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: [email protected]
*These authors contributed equally to this
work
Present addresses
Division of Population Science, Fox
Chase Cancer Center, Philadelphia, PA,

). (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

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

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-

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

(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

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

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

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

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

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

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

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
,
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.
References
1 Yancey PH, Clark ME, Hand SC, Bowlus RD &
Somero GN (1982) Living with water stress: evolution
of osmolyte system. Science 217, 1212–1222.
2 Borowitzka LJ (1985) Glycerol and other carbohydrate
osmotic effectors. In Transport Processes, Iono- and
Osmoregulation (Gilles R & Gilles-Baillen M eds)
pp. 437–453. Springer, Berlin.
3 Bolen DW & Baskakov IV (2001) The osmophobic
effect: natural selection of a thermodynamic force in
protein folding. J Mol Biol 310, 955–963.
4 Timasheff SN (2002) Protein–solvent preferential
interactions, protein hydration and the modulation of
biochemical reactions by solvent components. Proc Natl
Acad Sci USA 99, 9721–9726.
Functional stability and activity by osmolytes S. Jamal et al.
6030 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS
5 Uverski VN, Li J & Fink AL (2001) Trimethylamine-N-

Chem 274, 32970–32974.
13 Wang A & Bolen DW (1996) Effect of proline on
lactate dehydrogenase activity: testing the generality
and scope of the compatibility paradigm. Biophys J 71,
2117–2122.
14 Yancey PH (2004) Compatible and counteracting
solutes: protecting cells from the dead sea to the deep
sea. Sci Prog 87, 1–24.
15 Yancey PH (2003) Proteins and counteracting
osmolytes. Biologist 50, 126–131.
16 Lin TY & Timasheff SN (1994) Why do some
organisms use a urea–methylamine mixture as
osmolyte? Thermodynamic compensation of urea and
trimethylamine N-oxide interactions with protein
Biochemistry 33, 12695–12701.
17 Yancey PH & Somero GN (1979) Counteraction of
urea destabilization of protein structure by methylamine
osmoregulatory compounds of elasmobranch fishes.
Biochem J 183, 317–323.
18 Wang A & Bolen DW (1997) A naturally occurring
protective system in urea rich cells: mechanism of
osmolyte protection of protein against urea
denaturation. Biochemistry 36, 9101–9108.
19 Baskakov I, Wang A & Bolen DW (1998)
Trimethylamine-N-oxide counteracts urea effects on
rabbit muscle lactate dehydrogenase function: a test
of the counteraction hypothesis. Biophys J 74,
2666–2673.
20 Burg MB, Peters EM, Bohren KM & Gabbay KH
(1999) Factors affecting counteraction by methylamines

Biophys Acta 1794, 929–935.
26 Crook EM, Mathias AP & Rabin BR (1960)
Spectrophotometric assay of bovine pancreatic
ribonuclease by the use of cytidine 2¢:3¢-phosphate.
Biochem J 74, 234–238.
27 Santoro MM, Liu Y, Khan SM, Hou LX & Bolen DW
(1992) Increased thermal stability of proteins in the
presence of naturally occurring osmolytes. Biochemistry
31, 5278–5283.
28 Shahjee HM, Rishi V & Ahmad F (2002) Effect of
D-amino acids on the functional activity and
conformational stability of ribonuclease-A. Indian J
Biochem Biophys 39, 368–376.
29 Fersht AR (1999) Structure and Mechanism in Protein
Science: a Guide to Enzyme Catalysis and Protein
Folding. W.H. Freeman, San Francisco.
30 Bowlus RD & Somero GN (1979) Solute compatibility
with enzyme function and structure: rationales for the
selection of osmotic agents and end-products of anaero-
bic metabolism in marine invertebrates. J Exp Zool 208,
137–151.
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 6031
31 Cayley S, Lewis BA & Record MT Jr (1992) Origins of
the osmoprotective properties of betaine and proline in
Escherichia coli K-12. J Bacteriol 174, 1586–1595.
32 Gulotta M, Qium L, Desamero R, Ro
¨
sgen J, Bolen
DW & Callender R (2007) Effects of cell volume

namic parameters of proteins in the presence and
absence of trimethylamine N-oxide. J Biol Chem 280,
1035–1042.
40 Monterroso B & Minton AP (2007) Effect of high
concentration of inert cosolutes on the refolding of an
enzyme: carbonic anhydrase B in sucrose and ficoll 70.
J Biol Chem 282, 33452–33458.
41 Auton M & Bolen DW (2004) Additive transfer
free energies of the peptide backbone unit that
are independent of the model compound and the
choice of concentration scale. Biochemistry 10,
1329–1342.
42 Yancey PH (2005) Organic osmolytes as compatible,
metabolic and counteracting cytoprotectants in high
osmolarity and other stresses. J Exp Biol 208,
2819–2830.
43 Burg MB (1995) Molecular basis of osmotic regulation.
Am J Physiol 268, F983–F996.
44 Burg MB (2000) Macromolecular crowding as a
cell volume sensor. Cell Physiol Biochem 10, 251–256.
45 Bigelow CC (1960) Difference spectra of ribonuclease
and two ribonuclease derivatives. C R Trav Lab
zCarlsberg 31, 305–309.
Functional stability and activity by osmolytes S. Jamal et al.
6032 FEBS Journal 276 (2009) 6024–6032 ª 2009 The Authors Journal compilation ª 2009 FEBS