proteins whose unfolding curves ®t the two-state model in
fact give intermediates in their unfolding/folding reactions
[34].
The larger values calculated for the conformational
stabilities of ¯avodoxin and its apoprotein in phosphate,
compared with NaCl, and the smaller rate of unfolding of
apoprotein in NaCl, indicates that the increased stabiliza-
tion of the protein by salts cannot be explained simply by
a s hielding of charged g roups that might o therwise
destabilize the protein. The decrease in the m value for
¯avodoxin with NaCl is unusual but not unique because a
similar effect has been reported on the m value for an
equilibrium intermediate in th e unfolding of apomyoglobin
from Aplysia limacina [34]. I n this case it was suggested
that the large decrease (% threefold) in the m value with
KCl is due to deviations from a proposed three-state
model.
trations of salt are probably due to a combination of factors
including the preferential binding of salt ions to the folded
protein, ionic strength e ffects, as well as to a change in the
physical properties of the solvent.
A detailed study of unfolding of the h oloprotein of a
¯avodoxin has been reported for one other protein, namely
¯avodoxin from D. desulfuricans [10]. This protein was
unfolded with guanidine HCl in 3 m
M
phosphate pH 7. The
reactions differed in several ways from the unfolding
reactions of D. vulgaris ¯avodoxin described above. First,
the reactions with the D. desulfuricans holoprotein were
complete in less than 1 min rather than requiring hours t o
reach completion. Second , the protein ¯uorescence i ncreased
at low c oncentrations of denaturant without a change in the
¯avin ¯uorescence. Third, the FMN w as found to be bound
tightly after the protein had been unfolded (calculated
K
d
0.2 n
M
[10]). The changes in protein ¯uorescence were
found to occur at smaller c oncentrations than changes in the
far UV c ircular dichroism of the protein, leading to the
conclusion that unfolding of this protein does not ®t a two-
state model, but rather that it occurs through an interme-
diate partly unfolded state in which the FMN is still tightly
bound to the apoprotein.The conformational stabilities
determined for ¯avodoxin and apo¯avodoxin from
M
FMN. It is clear that a K
d
value as small
as 0 .1 n
M
could not be measured by this method.
Recalculation of K
d
from the experimental points that lie
off the straight lines of Fig. 4 in [41] indicates that its value
is 0.14 0.1 l
M
.IftheK
d
value for the oxidized
D. desulfuricans protein is indeed % 700 times greater than
that of D. vulgaris ¯ avodoxin, differences in the m echa-
nisms of unfolding of the two proteins might be under-
standable. The conformations of the two loops of protein
that envelop the FMN are different in t he two proteins
[11,12]. As a result, the carbonyl of glutamate 99 in
D. vulgaris ¯ avodoxin points towards the solvent, while in
the D. desulfuricans protein it points towards the ¯avin
and is 0.29 nm from O(4) o f the isoalloxazine structure. As
was noted by others [12], the orientation in the D. desul-
furicans protein should lead to O-O repulsion and to a less
stable ¯avin±protein complex. It should be noted further
that neither the published K
d
the holoprotein can occur by two routes. One of these
involves unfolding o f apoprotein ( reaction I) and a conse-
quent perturbation of the holop rotein/apoprotein equilibria
(reactions A/B and G/H). Note that the equilibria A/B and
G/H do not depend directly on the concentration of urea.
The other route involves further interaction of urea with the
holoprotein complex (urea-apo-FMN) and the direct
unfolding of this complex (reaction L).
When the urea concentration is low, the small concen-
tration of apoprotein (apo-urea) that is in equilibrium with
the holoprotein, unfolds rapidly to a new equilibrium that
includes completely unfolded protein [(urea)
x
-apo*-urea],
the two species of folded protein ( apo-urea and urea-apo-
FMN), free FMN and urea. The FMN prevents the
apoprotein f rom unfolding completely and maintains a high
equilibrium concentration o f apo-urea-FMN. As the direct
unfolding/folding reactions of the holoprotein complex
(reactions K/L) are very slow, the protein unfolding/folding
occurs mainly via the apoprotein routes through the apo-
urea complex.
The scheme of Fig. 8 provides a working hypothesis for
the overall unfolding/folding reactions of D. vulgaris
apo¯avodoxin and ¯avodoxin, and it forms a b asis for
further experimentation. It does not account for all of the
experimental observations on the system, in particular the
different e ffects of salt on the two forms of the protein that
cause the conformational stability of t he apoprotein in
high salt to be greater than that of the holoprotein. It is pos-
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2. Mayhew, S.G. & Tollin, G. (1992) General properties of
¯avodoxins. In Chemistry and Biochemistry of Flavoenzymes
(Mu
È
ller, F., ed.), Vol. 3, pp. 389±426. CRC Press, Boca Raton, FL.
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