J. Mol. Biol.

(1989) 205, 731-750

Heat Capacity and Conformation of Proteins in the

Denatured State
P. L. Privalovl, E. I. Tiktopulol, S. Yu. Venyaminovl,
Yu. V. Grikol, G. I. Makhatadzel and N. N. Khechinashvili’

‘Institute of Protein Research and 21nstitute of Biological

Physics, Academy of Sciences of the U.S.S.R., 142292
Pushchino, Moscow Region, U.S.S.R.

(Received 20 July 1988, and in revised form 14 October 1988)

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins

(pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin
and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured,
with and without disulfide crosslinks or a heme) have been studied experimentally over a
broad range of temperatures. It is shown that the partial heat capacity of denatured
protein significantly exceeds the heat capacity of native protein, especially in the case of
globular proteins, and is close to the value calculated for an extended polypeptide chain
from the known heat capacities of individual amino acid residues. The significant residual
structure that appears at room temperature in the denatured states of some globular
proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat
capacity, probably due to partial screening of the protein non-polar groups from water. The
heat capacity of the unfolded state increases asymptotically, approaching a constant value
at about 100°C. The temperature dependence of the heat capacity of the native state,
which can be determined over a much shorter range of temperature than that of the
denatured state and, correspondingly, is less certain, appears to be linear up to 80°C.
Therefore, the denaturational heat capacity increment seems to be temperature-dependent
and is likely to decrease to zero at about 140°C.

guanidinium hydrochloride (Gu * HCl) or urea, while

1. Introduction
The denatured states of proteins have attracted
increasing attention. This interest arises from the
realization that the denatured state is the only
experimentally achievable state of a protein that
can be taken as an initial reference in considering
the mechanism of folding and stabilizing the native
protein structure, However, a denatured state can
play this role only if it can be associated with a
completely unfolded, random coil conformation of
polypeptide chain. The latter, as is clear, is an
idealized model that is never realized in practice,
since real polypeptides have too many groups
interacting in very different ways with each other
and with the solvent. However, it is unclear how far
the real situation is from the ideal, and how large
an error arises from equating a random coil with the
denatured state, particularly when we estimate
thermodynamic parameters for folding of the native
protein structure.
For many years it has been accepted that the
best approximation of a random coil polypeptide is
a denatured protein in concentrated solutions of
737
0022-2836/89/040737-14 $03.00/0
a heat or acid-denatured protein contains too much
residual structure to be regarded as a completely
unfolded, unstructured state (see Tanford, 1968).
This conventional view was shaken first when it was
shown, by direct calorimetric measurements, that
the enthalpies of lysozyme denaturation by heat
and GuoHCl are identical if the denaturant
solvation is correctly taken into account (Pfeil &
Privalov, 1976a). It was found also that the partial
heat capacities of the heat and GueHCl denatured
lysozyme are indistinguishable.
The importance of the partial heat capacity in
specifying the denatured state of proteins follows
from the fact that this parameter is a sensitive
index of the completeness of protein unfolding. The
exposure of the internal non-polar groups to water
should result in a heat capacity increment, since the
transfer of non-polar compounds to water is
associated with a significant increase of the heat
capacity (Kauzmann, 1959). On the other hand, the
heat capacity is the temperature derivative of a
basic thermodynamic function, the enthalpy There-
fore, the denaturational heat capacity increment
0 1989 Academic Press Limited
 P. L. Privalov et al
determines the temperature dependence of the
enthalpy and, hence, of the entropy of denatur-
ation, i.e. the parameters that determine the native
state stability.
The first more or less circumstantial study of the
denaturational heat capacity increment of globular
proteins was carried out by Privalov & tion from “Reanal” (Hungary), was recrystallized 3 times
Khechinashvili (1974), who showed that its value is
likely to be specific for a given protein and does not
depend on temperature. However, in that study,
the heat capacity was measured over a limited
temperature range from 20 to 90°C and calorimetric
experiments were not accompanied by studies of
the conformation of the denatured proteins. There-
fore, one cannot exclude the possibility that heat
capacity increment is caused by gradual melting of
the residual structure upon increasing temperature
and that, over a broader range of temperature,
there will not appear some temperature dependence
of the denaturational heat capacity increment on
temperature. To clarify these points, it was
necessary to make calorimetric measurements in a
much broader range of temperature than had been
done before, and to accompany the measurements
with studies of protein conformation.
Here, we report the results of a detailed
calorimetric study of the heat capacity of a number
of globular proteins in a temperature range from
-5 to 13O”C, and the results of viscosimetric and
spectropolarimetric studies of these proteins. Such
an extension of the temperature range of calori-
metric measurements became possible due to a new
-model of the precision scanning microcalorimeter
with capillary cells, which permits measurements
under considerable excess pressure at temperatures
above 100°C and scanning down the temperature
scale to measure aqueous solutions in a supercooled
state.
We chose five globular proteins that are most
popular among physical chemists; pancreatic ribo-
nuclease A, staphylococcal nuclease, hen egg-white
lysozyme, sperm whale myoglobin and horse heart
cytochrome c. These proteins were examined with
the disulfide crosslinks intact or disrupted and with
the heme groups in place or removed (apo-form). The
sixth globular protein used in this study was cata-
lase from thermophilic micro-organisms, which was
included for its extreme thermostability, in order to
clarify aspects of the heat capacity of the native
state. The seventh example, collagen, was chosen to
represent a quite different class of fibrillar proteins.
2. Materials and Methods
Metmyoglobin (Mb?) was isolated from frozen sperm
whale muscle according to Hapner et al. (1968). The
procedure for its further purification was as described by
Privalov et al. (1986).
Apomyoglobin (aMb) was obtained from sperm whale
metmyoglobin by precipitation with acetone according to
Rossi Fanelli et al. (1958).
Cytochrome c (Cyt) was isolated from bovine heart as
described by Margoliash & Walashek (1967).
Apocytochrome c (aCyt) was obtained according to
Fisher et al. (1973).
Ribonuclease A (RNase) was chromatographically
purified from the commercial preparation of bovine
pancreatic ribonuclease (“Reanal”, Hungary) using the
procedure described by Crestfield et al. (1962).
Hen egg-white lysozyme (Lys), a commercial prepara-
before use.
Staphylococcal nuclease (Nase) was isolated from a
homogenate of Escherichia coli cells that had been
transformed with a recombinant plasmid containing the
gene for the enzyme by Dr Robert Fox (Yale University)
and kindly provided for this study by Professor J.
Sturtevant (Yale University). This construction results in
the synthesis of a modified nuclease in which the
heptapeptide Met-Asp-Pro-Thr-Val-Tyr-Ser is appended
to the amino-terminal end of the nuclease; i.e. its
molecular mass is 17,600 and not 16,807 Da (for a
detailed description of this preparation, see Calderon et
al., 1985).
Catalase from Thermus therwwphilus (CTT) was a kind
gift from Dr Barynin from the Institute of Crystallo-
graphy of the USSR Academy of Sciences, who isolated it
from micro-organisms and purified as described by
Barynin & Grebenko (1986).
Collagen from pike skin (Coll) was purified from an
acid-soluble extract according to Glimcher et al. (1964).
The heptapeptide Glu-Lys-Lys-Leu-Glu-Gln-Ala
(Lys = lysine) was synthesized by Potekhin et al. (1988) in
our Institute and kindly provided for our experiments.
The purity of each preparation was checked electro-
phoretically in denaturing (Laemmli, 1970) and non-
denaturing (Ornstein, 1964) conditions, which showed
that the amount of impurities was less than 2%.
Disulfide crosslinks in RNase and Lys were disrupted
by reducing cysteine with thioglycolic acid in the
presence of 8.5 M-urea at 20°C. The SH groups of cysteine
residues were then carboxamidomethylated by iodoaceta-
mide according to the method of Sela et al. (1959). The
t Abbreviations used: Mb, metmyoglobin; aMb,
apomyoglobin; aMb”, apomyoglobin previously heated; Cyt,
cytochrome c; aCyt, apocytochrome c; RNase, ribonuclease A;
RNase-“, ribonuclease A with disrupted S-S crosslinks; Lys,
hen egg-white lysozyme; Lys-“, hen egg-white lysozyme with
disrupted S-S crosslinks; Nase, staphylococcal n&ease; C!l!T,
cat&se from Thrmua them$iZus; Coil, collagen from pike
skin; c.d., circular dichroism; EH, 3ro helix; Gu, guanidinium.
AC’“s(T) pl,aol,~lv, calorimetrically recorded apparent difference
in the heat capacity of the protein solution and solvent.
C (Thzo, spe cific heat capacity of water at temperature T.
C$T) partial specific heat capacity of native protein.
C$T)z: partial sp cific heat capacity of denatured protein.
AC&, = q,af - Cv,s, denaturational increment of protein heat
capacity.
A$f(T) = HD(T) - HN(T), the enthalpy difference of the
denatured and native states of protein.
A$S(T)=SD(T)-SN(T), the entropy difference of the
denatured and native states of protein.
V(T),,, partial specific volume of the protein in solution at
temperature T.
V(T),,,, specific volume of water at temperature T.
w(T),,, protein concentration in the solution at temperature
T.
$yd.ptein intrinsic viscosity at temperature T.
molecular elliptioity at the wavelength 2.
m.l, mean
’ molecular mass of an amino acid residue of a
protein.
A, number of residues in the polypeptide chain of protein.
 Heat Capacity of Proteins 739

amount of reduced SH groups was tested &a described by
Boyer (1954).
All the measurements were carried out in 10 mM-salt-
free glycine or phosphate buffer solutions of acidic pH
since, under these conditions, the proteins studied are
most soluble and no aggregation is observed upon
heating. The presence of aggregation was tested by high-
pressure liquid chromatography at various temperatures.
After heating experiments, the preparation was checked
electrophoretically under reducing conditions to ensure
the absence of hydrolysis of the polypeptide chain at
elevated temperatures.
Prior to measurements, solutions were carefully
dialyzed against solvent. Several replacements of the
solvent were used to achieve complete equilibration of
low molecular weight compounds in the solvent and
solution used in differential measurements. After dialysis,
solutions were centrifuged for 40 min at 24,000 g. Protein
concentrations in solution were determined spectrophoto-
metrically with correction for light-scattering (Winder &
Gent, 1971). The extinction coefficients were found, in
separate experiments, by measuring the nitrogen content
in stock solutions according to Jaenicke (1974). For
solutions of pH 2.0 to 5.0, the following extinction
coefficients were used: Er&,., = 176 for Mb, 9.0 for aMb,
26.9 for intact Lys, 25.25 for Lys with reduced disulfides,
10.0 for CTT, 9.39 for Nase, 9.2 for aCyt, E:%,,snm = 7.32
for intact RNase, 7.25 for RNase with reduced disulfides,
and Eaon, = 9-06 for Cyt.
Calorimetric measurements were peformed using a
DASM-4A capillary scanning microcalorimeter equipped
with gold cells of 1.0 ml volume (Bureau of Biological
Instrumentation of the Academy of Sciences of the
USSR). To extend the heating range to 13O”C, all
measurements were performed under an excess pressure
of 506,625 Pa. For measurements below O”C, the
solutions were supercooled as described earlier (Privalov
et al., 1986). The heating rate was from 1.0 to
2.0 K min- ‘. The protein concentration in the solutions
used for the experiments varied from I.0 to 5.0 mgml-‘.
The partial specific heat capacity of the protein in the
solution was determined as described by Privalov &,
Potekhin (1986) from the calorimetrically recorded
apparent difference in the heat capacity of the protein
solution and solvent, ACBpPP(T)pr.sollsol, and the apparent
difference in the heat capacities of the solvent and water,
where v(T) is the operational volume of the calorimetric
cell at temperature T, w(T),, is the protein concentration
in the cell at this temperature in gml-i, V(T)H20 and
C,(T),,, are, respectively, the specific volume and
specific heat capacity of water at this temperature and
UT),, is the partial specific volume of protein at this
temperature. For the specific volume and specific heat
capacity of water, data tabulated in standard handbooks
(Kell, 1971) were used. The partial specific volumes of
proteins were determined from the density of the protein
solution, P(T)~~.~~, and that of the solvent, P(T)~~~“,
measured by a vibrational densimeter DMA-2 (Anton
Paar, Austria), using the equation (Masterton, 1954):
WY,, = PICwm,rIH~ - ~P~~~,,.,,,l/~P~~~~O,“l
+ b4m/wh,,4. (2)
It should be noted that, at the protein solution
concentrations (20) studied, the values determined by
eqns (1) and (2) do not show any concentration
dependence and can be considered as corresponding to
infinitely dilute solutions; i.e. Cp,pr = CEpr and VP, = VE
(Fig. 1).
The partial specific volume of the protein was
determined over a temperature range of 2 to 8O”C, where
it exhibited a linear temperature dependence (Fig. 2). For
heat capacity determinations at higher temperatures, we
used the extrapolated values of the partial specific
volume. At the accuracy with which partial specific
volumes could be determined in these experiments
(*0403 cm3g-‘), no difference was noticed between the
partial specific volumes of the native, denatured and
completely unfolded forms of a protein at a given
temperature.
The solution viscosity was measured in an Ostwald
viscosimeter with a water flow-time of about 200 s at
25°C and in a VBA-1 automatic rotational viscosimeter
(Bureau of Biological Instrumentation of the USSR
Academy of Sciences). A great advantage of the second
instrument is that the protein solution does not contact
air and is under an excess pressure. Therefore, the protein
solution does not foam during experiments, as occurs in
the usual capillary instruments at elevated temperatures.
The protein intrinsic viscosity [q]r was determined by
extrapolating the reduced viscosity determined from the
solution flow-time (or period of rotation) t to zero

ACPpPpV)so,v,u~o: concentration:
tvl~ = lim [@--WM (3)
W-+0

where the protein concentration w (in gml-‘) and the

T
m 20 -A+.--.- 7-----*---R-----+
7 #
Y
2
& 1.5-
d,- -o-e- AL?
G - o--o----o
I.O-
1 I I 1 I 1 I I I
I.0 3.0 5.0 7.0 9.0
Concentration (mg mPl

Figure 1. Concentrational dependence of the partial specific heat capacity of Lys (0) and LYS-‘~ (0) in 10 mM-
glycine (pH 2.5) at 25°C.
740 P. L. Privalov et al.

0.65’ 1 I I I I I I I I
0 20 40 60 80
Temperature PC)

Figure 2. Temperature dependence of the partial specific volume of Lys (O), RNase (n), Mb (A) and Cyt (0) in
10 mru-glycine (pH 2.5). Filled symbols correspond to proteins with disrupted S-S crosslinks and apo-forms.

solvent flow-time to are measured at the same tempera- unfolded conformation. This is a very important
ture, T. The extrapolation was done using at least 5 circumstance for further interpretation of all the
measurements on solutions with different concentrations experimental results obtained in this study.
over the range of 1 to 5 mg/ml-’ (Fig. 3). The intrinsic viscosity of the native protein at
The circular dichroism spectra in the range of 183 to
10°C is low and is almost independent of pH
320nm were measured with a Jasco-41A spectropolari-
meter (Japan) in thermostated cells with a pathlength throughout the pH region in which this protein is in
from 0.15 to 0.9 mm using solutions with -protein the native state (Fig. 4). For Lys and RN&se with
concentration varying from 0.2 to 2.0 mg ml . The intact S-S crosslinks, the native state is realized
molar ellipticity was determined as throughout the entire acidic pH range at 10°C.
However, in the case of much less stable proteins
[e]pw = (4%+ ~aA/W) (4) that denature at acidic pH values, the intrinsic
where w is the protein concentration (in g ml-‘), 1 is the viscosity increases considerably at decreasing pH.
light pathlength in the cell (in mm), e1 is the measured The observed denaturational increment of intrinsic
ellipticity (in degrees) at a wavelength 1, and g,, is the viscosity is especially large in cases where the
mean molecular mass (in daltons) of an amino acid
polypeptide chain is not crosslinked by disulfide
residue of a protein determined from the known sequence
of the given protein. For the proteins studied, this
bonds, as occurs for Mb. Upon disruption of S-S
quantity varied from 93 for collagen to 116 for crosslinks, intrinsic viscosities of Lys and RNase
apomyoglobin (for details of the experimental procedure, increase at low pH to the same extent as that of
see Venyaminov & Gogia, 1982; Brzeska et al., 1983). Nase, Mb and Cyt. It is notable that with increasing
pH, the intrinsic viscosities of these polypeptide
chains decrease, though not to the level of the
native compact proteins. A similar situation is
3. Results observed with previously heated apomyoglobin
(a) Viscosimetric study @Mb”), which does not regain its native-like
compact structure (Griko et aZ., 1988), but its
As seen in Figure 3 for lysozyme with disrupted intrinsic viscosity drops to a remarkably low level
S-S crosslinks (Lys-““), the reduced viscosity of the at neutral pH values. It appears that heating of
unfolded polypeptide does not depend significantly aMb to 100°C leads to such a chemical modification
on the protein concentration in solution at pH 2.2 of some of its amino acid residues that its
at any temperature studied. This shows that, under polypeptide chain cannot fold into a native
the chosen solvent conditions, the interaction structure (on this aspect, see Zale & Klibanov,
between protein molecules is rather low even in the 1986).

I I I I I
I.0 2.0 3.0 4.0 5.0
Concentration (mg ml-‘)

Figure 3. Concentrational dependence of the reduced viscosity of Lys e-S’ in 10 mM-glycine (pH 25) at various
temperatures (“C).
 Heat Capacity of Proteins 741

-I
Table 1
(a) Intrinsic viscosity of unfolded proteins at 25°C
(RNase and Lys without S-S crosslinks and Mb and
Cyt without the heme in solutions with pH 2.2 and
6 M-Cu. HCI)

[11 rtl1t W’c

pH 2.2 6 M-Gu.HCI for random
Protein 12 (cm’ g-l) coil

acyt 11,702 104 15.0 149

RNase-‘” 13,683 124 15.6 16.1 16.6
14,396 129 17.0 17.6 17.0
17,170 153 20.0 20.1 19.1

M, molecular mass; 12,number of amino acid residues; [q],

intrinsic viscosity; [q]c”c, values for the random coiled
polypeptide calculated according to Tanford (1968) by eqn (5).
t Values from Ahmad & Salahuddin (1974).

last column of Table 1. As is seen, the experimental

values of intrinsic viscosity of the considered
polypeptide chains without S-S crosslinks or a
heme are rather close to the value calculated for a
random coiled polypeptide. Thus, one can suppose
that in solution at pH 2.0, the polypeptide chains of
0' 1 , I I I I all the proteins studied are in a random coil
2.0 4.0 6.0 conformation at 25°C if they are not fixed by S-S
PH crosslinks.
Figure 4. pH dependence of the intrinsic viscosity of An increase of temperature leads to a significant
proteins at 10°C. (a) Lys and RNaae with intact and decrease of the intrinsic viscosity of the unfolded
disrupted S-S crosslinks and aCyt; (b) Mb with the heme polypeptide chain (Fig. 5). The effect of tempera-
groups in place and removed. Symbols: Lys (O), Lys-” ture on the intrinsic viscosity of unfolded poly-
(01, R&se (Cl), RNase-w (ml, Cyt (01, aCyt (+), Mb peptide chains was observed by Ahmad &
(AL aMb (A), aMbh (A). Salahuddin (1974) for proteins in concentrated
solutions of Gu.HCl; but, in that case, the
temperature influence is even more pronounced
than in the case where unfolding of the polypeptide
With decreasing pH, the intrinsic viscosity of a chain is caused by extreme pH, i.e. by the charges
non-crosslinked protein rises to some constant level, on the polypeptide chain.
which is specific for a given protein at a given The temperature influence on the intrinsic
temperature and ionic strength of the solution. viscosity of the unfolded polypeptide chain can be
These maximal values of intrinsic viscosity, which
are achieved at pH below 3-O at 25”C, are
summarized in Table 1 for RNase and Lys without
SS crosslinks and for the apo-forms of Mb and Cyt.
The Table presents values for the intrinsic viscosity
of the same proteins in 6 M-Gu.HCl solution
obtained by Ahmad & Salahuddin (1974). As can be
seen, they are in rather good correspondence with
our values obtained in acidic solutions without a
denaturant. There is a clear correlation of intrinsic
viscosity values with the molecular weight of the
corresponding protein or with the number of amino
acid residues in the polypeptide chain.
According to Tanford (1968), the intrinsic
viscosity of the polypeptide chain in a random coil
conformation at 25°C can be expressed as:
[q] = 77.0 n”.666/iiZ5.r, (5) Figure 5. Temperature dependence of the intrinsic
viscosity of proteins: (1) RNase, pH 3.0; (2) RNase,
where iii,, is the mean molecular mass of an amino pH 4.0; (3) Lys, pH 3.0; (4) Mb, pH 5.0; (5) Lys, pH 4.0;
acid residue and n is the number of residues in the and of unfolded polypeptide chains: (6) aMb and Mb,
polypeptide chain. The values of intrinsic viscosity pH 3.0: (7) Lys-““, pH 2.2; (8) RNase-=, pH 2.2. The
calculated bv eauation (5) frnlcslc) are given in the
Y 1 T I \L ,J I 0
symbols are the same as for Fig. 4.
 742 P. L. Privalov et al.

explained by the increase of flexibility of the chain heat-denatured state is almost the same as that of
caused by the increase of rotational freedom of the random coil state.
backbones at increasing temperature. Clearly,
increased flexibility of the chain should lead to a
(b) Circular dichroism study
decrease in the hydrodynamic volume occupied by
this chain (Ahmad & Salahuddin, 1974). The Figure 6 presents c.d. spectra of the studied
decrease of the hydrodynamic volume of the coil proteins in various states; native, acid-denatured,
might be caused also by an increase of the heat-denatured and denatured by GuaHCl. The
hydrophobic pressure; i.e. the random hydrophobic spectra for all proteins in the concentrated solution
interactions between the non-polar groups of a of GueHCl are similar to those found by Dearborn
chain, since the hydrophobic interactions are known & Wetlaufer (1970), Tiffany 6 Krimm (1973) and
to increase at increasing temperature (Privalov et Cortijo et al. (1973).
al., 1986). The spectra of heat-denatured proteins are rather
It follows from the above that the con- close to the spectra of unfolded polypeptide chains
formation of a heat-denatured protein does not without disulfide crosslinks or heme at acidic pH,
differ greatly from that of a random coil, as it is but deviate significantly from the spectra of
supposed to be when the intrinsic viscosity of a proteins in 6 M-Gu.HCl. This deviation is especially
heat-denatured protein as measured at 80°C is clear at 222 nm, lO”C, but with a temperature
compared with that of Gu.HCl-denatured protein increase it decreases and is likely to disappear
measured at 25°C. Judging by Figure 5, the above 80°C (Fig. 7). It is notable that the deviation
intrinsic viscosity of a random coil polypeptide decreases due to a considerable temperature
chain at 80°C is only twice as large as that of the dependence of the ellipticity of protein in the
corresponding heat-denatured protein with intact presence of Gu. HCl.
S-S crosslinks, while for proteins without S-S The ellipticity of polypeptide chain at 222 nm is
crosslinks, such as Mb, the intrinsic viscosity of a usually considered as an index of its secondary

-15 ’ i
,‘.j, , Cyt., pH , 3.0 , , , I Collagen,
, ( pH, 3.5 (
200 220 240 200 220 240
Wavelength km)

Figure 6. Circular dichroism spectra of proteins in the native state at 10°C (continuous line) and temperature-
denatured state at 80°C (dot-and-dash line), both at the pH indicated in the boxes; in the acid-unfolded state with
disrupted S-S crosslinks or removed heme, at 10% pH 2.5 (dashed line), and in the presence of 6 M-GU . HCl at 10°C
(dotted line).
 Heat Capacity of Proteins 743

o ,+ ( , , , 1 43 . . . . . . . ,...‘..’ , I , , /
20 40 60 60
2.0 4.0 6.0
PH Temperature PC)

Figure 7. Dependence of the ellipticity of proteins at 222 nm on (a) pH at 10°C and (b) on temperature at various pH
values. The symbols are the same as for Fig. 4. The crossed symbol corresponds to the results in the presence of 6 M-
Gu.HCl.

structure (Chen et al., 1972). If one accepts that the
polypeptide chain is in a completely random coiled
conformation in a concentrated solution of Gu . HCl
(see Tanford, 1968), then it follows from Figures 6
and 7 that heat-denatured protein has considerable
residual secondary structure but its content
decreases upon a temperature increase and practi-
cally disappears at about 100°C. However, the
question is whether one can use the cd. spectra of
proteins in concentrated solutions of Gu.HCl as a
reference for the random coil conformation of the
polypeptide chain?
Our experiments with short synthetic poly-
peptides that could hardly have any helical
conformation in aqueous solutions showed that the
presence of Gu . HCl induces significant changes in
ellipticity (Fig. 8). This is not unexpected, as it is
known that Gu * HCI interacts with peptide groups
with a large negative enthalopy (Lee & Timashelf),
1974; Pfeil & Privalov, 1976a). According to
Robinson & Jencks (1965), one molecule of Gu . HCl
links with two peptide bonds; therefore, one should
expect that it can affect the conformation of the
polypeptide group. According to Tiffany & Krim
(1972, 1973), the high concentration of Gu . HCl and
urea favors the locally extended 3,,-helix
conformation (EH) of polypeptides. If so, the
effect of temperature on the ellipticity of proteins
in concentrated Gu * HCl solutions could be
explained by the decrease of the content of the EH
conformation at a temperature increase. This seems
quite likely, as increasing temperature should lead
to desolvation of Gu * HCl bound to polypeptide if
its binding enthalpy is negative. It follows then,
that proteins in concentrated solutions of Gu * HCI
attain a random coil conformation only at a rather
high temperature.
Another possibility that should not be excluded is
that GuaHCl can influence the intrinsic optical
properties of the individual amino acid residues,
and this influence is temperature-dependent (see
Hvidt et al., 1985). In this case, the observed optical
effect cannot be interpreted in conformation terms.
The above leads us to conclude that the
conformation of protein denatured by Gu. HCI or
acid is certainly close to a random coil only at
temperatures about 100°C. At lower temperatures,
there is some difference in the ellipticities of the
Gu*HCl and acid-unfolded polypeptides, which in
some cases amounts to 5666 deg cm2 dmol-‘; that
is, to about 15% of the value specific for the 166%
a-helix. However, this difference is hardly interpret-
able with certainty as evidence for the residual
helicity in the acid-unfolded polypeptide chain,
744 P. L. Privalov et al.

0 20 40 60 80
Temperature PC)

10°C
v , ( , ,
200 220 240
Wovelength (nm)

Figure 8. Ellipticity of the heptapeptide Glu-Lys-Lys-Leu-Glu-Gln-Ala (Lys = lysine) in 100 mM-sodium phosphate,
pH 7-O (continuous line) and in the presence of 6 M-GU. HCl (dotted line) at different temperatures.

since the nature of this systematic difference in the
ellipticities is unclear. Therefore, in considering the
conformation of a polypeptide chain we can only
neglect this difference (especially when it is small)
and suppose that the conformation of the acid and
GueHCl-unfolded polypeptide chains are both close
to a random coil.
For some of polypeptide chains, such as RNase-“”
and aCyt, the random-coil conformation is likely to
persist at higher pH up to 7.0 (Fig. 7). In the case
of Nase, whose unfolding and folding is a perfectly
reversible process, an increase of pH leads to folding
of its polypeptide chain into the native structure.
The increase of pH leads to an increase of negative
ellipticity at 222 nm of Lys-“’ and aMb. If lOOo/o
a-helicity disposes the ellipticity of 34,500 deg cm2
dmol- ’ at 222 nm, as reported by Chen et al. (1972),
then one can suppose that at pH 5.0 the content of
u-helicity reaches 8% in Lys-“’ and 25% in
preliminarily heated aMb’. This increase of the
a-helicity is accompanied by a decrease of the
intrinsic viscosity of these polypeptide chains (see
Fig. 4).
It is remarkable that, with increasing
temperature, the high ellipticity of Lys-“” drops to
the initial value within a temperature range from
30°C to 70°C (Fig. 7). In contrast to Lys-“, the
ellipticity of the preliminarily heated aMb6 is not
very sensitive to temperature. Thus, it does not
seem to be associated with an extended co-
operative structure.
(c) Calorimetric study
Figure 9 represents the partial specific heat
capacity of the proteins studied here in solutions
with various pH values as determined calorimetri-
cally over the temperature range -5°C to 130°C.
To expand the y-axis, we have omitted the tops of
the denaturational heat absorption peaks as, in this
paper, we are not interested in the denaturational
process itself, but in the heat capacities of the
native and denatured states. As can be seen,
increasing pH shifts the denaturation temperatures
to higher values, but does not noticeably change
either the heat capacity of the native state or the
heat capacity of the denatured state. These two
quantities are equal to within the experimental
error of kO.04 J K-r g- 1 with only two exceptions
(considered below).
However, the most remarkable observation is
that RNase and Lys without S-S crosslinks in
solutions of pH below 3.0, in which they are in
completely unfolded, random coil conformations,
have the same heat capacity as the denatured states
of these proteins with intact S-S crosslinks. A
similar situation is observed with Mb and Cyt; in
solutions with pH below 3.0, the denatured states of
 Heat Capacity of Proteins 745

completely unfolded state. Therefore, one can

suppose that the structure formed by Lys-” at pH
values close to neutral is quite open. This is
RNase confirmed by viscosimetric studies, which show that
the intrinsic viscosity of Lys-“” at pH 5-O is about
10.
In contrast to the structure formed by Lys-“‘,
the structure formed by the preliminarily heated
aMbh at pH above 4.0, as mentioned in the previous
section, does not change significantly with
increasing temperature (Fig. 7). Correspondingly,
we do not observe a clear heat effect that can be
Mh interpreted as additional heat absorption, although
there is some difference between the heat capacity
functions found at pH 5.0 and pH 2.5 (Fig. 9). This
difference amounts to O-1 J K-‘g-’ at a low
temperature (i.e. is about a 5th of the heat capacity
difference between the native and denatured states)
and gradually disappears upon heating from 40°C

2Jz#Y-- Nase to 70°C.

In all cases, the heat capacities of the native
protein are much lower than that of the denatured
I-’ 14 I I I I I / protein, and they differ considerably, providing a
0 20 40 60 80 too 120
large variation between the values of the denatura-
Temperature PC)
E;nal heat capacity increment A$?,,,, =
Figure 9. Temperature dependence of the partial P-PI - CF,PI for the proteins studied here (Table 2).
specific heat capacity of proteins with intact and The A% N p,pr values obtained at 25°C agree well with
disrupted disulfide crosslinks and with or without the previous findings (Privalov & Khechinashvili,
heme, in solutions with different pH values (indicated on 1974).
the curves). It is remarkable that the temperature depen-
dences of the heat capacity of all the proteins

these molecules have the same heat capacities as Table 2

the apo-forms of these proteins, which, according to Partial speci$c capacities of proteins in native (N)
viscosimetric and c.d. studies, are in the unfolded and denatured (D) states at 25°C measured
conformation close to a random coil. calorimetrically (exp) and calculated (talc) for the
The influence of pH on the heat capacity of the unfolded polypeptide chain (U) from the heat
denatured state is observed only for Lys-“” and capacities of constituting amino acid residues
aMbh. As shown above, with increasing pH above
4.0, both these polypeptides, and especially aMbh, Protein,
condition Cr.,, (exp)t CLpr (exp)t c;,r (calcH
form structures that are characterized by consider-
able helicity. Upon heating Lys-“” in pH 5.0 Nase,
solution, its additional helicity disappears over the pH 7.0 1.45
temperature range 30°C to 70°C (Fig. 7) and, in the pH 2.0-3.0 1.90 2.15
same temperature range, an extra heat absorption RNaN!,
pH 4.0 1.52
is observed that appears as a broad and flat peak, RNaWZ-““,
shown by a dotted line in Figure 8. The area of this pH 2.fS5.0 1.90 1.85
peak is about 5 Jg-’ (70 kJ mol-‘), which is less LYS,
than 20% of the enthalpy of lysozyme pH 2Xk5.0 1.40
denaturation. The van? Hoff enthalpy, determined LysC”,
pH 2.0-4.0 1.88 1.96
from the breadth of this peak, is of the same order. Mb,
This shows that the structure formed by Lys-‘” at pH 4+6.0 1.36
pH 5.0 is co-operative. From this point of view, this pH 2.C3.0 1.98
structure resembles the native structure of lyso- aMb,
pH 5.Ck6.0 1.60 1.90
zyme with intact crosslinks, the more so because pH 2.0-4.0 2.02 2.28
they are also comparable in ellipticity. However, Cyt,
these two structures differ essentially in the pH 3.0-6.0 1.42 -
enthalpy of formation and in heat capacity. The aCyt,
heat capacity of Lys-” at pH 5-O and 25”C, where pH 2G6.0 2.03 2.22
Coll,
temperature-induced melting of its structure does pH 3.0 1.35 1.55 1.58
not occur yet judging by its ellipticity, is almost the
same as that of Lys-” at pH 2.5, which is in a f c,.,r in JK-‘g-l.
746 P. L. Privalov et al.
40
Temperature PC)
Figure 10. Temperature dependence of the heat capacity of RNase (pH 4.0), Lys (pH 3.5), Mb (pH 4.4), C!l!T (pH 8.5)
and RNaseWsS,Lys-“, aMb ai pH 2.5.
studied here in the completely unfolded state are
represented by rather similar functions. Over the
broad temperature range of - 5°C to 13O”C, this
function does not appear linear, as it appeared
when examined over the smaller temperature range
from 20°C to 90°C (Privalov t Khechinashvili,
1974). For the native protein, one cannot draw the
same conclusion, since the temperature range over
which the native state can exist is much smaller.
Even for such a thermostable protein as catalase
from thermophilic micro-organisms (CTT), the pure
native state can be observed with certainty only up
to 80°C (Fig. 10).
4. Discussion
As has been shown above, the polypeptide chain
of protein without S-S crosslinks and the heme in
acidic solutions with pH below 3-O appears to be in
an unfolded state with little, if any, residual
structure.
An increase in temperature leads to some
squeezing of the hydrodynamic volume of an
unfolded polypeptide chain, but this does not at all
imply the formation of a compact structure. On the
contrary, temperature-induced squeezing appears
to be a general property of all polymers and, at high
temperature, the conformation of a polypeptide
chain is likely to be even closer to a random coil
judging by its ellipticity.
With increasing pH, polypeptide chains fold into
a structure that is characterized by some helicity
(Fig. 7). In some cases this is a compact, co-
operative native structure (as in the cases of Nase
and aMb); in other instances, this is a not quite
compact, but co-operative structure (as in the case
of Lys-““); and in some cases it is neither compact
nor co-operative, as with the preliminary heated
apomyoglobin, aMb”. In the latter case, the
enthalpy of the structure formation is found to be
close to zero (Griko et al., 1988). Therefore,
temperature does not affect this structure greatly.
60 80 100 120
Crosslinking of a polypeptide chain by disulfide
bonds results in a significant decrease of its intrinsic
viscosity, i.e. of the hydrodynamic volume occupied
by this chain, as expected. However, this does not
mean in itself that a crosslinked polypeptide chain
has a significant amount of residual structure.
Indeed, the ellipticity of heat-denatured Lys and
RNase with intact S-S crosslinks is very close to
the ellipticity of these polypeptide chains without
S-S crosslinks, which are in a completely unfolded
state, and, upon temperature increase, these two
values of ellipticity become closer (Fig. 7). Thus, it
appears that, at a sufficiently high temperature
(above SO’%), the conformation of the heat-
denatured protein in acidic solutions is rather close
to a random coil (i.e. crosslinked random coil).
Before discussing our experimental results on the
heat capacity of proteins, it is interesting to
consider what should be expected for the heat
capacity of an extended polypeptide chain in which
all the groups are in perfect contact with water, and
in which no residual structure is supposed to be
present. Let us assume, in the first approximation,
that each amino acid residue contributes addi-
tively to the integral heat capacity of such a
chain as if these residues have an identical peptide
group (CHCONH) and differ in the side-chains (R),
and there are two terminal groups (NH,) and
(CHCOOH) in the chain. The integral heat capacity
of the extended polypeptide can be represented as:
C ca1c _ c
P>PI - p,NH2 + (n- ~~‘P,CHCONH
+ f ‘p,r + cp,C”COOH. (6)
i=l
The partial heat capacities of all these groups were
calculated from the known partial heat capacities of
model compounds in dilute aqueous solutions, and
for some of the groups they were determined by
direct calorimetric measurements of the heat
capacity of tripeptide Gly-X-Gly in solution
(Makhatadze, Medvedkin & Privalov, unpublished
results). The heat capacity values used are listed in
 Heat Capacity of Proteins 747

Table 3 to neutral, does not affect significantly the heat

Partial heat capacities C,,i of polypeptide groups capacity function. Its deviation from the function
for a fully unfolded polypeptide does not exceed
7% of the value of the specific heat capacity of the
Procedure for protein, i.e. is less than 20% of the heat capacity
Group Analog calculating 6,
difference between the native and denatured states
NH, t -5 of a protein.
CHCOOH ql,CH+ c, co + C,,,, 36 It follows then, that the denaturational heat
CHCONH CH,CONHf Calorimetric(I 19 capacity increment cannot be explained by a
R WY) Hydrogen?’ 75 gradual melting of the residual structure in a
R (Ala) Methane5 c -c 168
denatured protein. It cannot be explained either by
R (Val) Propane§ CT;:::
- c;::: 293
R (He) 1-Butane§ CPA
- ql,” 395 the increase of the configurational freedom of the
R (Phe) Methylhenzene§ %A-c,,, 355 polypeptide chain upon the “disruption” of the
R (TY~) 4-MethylphenoQ GA- C&H 309 rigid native protein structure. According to
R (Ser) Methanol5 C&A
- C,,” 83
Sturtevant (1977) and Velicelebi & Sturtevant
R (Thr) Ethanols GA-c,., 183
(1979), the contribution of this effect to the
R (Asp) Acetic acid5 %A- C,.” 90
R (Am) Acetamide$ CPA
-c&H 85 observed denaturational increment of the protein
R (Glu) Propionic acids %A- C pH 178 heat capacity cannot be large. This was shown
R (Gln) Propionamide§ (I,,* - c,:, 179 experimentally by measuring the heat capacity
R (LYs) n-Butylamine§ ~P.A -(Jim 347
377 change upon melting of membranes, which can be
R (I-) G.“,, + %,H
R (Pro) Gly-X-Gig Calorimetric~~ 175 considered as a model of protein denaturation
R (HYP) G&X-Glj CalorimetricI] 168 without penetration of water inside the molecule,
R IMet)
~ Glv-X-Glv CalorimetricI/ 173 i.e. without unfolding of the hydrophobic core, but
R (His) GI;-X-G6 Calorimetric/l 176
CalorimetricI\ 332
with a significant increase of the configurational
R (Trp) Gly-X-Gly
R (Ax) n-Propyl- freedom (Bendzko et al., 1988). The inconsistency of
guanidinefr %4 - (kH 266 the explanation of the protein denaturation heat
R (CY#I (:p,t.,e,- q,.cH + %co + CF.,,” 17 capacity increment by the decrease of the
configurational freedom becomes especially evident
Heat capacities of groups CH,. CH, NH,, CO, OH, 0 and H
were taken from Gutrie (1977). in considering the cold denaturation phenomenon,
t Gutrie (1977). which induces a similar heat capacity increase as
$ Jolicoeur & Boileau (1978). heat denaturation, notwithstanding the fact that it
4 Cabani et al. (1981). is induced by decreasing temperature (Privalov et
1)Makhatadze et al., unpublished.
7 Carboxymethylated.
al., 1986).
Therefore, the only reasonable explanation for
the observed denaturational heat capacity incre-
Table 3, which gives also the model compounds ment is the assumption that it is caused mainly by
together with corresponding references to their heat hydration of the non-polar groups, which are
capacity values and to the procedure used to derive exposed to water upon unfolding of the compact
the heat capacity of a group from the heat capacity protein structure. This was first suggested by
of an analog. Kauzmann (1959), on the basis of the well-known
The values of the integral heat capacity of fact that the transfer of non-polar molecules to
extended polypeptide chains, calculated per gram of water is accompanied by a significant heat capacity
protein, are given in Table 2. As can be seen, they increase (Edsall, 1935; for a review, see Franks &
agree well with the experimental values of the heat Reid, 1973). One of the arguments confirming this
capacity of the denatured protein, but are some- assumption is that A$, is found to be proportional
what larger in all cases. The deviation, which does to the number of contacts between the non-polar
not exceed lo%, is not surprising, as the groups in native protein, i.e. actually to the surface
polypeptide chain, even in the random coil area of the non-polar groups that are exposed to
conformation, can hardly be considered as a dilute water upon folding of a compact protein structure
solution of the constituent groups. (Privalov & Khechinashvili, 1974; Privalov, 1979).
It was expected that a heat-denatured protein, One of the most important facts established by
which was supposed to be not as unfolded as the these calorimetric studies of protein performed over
Gu.HCl-denatured protein, should have a lesser a broader temperature range than that examined
heat capacity than the protein in a concentrated previously is that the partial heat capacity of a
solution of Gu. HCl (Tanford & Aune, 1970). denatured protein is not a linear function of
However, direct calorimetric measurements carried temperature. At temperatures above 6O”C, the
out on lysozyme, showed that, if the GueHCl slope of this function asymptotically decreases and
solvation effects are correctly taken into account, almost disappears at about 100°C. It is tempting to
no difference between the Gu.HCl and heat- explain the observed curvature of the heat capacity
denatured protein heat capacities can be observed function by extended heat absorption caused by
(Pfeil & Privalov, 1976a). Now we see that even a some gradual process (i.e. the melting of the
very extended residual structure, which persists in residual structure), which is superimposed on the
the preliminarily heated aMbh, at pH values close trivial temperature-induced increase of the heat
748 i? L. Privalov et al.

for the enthalpy and entropy functions we have:

T
A;H(T) = A;H(T,) + 1 A:C,dT (7)
T,

@W’) = C~~HV’,W,+ j N$&M’l dT, (8)

T,
where T, is transition temperature at which:
A;H(T,) - T,A$‘(T,) = 0.

I ,
If A$?, does not depend on temperature, the
50 100 150 enthalpy and entropy of protein denaturation will
Temperature PC) be increasing functions of temperature; namely, the
Figure 11. Denaturational increment of the partial
enthalpy will be a linearly increasing function (see
heat capacity of RNase, Lys and Mb. The dashed lines Privalov, 1979). However, if A$?, is temperature
represent parts of these functions that were obtained by dependent and decreases to zero at some tempera-
a linear extrapolation of the heat capacity of the native ture T,, then AEH and A$S will be functions that
state. The dot-and-dash lines show the behavior when the are asymptotically approaching some constant
values measured at 50°C are assumed to be temperature- value at To. It is most interesting that, for the
independent. specific enthalpy and entropy, this constant value
appears to be universal for all compact globular
proteins. This is shown in Figures 12 and 13 for two
proteins (RNase and Mb) that differ greatly in their
capacity of the polypeptide chain. However, the
denaturational heat capacity increments. As seen
universality of this effect, and the fact that it is
from these Figures, these asymptotic values are
observed under conditions when the unfolded very close in magnitude to the values at the
polypeptide chain hardly possesses any residual
intersection of the enthalpy and entropy functions
structure, urge us to consider the shape observed
derived on the assumption that AEC, is constant,
for the heat capacity function as an intrinsic
which takes place at about 110°C. However, while
property of the unfolded polypeptide chain in
the values at the intersection of these functions at
water, as we consider the effect of squeezing the
110°C do not have a direct physical sense (as upon
hydrodynamic volume of a random coil with
a further extrapolation above 110 “C these functions
increasing temperature.
The situation with the heat capacity function of
the native state is much less clear, since it can be
experimentally determined only to 80°C even for
the most thermostable protein, such as CTT
(Fig. 10). Within the region 0°C to SOY!, this
function appears to be linear but it is not
improbable that this linearity is only apparent,
being caused by a low-temperature tail of the
denaturation heat effect. Assuming this function to
be linear above 80°C we would conclude that the
denaturational heat capacity increment should be a
decreasing function of temperature, as shown in
Figure 11. The main argument for the correctness of
such an extrapolation is the fact that the heat
capacity increment of transfer of non-polar solutes
to water behaves in a similar way, asymptotically
decreasing with increasing temperature (Gill et al.,
1985; Makhatadze & Privalov, 1988). Then, such a I ,
behavior of the difference heat capacity function 50 100

seems to be much more natural since, if it is caused Temperature (‘73

by the influence of protein non-polar groups on the

Figure 12. Temperature dependence of the specific
state of surrounding water, one can expect that, at enthalpy of denaturation of Mb and RNase (per mol of
increasing temperature, this influence should amino acid residues) in solutions with pH and buffer
diminish and even disappear at a sufficiently high providing maximal stability of these proteins and
temperature. compensation of heat effect of ionization (see Privalov &
This is a very important conclusion for Khechinashvili, 1974). The broken extension of the
evaluating the temperature dependence of the continuous lines represents a region that is less certain
enthalpy and entropy of protein denaturation. due to uncertainty in the A$?, function (see Fig. 10). The
Since : dot-and-dash lines represent the functions calculated with
the assumption that the denaturation heat capacity
A$, = (aAEH)/aT and (A$Y,)/T = (aA$S)/aT, increment is temperature-independent.
 Heat Capacity of Proteins 749

r We are grateful to Drs S. J. Gill and B. G. Bar&s for

helpful discussion of the manuscript in its preparation.

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Edited by R. Huber