Báo cáo khoa học: Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes ppt

Relationship between functional activity and protein
stability in the presence of all classes of stabilizing
osmolytes
Shazia Jamal*, Nitesh K. Poddar*, Laishram R. Singh*,, Tanveer A. Dar*,à, Vikas Rishi§ and
Faizan Ahmad
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, India
Introduction
Both prokaryotic and eukaryotic cells, when subjected
to harsh environmental conditions such as water, salts,
cold and heat stresses, adopt a common strategy in
protecting their proteins by producing low molecular
weight organic substances called osmolytes [1,2].
Chemically stabilizing osmolytes (low molecular mass
organic compounds that raise the midpoint of thermal
denaturation) are divided into three classes: amino
Keywords
catalytic efficiency; denaturation equilibrium;
enzyme activity; osmolytes, protein
stability
Correspondence
F. Ahmad, Centre for Interdisciplinary
Research in Basic Sciences, Jamia Millia
Islamia, New Delhi, India 110025
Fax: +91 11 2698 3409
Tel: +91 11 2698 1733
E-mail:
*These authors contributed equally to this
work
Present addresses
Division of Population Science, Fox
Chase Cancer Center, Philadelphia, PA,

USA
àDepartment of Chemistry Biochemistry,
University of Montana, Missoula, MT,
USA
§National Cancer Institute, NIH, Bethesda,
MD, USA
(Received 29 May 2009, revised 10 August
2009, accepted 19 August 2009)
doi:10.1111/j.1742-4658.2009.07317.x
We report the effects of stabilizing osmolytes (low molecular mass organic
compounds that raise the midpoint of thermal denaturation) on the stabil-
ity and function of RNase-A under physiological conditions (pH 6.0 and
25 °C). Measurements of Gibbs free energy change at 25 °C(DG
D
°) and
kinetic parameters, Michaelis constant (K
m
) and catalytic constant (k
cat
)of
the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to
classify stabilizing osmolytes into three different classes based on their
effects on kinetic parameters and protein stability. (a) Polyhydric alcohols
and amino acids and their derivatives do not have significant effects on
DG
D
° and functional activity (K
m
and k
cat

). (b) Methylamines increase
DG
D
° and k
cat
, but decrease K
m
. (c) Sugars increase DG
D
°, but decrease
both K
m
and k
cat
. These findings suggest that, among the stabilizing osmo-
lytes, (a) polyols, amino acids and amino acid derivatives are compatible
solutes in terms of both stability and function, (b) methylamines are the
best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but
they apparently do not yield functionally active folded molecules.
Abbreviations
DG
D
°, Gibbs free energy change at 25 °C; DC
p
, constant pressure heat capacity change; T
m
, midpoint of thermal denaturation;
DH
m
, enthalpy change at T

m
; K
m
, Michaelis constant; k
cat
, catalytic constant; k
cat
⁄ K
m
, overall enzyme efficiency.
6024 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS
acids and their derivatives, polyhydric alcohols and
sugars, and methyl ammonium derivatives [1]. These
osmolytes are known not only to stabilize proteins
[3,4], but they also induce refolding of misfolded
proteins [5–8] and remove protein aggregation [9–12].
Mechanisms of protein osmolyte interactions, the
effect of osmolytes on protein stability, and how osmo-
lytes correct protein misfolding defects and remove
protein aggregation have been widely investigated.
It has been demonstrated that the unfavourable
interaction between the peptide backbone and the
osmolytes leading to the preferential hydration of the
protein domain is the driving force of protein stabiliza-
tion or folding [3,4]. Furthermore, the effect of
osmolytes on the functional activity of an enzyme has
also been investigated on a number of enzymes. Conse-
quently, this has led to the classification of osmolytes
into two classes: compatible or counteracting. Compat-
ible osmolytes increase protein stability against

denaturation with little or no effect on their function
under native conditions [1,13,14]. Representatives of
this class include certain amino acids (e.g. proline
and glycine) and polyols (e.g. trehalose, sucrose and
sorbitol). Counteracting osmolytes consist of the
methylamine class of osmolytes, which are believed to
have the special ability to protect intracellular
proteins against the inactivation ⁄ destabilization by
urea [14–17]. In contrast to compatible osmolytes,
counteracting osmolytes are believed to cause changes
in protein function that are opposite to the effects that
urea has on protein function [16–19].
Despite significant advances in understanding the
effect of osmolytes on protein stability, folding and the
activity of proteins and enzymes, the relationship
between protein stabilization by osmolytes and its con-
sequent effects on the activity of enzymes has not been
examined. It is not yet understood how well protein
stability and activity are coupled in the presence of an
osmolyte. This study was undertaken to investigate the
relationship between protein stability and activity
changes in the presence of a wide range of osmolytes.
For this we evaluated the protein stability (DG
D
°,
Gibbs free energy change at 25 °C) of RNase-A and
its activity parameters ( K
m
, Michaelis constant; k
cat

,
catalytic constant) in the presence and absence of
almost all naturally occurring osmolytes. We report
here that protein stability and activity are not largely
coupled in the presence of osmolytes. However, protein
stability and activity have a linear correlation in the
presence of methylamines and sugar osmolytes. This
study, in fact, has led to the classification of osmolytes
into three different classes based on their effects on
stability and activity parameters of RNase-A.
Results and Discussion
Protein stability and enzyme activity have a well-corre-
lated function. However, we do not know how this
relationship is maintained in the presence of stabilizing
osmolytes accumulated under stressed conditions.
Because stabilizing osmolytes do not have a direct
interaction with the protein domains per se,itis
expected that an increase in protein stability (DG
D
°)by
an osmolyte due to the shift in the denaturation equi-
librium, native state M denatured state, towards the
left, must increase the catalytic efficiency of the enzyme
and vice versa. The reason for saying this is that urea,
which decreases DG
D
°, is known to decrease the cata-
lytic efficiency of osmolytes [20, references therein].
Thus, it will be interesting to investigate how kinetic
parameters of the enzyme-catalyzed reaction change

upon modulation of protein stability (DG
D
°) by osmo-
lytes. To investigate the protein stability–activity rela-
tionship in the presence of osmolytes, we intentionally
chose two different groups of osmolytes. The first
group consists of polyols, amino acids and amino acid
derivatives, which have been reported to have no effect
on DG
D
° associated with the protein denaturation
equilibrium, native state M denatured state, under
physiological conditions. The second group consists of
methylamines and sugars, which are shown to increase
DG
D
° of proteins associated with the denaturation
equilibrium, native state M denatured state. The
observed effects of polyols, sugars and methylamines
and some amino acids on DG
D
° of RNase-A have been
reported previously [21–25], and DG
D
° values in the
presence of these osmolytes are given in Table 1. How-
ever, DG
D
° values of RNase-A in the presence of
alanine, serine, lysine, b-alanine, taurine and dimethyl-

glycine have not been published elsewhere. We have
therefore measured the thermodynamic parameters of
RNase-A in the presence of these amino acids and
amino acid derivatives, and values of DG
D
°, measured
in triplicate, are given in Table 1.
The effect of polyols on the kinetic parameters (K
m
and k
cat
) of the RNase-A mediated hydrolysis of cyti-
dine 2¢-3¢ cyclic monophosphate has been previously
reported [22]. Values of the kinetic parameters of this
protein in the presence of all other osmolytes were
determined and are presented in Table 1. It should be
noted that the value for each kinetic parameter repre-
sents the mean of three independent measurements
together with the mean error. These kinetic parameters
in the absence of the osmolytes, shown in Table 1, are
in excellent agreement with those reported previously
[26–28]. These agreements led us to believe that our
measurements of the enzyme-catalyzed reactions and
S. Jamal et al. Functional stability and activity by osmolytes
FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6025
Table 1. Stability and activity parameters of RNase-A in the presence of different classes of osmolytes at physiological conditions. Values are from triplicate measurements. DG
D
° values
were taken from [21] for class III, from [22,23] for class I and from [24,25] for class II.
Class III Class I Class II

[Sugars]
M
DG
D
°
(kcalÆmol
)1
)
k
cat
(s
)1
)
K
m
(mM)
[Polyols]
M
DG
D
°
(kcalÆmol
)1
)
k
cat
(s
)1
)
K

m
(mM)
[Amino
acids and
derivatives]
M
DG
D
°
(kcalÆmol
)1
)
k
cat
(s
)1
)
K
m
(mM)
[Methylamines]
M
DG
D
°
(kcalÆmol
)1
)
k
cat

(s
)1
)
K
m
(mM)
0.00 9.83 3.10 ± 0.08 1.05 ± 0.04 0.00 10.60 3.22 ± 0.35 1.33 ± 0.15 0.00 9.83 3.10 ± 0.08 1.05 ± 0.04 0.00 9.83 3.10 ± 0.08 1.05 ± 0.04
Glucose Sorbitol Alanine Sarcosine
0.50 10.31 3.07 ± 0.07 1.03 ± 0.02 0.55 10.67 3.17 ± 0.23 1.36 ± 0.24 0.25 9.92 3.07 ± 0.08 1.05 ± 0.11 0.25 10.25 3.39 ± 0.12 0.92 ± 0.02
1.00 10.79 2.62 ± 0.08 0.83 ± 0.04 1.10 10.72 3.28 ± 0.12 1.33 ± 0.14 0.50 9.96 2.97 ± 0.12 1.02 ± 0.15 0.50 10.70 3.71 ± 0.11 0.80 ± 0.05
1.50 11.32 2.39 ± 0.07 0.71 ± 0.03 1.65 10.57 3.25 ± 0.18 1.38 ± 0.18 0.75 11.20 4.03 ± 0.11 0.71 ± 0.04
2.00 11.77 2.00 ± 0.06 0.53 ± 0.03 2.20 10.65 3.22 ± 0.30 1.30 ± 0.21 1.00 11.60 4.21 ± 0.14 0.58 ± 0.03
Fructose Glycerol Proline Dimethylglycine
1.00 10.84 2.47 ± 0.11 0.81 ± 0.03 1.09 10.50 3.25 ± 0.22 1.25 ± 0.17 0.25 9.83 3.07 ± 0.07 1.03 ± 0.08 0.25 10.06 3.29 ± 0.10 0.99 ± 0.04
1.50 11.39 2.24 ± 0.09 0.69 ± 0.02 2.17 10.67 3.17 ± 0.17 1.34 ± 0.12 0.50 9.70 2.98 ± 0.10 1.01 ± 0.13 0.50 10.31 3.41 ± 0.09 0.95 ± 0.07
2.00 11.79 1.93 ± 0.07 0.58 ± 0.02 3.26 10.56 3.30 ± 0.28 1.31 ± 0.15 1.00 9.77 3.25 ± 0.09 1.11 ± 0.07 0.75 10.58 3.65 ± 0.12 0.88 ± 0.03
2.50 12.18 1.61 ± 0.05 0.42 ± 0.03 4.35 10.53 3.48 ± 0.42 1.43 ± 0.20 1.50 9.80 3.29 ± 0.09 1.07 ± 0.06 1.00 10.93 3.92 ± 0.12 0.79 ± 0.05
Galactose Xylitol Serine Betaine
0.50 10.31 3.05 ± 0.08 1.02 ± 0.03 0.25 10.49 3.15 ± 0.20 1.41 ± 0.16 0.25 9.74 2.91 ± 0.12 1.00 ± 0.15 0.25 9.96 3.22 ± 0.11 1.02 ± 0.03
0.75 10.55 2.87 ± 0.07 0.91 ± 0.04 0.50 10.57 3.32 ± 0.17 1.32 ± 0.19 0.50 9.84 3.02 ± 0.08 1.05 ± 0.10 0.50 10.19 3.37 ± 0.11 0.99 ± 0.04
1.00 10.74 2.68 ± 0.06 0.81 ± 0.03 0.75 10.61 3.22 ± 0.12 1.35 ± 0.08 0.75 10.40 3.46 ± 0.10 0.92 ± 0.03
1.00 10.67 3.25 ± 0.40 1.39 ± 0.19 1.00 10.81 3.62 ± 0.12 0.83 ± 0.06
Sucrose Adonitol Lysine Trimethylamine N-oxide
0.50 10.55 3.01 ± 0.10 1.00 ± 0.04 0.25 10.41 3.12 ± 0.20 1.41 ± 0.13 0.25 9.82 3.05 ± 0.15 1.06 ± 0.18 0.25 10.07 3.34 ± 0.09 0.96 ± 0.03
1.00 11.17 2.50 ± 0.11 0.78 ± 0.02 0.50 10.68 3.18 ± 0.30 1.29 ± 0.16 0.50 9.87 3.08 ± 0.10 1.04 ± 0.12 0.50 10.54 3.61 ± 0.12 0.87 ± 0.04
1.50 11.94 2.10 ± 0.06 0.61 ± 0.03 0.75 10.64 3.33 ± 0.17 1.33 ± 0.11 0.75 10.95 3.83 ± 0.09 0.76 ± 0.03
1.00 10.75 3.15 ± 0.33 1.32 ± 0.09 1.00 11.48 4.13 ± 0.14 0.65 ± 0.05
Raffinose Mannitol Glycine
0.10 10.00 3.07 ± 0.04 1.03 ± 0.02 0.25 10.51 3.25 ± 0.20 1.36 ± 0.13 0.25 9.89 3.17 ± 0.07 1.04 ± 0.05
0.20 10.19 2.94 ± 0.03 0.93 ± 0.02 0.50 10.54 3.18 ± 0.15 1.30 ± 0.12 0.50 10.03 3.11 ± 0.10 1.05 ± 0.04

0.30 10.38 2.85 ± 0.05 0.87 ± 0.03 0.75 10.64 3.25 ± 0.20 1.37 ± 0.16 1.00 10.17 3.05 ± 0.10 1.07 ± 0.07
0.40 10.50 – – 1.00 10.62 3.23 ± 0.32 1.33 ± 0.08
Stachyose b-Alanine
0.25 10.38 2.81 ± 0.07 0.91 ± 0.03 0.25 9.85 3.14 ± 0.08 1.07 ± 0.06
0.50 10.86 2.61 ± 0.08 0.76 ± 0.04 0.50 9.87 3.10 ± 0.07 1.04 ± 0.05
0.75 11.41 – – 1.00 9.90 3.05 ± 0.09 1.05 ± 0.05
Taurine
0.25 9.80 3.18 ± 0.08 1.08 ± 0.05
0.50 9.86 3.09 ± 0.10 1.03 ± 0.06
Functional stability and activity by osmolytes S. Jamal et al.
6026 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS
the analysis of the progress curves for kinetic parame-
ters are accurate. It can be seen in Fig. 1 (see also
Table 1) that sugars and methylamines affect both the
thermodynamic (DG
D
°) and the kinetic (K
m
and k
cat
)
properties, whereas polyols, amino acids and amino
acid derivatives do not have any significant effect on
these parameters. In fact, based on the effects that the
osmolytes have on both DG
D
° and the catalytic prop-
erties of RNase-A (Table 1), we can distinctly classify
osmolytes into three different classes. (a) Class I
includes polyhydric alcohols (sorbitol, glycerol, xylitol,

adonitol, mannitol) and amino acids and derivatives
(glycine, alanine, proline, serine, lysine, b-alanine and
taurine) that have no significant effects on both DG
D
°
and k
cat
. (b) Class II represents methylamines (sarco-
sine, dimethylglycine, betaine, trimethylamine N-oxide)
that increase both DG
D
° and k
cat
, but decrease K
m
. (c)
Sugars (glucose, fructose, galactose, sucrose, raffinose,
stachyose) that increase DG
D
°, but decrease both K
m
and k
cat
belong to class III.
k
cat
alone does not absolutely define the overall cata-
lytic activity of an enzyme, as it is a first-order rate
constant that refers to the properties and reactions of
the enzyme–substrate, enzyme–intermediate and

enzyme–product complexes [29]. On the other hand,
k
cat
⁄ K
m
is an apparent second-order rate constant that
refers to the properties and the reaction of the free
enzyme and free substrate [29]. We have therefore esti-
mated k
cat
⁄ K
m
values of all the reactions in the pres-
ence and absence of all classes of osmolytes. It can be
Fig. 1. Effect of osmolytes on enzyme kinetic parameters. Plot of Dk
cat
of RNase-A versus [osmolyte] (left panels) and DK
m
of RNase-A
versus [osmolyte] (right panels).
S. Jamal et al. Functional stability and activity by osmolytes
FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6027
seen in Fig. 1 that class I osmolytes (polyhydric alco-
hols, amino acids and amino acid derivatives) do not
significantly perturb kinetic parameters (K
m
and k
cat
)
and, hence, the overall catalytic efficiency (k

cat
⁄ K
m
)of
RNase-A. This observation on the effect of polyols
and amino acids on RNase-A is in agreement with that
on other enzymes (lactate dehydrogenase, lysozyme,
pyruvate kinases) reported previously [13,22,30]. It has
been argued that these compatible osmolytes affect the
association of the substrate with the enzyme in any
one of several ways, e.g. through solvation effects on
substrates or enzyme active sites and through their
effects on the thermodynamic activity of substrates
and enzymes [13,30,31]. Thus, a lack of effect on both
enzymatic parameters (K
m
and k
cat
) of RNase-A sug-
gests that polyols, amino acids and amino acid deriva-
tives have little or no effect on the solvation properties
of the substrate and the enzyme active sites or on their
thermodynamic activities. Another explanation for
these observations comes from our DG
D
° measure-
ments. Because of perfect enthalpy–entropy compensa-
tion, DG
D
° is unperturbed in the presence of class I

osmolytes (see Table 1), i.e. the denaturation equilib-
rium, native state M denatured state, of RNase-A is
unperturbed and, hence, no change in the functional
activity of the enzyme in the presence of such osmo-
lytes (see Fig. 1).
If our explanation is correct, an increase in protein
stability (DG
D
°) by osmolytes must result in an
increase in the number of N molecules due to a shift
in the denaturation equilibrium, native state M dena-
tured state, towards the left. Consequently, both k
cat
and k
cat
⁄ K
m
are expected to increase in the presence of
such osmolytes, as k
cat
⁄ K
m
refers to the reaction of
free (active) enzyme [29]. Data presented in Table 1
and Fig. 2 for the effect of methylamines (class II) on
DG
D
° and kinetic parameters show that this is indeed
true. It is noteworthy that our observation of the effect
of methylamines on RNase-A is also in agreement with

previous reports on many other enzymes, such as rab-
bit muscle lactate dehydrogenase, triose phosphate
isomerase, pyruvate kinase, creatine kinase, A4-lactate
dehydrogenase, glutamate dehydrogenase, argininosuc-
cinate lyase, porcine arginosuccinase [17,19,32–35].
However, it should be noted that both K
m
, the overall
dissociation constant of all enzyme bound species [29],
and k
cat
are decreased in the presence of sugar (class
III) osmolytes (see Fig. 1, Table 1). One possible
explanation for this observation is that the original
native state ensembles and ⁄ or the refolded protein
molecules in the presence of sugars undergo a subtle
change in conformation, yielding all or some enzyme
bound species that are more stable than those in the
absence of sugars, i.e. K
m
is decreased. On the other
hand, this change in conformation results in a decrease
in k
cat
, the turnover number of the enzyme in the pres-
ence of sugars, i.e. the maximum number of substrate
molecules converted to product per active site per unit
time is decreased. A subtle change in the enzyme active
site that occurs in the presence of sugars may be a pos-
sible cause for the observations on K

m
and k
cat
of
RNase-A in the presence of class III osmolytes.
To evaluate if all the refolded protein fractions
produced by an osmolyte are in functionally active
conformation, we determined the relationship between
changes in protein stability (DDG
D
°) and overall
catalytic efficiency (Dlog(k
cat
⁄ K
m
)) in the presence of
Fig. 2. Relationship between protein stability and catalytic effi-
ciency. Plot of Dlog(k
cat
⁄ K
m
) versus DDG
D
° of RNase-A obtained in
the presence of various osmolytes.
Functional stability and activity by osmolytes S. Jamal et al.
6028 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS
different concentrations of each osmolyte (Fig. 2). It
can be seen in Fig. 2 that for class I osmolytes (poly-
ols, amino acids and amino acid derivatives) the slope

is nearly 0. This is an expected result, as there is no
perturbation of the denaturation equilibrium and,
hence, there is no increase in catalytic efficiency in the
presence of this group of osmolytes. Interestingly,
there is a linear relationship between DDG
D
° and
Dlog(k
cat
⁄ K
m
) in the presence of methylamines and
sugar. However, the slopes of the plot (Dlog(k
cat
⁄ K
m
)
versus DDG
D
°) are very different. In fact, the slope in
the presence of sugar osmolytes is 10 times less than
that in the presence of methylamines. A higher slope
in the case of methylamines will mean that the total
refolded protein fraction generated by the methylam-
ines is more active than those generated by sugars.
Taking these observations and k
cat
values of RNase-A
in the presence of class II and III osmolytes, it seems
that the refolded protein fraction in the presence of

sugars is not as active as the original native molecules,
whereas it is opposite in the presence of methylamines.
We therefore conclude that equilibrium shift is not the
only ultimate step to increase the activity of an enzyme
in the presence of osmolytes.
In general, two thermodynamic models are used to
explain the effect of osmolytes on protein stability [36,
references therein]. The binding model claims that an
increase in the osmolyte-induced stability arises from
the preferential hydration (or exclusion of the osmo-
lyte) leading to a shift in the denaturation equilibrium,
native state M denatured state, towards the left. The
excluded volume model focuses on the fact that osmo-
lytes limit the conformational freedom of proteins by
driving them to their most compact native state (cata-
lytically most efficient form). The decrease in confor-
mational freedom arises from steric repulsions between
the protein and the osmolyte. The latter model
assumes that the native state of a protein consists of
inter-converting high (most compact) and low (less
compact) activity state ensembles and also demon-
strated that the presence of osmolytes shifts the native
conformational equilibria towards the most compact
protein species within native state ensembles [32,37,38].
The variation in the effect of stabilizing osmolytes in
modulating the catalytic efficiency of RNase-A in the
presence of each class of osmolyte may best be
explained by the combination of both thermodynamic
models. Our results suggest that: (a) methylamines not
only decrease conformational freedom, but also

increase preferential hydration, which consequently
generates more active protein molecules; (b) sugar
osmolytes affect the conformational freedom and
preferential hydration in such a way that it produces
catalytically less competent species; and (c) class I
osmolytes have no significant effects on both the
conformational freedom and the preferential hydration
of the protein. In agreement with the explanation
on methylamines, previous reports on trimethylamine
N-oxide indicate that it not only produces more active
molecules by shifting the denaturation equilibrium
[24,25,36,39], but also affects the native state by con-
verting the low activity ensembles to the high activity
ensembles [37]. Very interestingly, a recent refolding
kinetic study of carbonic anhydrase II in sucrose
showed that the sugar significantly accelerates the rate
of refolding of the enzyme to the native or compact
near-native conformations, but decreases the fraction
of catalytically active enzyme recovered [40].
It has already been reported that osmolytes indepen-
dently affect proteins and, hence, their effects are
algebraically additive [21,41]. Based on our results
given in Table 1, one can speculate that: (a) the poly-
ols–amino acids (or amino acid derivatives) system is
an exclusive mixture that is compatible both with
thermodynamic stability ( DG
D
°) and function, and (b)
sugar–methylamine mixtures are attractive candidates
to yield amazingly enhanced protein stability and

function. Thus, different osmolyte mixtures may serve
as post-translational modulators of stability and ⁄ or
function of many enzymes. This may perhaps be the
main reason why many organisms use multi-osmolyte
systems [1,14,15,42–44].
Furthermore, the osmolyte-induced folding of pro-
teins is determined by interactions of the osmolyte
with all protein groups (peptide backbone and side
chains) exposed on denaturation. For various osmo-
lytes, Bolen & Baskakov [3] have shown that: (a) the
main driving force for the folding is the unfavourable
interaction between the osmolyte and the peptide back-
bone, and (b) the total contribution of side chains to
the stability of the native state, which may interact
differently with different osmolytes, is very small.
These conclusions are supported by our measurements
of DG
D
° of RNase-A in the absence and presence of
sugars and methylamines. It is seen in Table 1 that, on
the molar scale, these osmolytes, which are chemically
different, have, within experimental errors, almost
identical effects on DG
D
°.
We are confident of three findings: (a) Polyols,
amino acids and amino acid derivatives are ideal
osmolytes, for they neither perturb the denaturation
equilibrium nor affect the functional activity under
native conditions. However, they have the ability to

protect proteins from denaturing stresses. (b) Methyl-
amines not only stabilize proteins, but also refold the
denatured protein to a more active state under native
S. Jamal et al. Functional stability and activity by osmolytes
FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS 6029
conditions. (c) Sugar osmolytes stabilize proteins, but
they convert the denatured protein molecule to a less
active form under native conditions. These findings
make these chemical chaperones aptly suitable for
structure–function studies of proteins, as each class of
osmolytes (classes I–III) can modulate the stability
and ⁄ or function of a protein differently.
Experimental procedures
Chemicals
Commercial lyophilized preparations of RNase-A (type
III-A) were purchased from Sigma Chemical Company
(St Louis, MO, USA). d-glucose, d-fructose, d-galactose,
d-sucrose, d-raffinose, d-stachyose, glycine, l-alanine,
l-proline, l-lysine, l-serine, b-alanine, taurine, sarcosine,
dimethylglycine, glycine betaine, trimethylamine N-oxide,
and cytidine 2¢-3¢ cyclic monophosphate were also obtained
from Sigma. These and other chemicals, which were of
analytical grade, were used without further purification.
Dialysis and the determination of the
concentration of protein
An RNase-A solution was dialyzed extensively against
0.1 m KCl solution at  4 °C. Protein stock solutions were
filtered using 0.45 lm Millipore filter paper. The protein
gave a single band during the native and SDS poly-
acrylamide gel electrophoresis. The concentration of the

protein stock solution was determined experimentally using
a value of 9800 at 277.5 nm for e, the molar absorption
coefficient (m
)1
Æcm
)1
) [45]. All solutions for activity
measurements were prepared in 0.05 m cacodylic acid buffer
containing 0.1 m KCl. Because the pH of the protein solu-
tion may change on the addition of the osmolytes, the pH
of each solution was also measured after each measurement.
It was observed that the change in pH was not significant.
Activity measurements
In order to see the effect of an osmolyte on the kinetic
parameters (K
m
and k
cat
) of RNase-A, the substrate and
the enzyme were preincubated in a given concentration of
each osmolyte. Following the procedure described previ-
ously [22], RNase-A activity using cytidine 2¢-3¢ cyclic
monophosphate as a substrate was measured. The progress
curve for RNase-A mediated hydrolysis of cytidine 2¢-3¢
cyclic monophosphate in the concentration range 0.05–
0.50 mgÆmL
)1
in the absence and presence of a given
concentration of each osmolyte was followed by measuring
the change in absorbance at 292 nm for 20 min in a Jasco

V-560 UV ⁄ Vis spectrophotometer (Hachioji, Tokyo,
Japan). Sample and reference cells were maintained at
25.0 ± 0.1 °C. From each progress curve at a given sub-
strate concentration and in the absence and presence of a
fixed osmolyte concentration, initial velocity (m) was deter-
mined from the linear portion of the progress curve, usually
30 s. The plot of initial velocity (m) versus [S] (in mm)at
each osmolyte concentration was analysed for K
m
and k
cat
using Eqn (1).
v ¼ k
cat
½S=ðK
m
þ½SÞ ð1Þ
Thermal denaturation measurements
Thermal denaturation studies were carried out in a Jasco
V-560 UV ⁄ Vis spectrophotometer equipped with a Peltier-
type temperature controller (ETC-505T), with a heating
rate of 1 °CÆmin
)1
. The change in absorbance with increas-
ing temperature was followed at 287 nm for RNase-A.
Approximately 650 data points of each transition curve
were collected. The raw absorbance data were converted
into (De
287
), the difference molar absorption coefficient

(m
)1
Æcm
)1
). Each heat-induced transition curve (plot of
De
287
versus temperature; not shown) was analysed for T
m
(midpoint of denaturation) and DH
m
(enthalpy change at
T
m
) using a nonlinear least squares analysis according to
the relationship described earlier (see equation (1) in [25]).
Using a value of 1.24 kcalÆmol
)1
ÆK
)1
for DC
p
(the constant
pressure heat capacity change in RNase-A; [39]), DG
D
(T),
the value of DG
D
at any temperature T was estimated using
the Gibbs–Helmholtz equation with known values of T

m
,
DH
m
and DC
p
using the relationship described previously
(see equation (2) in [25]).
Acknowledgements
FA is grateful to the Department of Science and
Technology (India) and the Council of Scientific and
Industrial Research (India) for financial support.
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