J. CHEM. SOC. FARADAY TRANS., 1993, 89(15), 2769-2774 2769

Effect of Osmoregulatory Solutes on the Stability of Proteins

Swarita Gopal and Jagdish C. Ahluwalia" Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi- 1 10 0 16, India

The thermal denaturation of ribonuclease A and lysozyme in aqueous solutions of osmoregulatory solutes methylamines [trimethylamine N-oxide, betaine (trimethylammonioacetate) and sarcosine] and urea have been investigated by differential scanning calorimetry (DSC). The transition temperature, heat capacity and enthalpy of denaturation in aqueous solutions of ribonuclease A and lysozyme at pH 6.00 have been determined by a least-squares fit of the excess heat capacity data to the two-state model. Thermodynamic functions of denatur- ation, AGO, AHo and ASo at various temperatures have also been evaluated. The methylamines increase the thermal stability of ribonuclease A and lysozyme in the order: trimethylamine N-oxide > sarcosine > betaine. The stabilizing effect of methylamines when compared with that of amino acids follows the order: tri- methylamine N-oxide > glycine > 6-alanine > y-aminobutyric acid > sarcosine > serine > a-alanine > betaine > proline. The structure-stabilizing effect of methylamines and structure-destabilizing effect of urea on ribonu- clease A are nearly additive. No net effect on the stability of ribonuclease A is observed in a 2 : 1 mixture of urea and methylamines.

The interaction between globular proteins and solvent plays a central role in determining the native conformation of pro- teins which is usually regarded as the conformation exhibited within the cellular environment or by isolated proteins at their maximum activity.' The growth, development and yield of organisms are adversely affected by hostile environmental conditions. Yancey et aL2 observed that cells under stress accumulate low-molecular-weight substances (polyhydric alcohols, amino acids, methylamines and urea) known as osmolytes. Marine organisms produce (10-100) x mol dm-3 intracellular osmolytes in order to withstand the denaturing stresses2

Urea is an important product of nitrogen metabolism and is accumulated by some Epecies as the major blood and intra- cellular ~ s m o l y t e . ~ - ~ In cartilaginous marine fishes and the coelacanth, urea is present up to ca. 0.4 mol dm-3. Aqueous urea solutions even at such relatively low concentrations have a strong perturbing effect on macromolecules. In such urea-accumulating species, the other major class of organic osmolytes present are the methylamines : trimethylamine N-oxide (TMAO), sarcosine and betaine.2-5 Methylamines display either non-perturbing or in some cases, favourable effects on enzyme-substrate and enzyme-co-factor complex formation, catalytic velocity and protein structural stabil-

and these are highly compatible with proper enzyme function and structure.

In the presence of urea alone, the protein can,become too labile to function.6 In the cellular osmolyte system, the pres- ence of methylamines alone is also as deleterious; since high concentrations of methylamines can stabilize proteins to such an extent that they make them too rigid to function effec- tively.2 The inhibitory effect of urea on the actions of a number of enzymes is largely or entirely overcome by TMAO and other me thy la mine^.^ Marine cartilaginous fishes and the coelacanth have urea and methylamines present in their cel- lular systems in a ratio of ca. 2 : l.4*5 The use of such a bal- anced mixture of urea and methylamine solutes reflects a selection for a solute system which preserves an optimal degree of protein conformational m~b i l i t y .~ Adoption of this osmolyte system has obviated the necessity for development of specially modified proteins for function in the presence of high urea or methylamine ~oncent ra t ion .~ .~

Studies of specific solvent effects of urea and methylamines may contribute to our understanding of the interactions and forces which stabilize the tertiary structure of biological

macromolecules. Despite numerous report^,^. - the mech- anism of the denaturation of proteins by urea has not been defined unequivocally. Urea is believed to function by increasing the aqueous solubility of the hydrophobic portions of proteins,' O?' ' while maintaining the hydrogen-bonding capability of the aqueous solvent^.'^*'^ St~dies'~-' ' involv- ing the transfer of solutes with non-polar moieties from water to aqueous urea solutions also indicate that the addition of urea to water destabilizes the water structure, causing dis- ruption of the hydrophobic bonds." Urea has a strong ten- dency to form hydrogen bonds with proton donors as well as proton acceptors and it has also been suggested that the effect of urea upon the relative conformation of proteins occurs either through the perturbation of the solvent struc- ture, affecting the hydrophobic interaction, or through the specific binding of the protein molecules, or both.7 Urea is

to interact especially with the amide and the peptide groups of the proteins apart from the hydrophobic effect on the non-polar groups.

Amongst trimethylamine N-oxide (TMAO), sarcosine (N- methylglycine) and betaine (trimethylammonioacetate), TMAO is uncharged but the other methylamines are zwitter- ions in the physiological pH range. The mechanism of the effect of these ions is not fully understood, but it appears to involve general aspects of solute and protein interaction with water ~ t ruc tu re .~*~ '

The thermal denaturation of ribonuclease A and lysozyme in aqueous solutions has been extensively The reversible nature of the denaturation of both proteins is well established. The study of thermal transition in the presence of urea and methylamines could provide a better understanding of specific solvent effects on these proteins. Here we report measurements of the transition temperature, q, enthalpy, Ad H, and heat capacity Ad C, of denaturation of ribonuclease A (RNase A) and lysozyme in the presence of urea and methylamines : TMAO, betaine and sarcosine.

Experimental The methylamines were procured from Sigma and were of 99% stated purity. They were dried over P20, in a vacuum desiccator for 72 h prior to use. Urea of analytical-reagent grade from BDH (India) was used after drying it in an oven at ca. 333 K for 12 h.

Bovine pancreatic ribonuclease A (lot no. 93F-0498, type

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online / Journal Homepage / Table of Contents for this issue

2770 J. CHEM. SOC. FARADAY TRANS., 1993, VOL. 89

R-4875) and lysozyme (lot no. 39F-8211, type L-6876) were also procured from Sigma. These proteins were exhaustively dialysed against distilled and deionized water at 277 K for over 24 h and lyophilized. The millimolar protein solutions were made in 0.02 mol dm-' NaCl and adjusted to the desired pH 6.00 with HCl or NaOH. The molecular weight of RNase A was taken to be 13 700 and that of lysozyme 14 600. The protein concentrations were measured spectrophoto- metrically by using absorbance values of 0.738 at 278 nm for 0.1 mass % RNase A and 2.635 at 280 nm for 0.1 mass % l y ~ o z y m e ~ ' * ~ ~ in 0.2 mol dm-3 Na2HP04-NaH2P04 buf- fered at pH 7.00.

Calorimetric measurements were performed on differential scanning microcalorimeter (SETARAM) at a scan rate of 0.6 K min-' from 298 to 371 K with a sample mass of 0.8-0.85 g at 25 pV amplification f.s.d. To analyse a DSC experiment, the instrumental baseline determined with buffer in both cells was substracted from the results obtained with the sample. All the excess power thermal scans were converted to excess heat capacity us. temperature scans, following the procedure described by Schwarz and Kirchhoff.26 Thermodynamic func- tions of protein denaturation, the transition temperature Td , heat capacity Ad C, and the Ad H of denaturation of RNase A and lysozyme in aqueous solutions of 0-2 rnol kg- methyl- amines and urea at pH 6.00 (0.02 mol dm-3 NaCl-NaOH) were determined from the least-squares fit of the excess heat capacity data to the two-state rn0de1.~'

The two-state model provides a functional dependence of the measured heat capacity on temperature of the following form

where a + b(T - Td) is the linear fit of the pre-transition baseline to T, and Aa + Ab (T - q) is the difference between extrapolation of the pre- and post-transitional baselines at T. Cp,s is the sigmoidal baseline extrapolated under the tran- sition curve and a is the fractional area under the transition curve at T . The transition enthalpy ddH is determined from

. the area under the transition curve and from the number of moles of protein in the cell.

Results A single reversible endothermic peak was obtained for lysozyme and RNase A under all conditions employed except

6.92 r c

I Y 7

> 0

E \

U .-

0

Q) c fn

c

8 X

-7.801 I i , I

322 332 342 352 362 , 372 T/K

Fig. 1 DSC thermal scan of lysozyme in 1.0 mol kg-' sarcosine at pH 6.00 (0.02 mol dm-3 NaC1-NaOH) at scan rate of 0.6 K min-'. Td = 353.08 K, A,,€€ = 503 kJ mol-'

in the case of lysozyme in the presence of TMAO, where the precipitation of solution was observed. A typical DSC ther- mogram of lysozyme in 1.0 mol kg-' sarcosine is shown in Fig. 1. Thermodynamic parameters obtained from the analysis of DSC scans of RNase A and lysozyme in the pres- ence and absence of methylamines and urea are summarized in Tables 1 and 2. Each value represents an average of three or four measurements and the uncertainty represents the standard error of the mean. The & values have an experi- mental error of k0.1 K and the AdH values have maximum expected error of +5% including errors in sample prep- aration, calibration constant and reproducibility. The co- operativity, which is the ratio of transition enthalpy to van't Hoff enthalpy for each measurement was determined by the approximate relation q = Ad H2/4RT,2 cd , where c d is the height of the transition peak per mole of protein at G. The G of RNase A and lysozyme in aqueous solutions of methyl- amines are illustrated in Fig. 2 and 3. A few experiments were carried out on the thermal transition of RNase A in the solvent system of urea-methylamine in a ratio of ca. 2 : 1 (Table 3). In Fig. 2 are also shown the of RNase A in mixture of urea and methylamines (2 : 1). A better estimation of the effect of methylamines and urea on protein stability is obtained by a comparison of the thermodynamic functions of denaturation, AGO, AHo and AS" at various temperatures. These values are the calculated differences in thermodynamic

Table 1 Thermodynamic parameters obtained from DSC scans on RNase A in the presence of varying concentrations of methylamines and urea

~~

0 0.25 1 0.500 1.009 2.002

0.500 1.001 2.012

.0.5 15 1.003 2.002

0.500 1.006 1.997

335.1 336.9 338.8 340.4 345.1

336.2 338.4 342.3

336.0 337.4 340.2

333.4 332.0 328.5

- 1.8 3.7 5.3 10.0

1.1 3.3 7.2

0.9 2.3 5.1

- 1.7 -3.1 - 6.6

trimethylamine N-oxide 441 442 448 483 505

440 441 444

betaine 442 445 457

sarcosine

urea 405 398 368

4.6 f 0.8 4.5 f 0.3 4.6 f 0.4 5.5 f 0.3 5.9 f 1.4

5.3 f 0.1 4.4 f 0.6 3.9 f 0.8

4.8 0.5 4.7 L- 0.6 3.7 * 0.2 4.8 f 1.1 3.7 f 0.7 3.3 f 1.2

0.963 f 0.010 1.019 f 0.022 1.010 k 0.025 1.004 f 0.004 1.003 f 0.021

1.005 f 0.011 1.021 f 0.003 0.989 f 0.021

0.993 f 0.032 1.013 f 0.016 0.999 f 0.004

0.879 _+ 0.011 1.004 f 0.024 1.01 8 f 0.020

~~

[RNase A] = 1.44 x mol dm-3, scan rate = 0.6 K min-'. pH 6.00 (0.02 mol dm-3 NaC1-NaOH).

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online

J. CHEM. SOC. FARADAY TRANS., 1993, VOL. 89 2771

Table 2 Thermodynamic parameters obtained from DSC scans on lysozyme in the presence of varying concentrations of methylamines and urea

m/mol kg-' T& ATJK Ad H/kJ mol-' Ad CJkJ K - ' mol-' tl

0 0.250 0.510 1.090

0.500 0.999 2.000

0.510 1.002 2.001

0.498 1.006 1.999

349.2 ppt. at 356 ppt. at 339 ppt. at 349

350.8 353.1 357.6

349.8 350.7 352.9

346.6 344.9 342.8

-

1.6 3.9 8.4

0.6 1.5 3.7

- 2.6 - 4.3 - 6.4

trimethylamine N-oxide 480

sarcosine 494 502 536

494 505 527

473 469 435

betaine

urea

6.1 f 0.7

3.1 f 0.3 4.7 f 1.0 1.3 f 0.4

1.3 f 0.2 7.0 f 1.5 2.9 & 0.2

5.9 f 0.1 3.7 * 1.0 2.4 f 1.4

0.998 f 0.021

1.004 +_ 0.004 0.985 f 0.021 i.100 f 0.027

1.073 f 0.021 1.002 f 0.012 0.999 f 0.032

1.017 f 0.011 1.002 f 0.021 0.999 f 0.009

[Lysozyme] = 1.40 x mol dm-3, scan rate = 0.6 K min-', pH 6.00 (0.02 mol dma3 NaCl-NaOH).

350 a Y, I-."

1:0.5 I

330 t 325

0 0.5 1 .o 1.5 2.0 rnlmol kg -

Fig. 2 Denaturation temperature of ribonuclease A as a function of methylamines and urea concentration. 0, TMAO; 0, sarcosine; A, betaine; 0, urea; 0, urea-TMAO (2:l); +, urea-sarcosine; A, urea-betaine.

355

5 350 I-."

345

340 I I 1 1 1

0 0.5 1 .o 1.5 2.0 rnlmol dm-3

Fig. 3 Denaturation temperature of lysozyme as a function of methylamine and urea concentration. 0, sarcodine; A, betaine; 0, urea.

Table 3 Thermodynamic parameters obtained from DSC scans of denaturation transition of RNase A in the presence of urea + methylamine

urea 0.0 0.502 0.997 1.963

urea 1.004

urea 1.006

TMAO - 0.0 335.1

0.25 1 335.2 0.1 0.499 335.0 -0.1 0.985 334.1 - 1.0

0.502 334.4 - 0.7

0.498 333.9 - 1.2

sarcosine

betaine

441 425 420 390

425

427

4.6 f 0.8 5.4 f 1.6 4.8 f 0.8 4.2 f 0.2

3.3 f 0.2

5.5 +_ 0.4 ~~

[RNase A] = 1.44 x mol dm-3, scan rate = 0.6 K min-'. pH 6.00 (0.02 mol dm-' NaCl-NaOH).

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online

2772 J. CHEM. SOC. FARADAY TRANS., 1993, VOL. 89

functions of the protein in the native and denatured states, at different temperatures for the transition of proteins in each solvent system. The values of AGO, AH" and ASo thus calcu- lated from eqn. (1)-(3) for the thermal unfolding of lysozyme in aqueous solution of sarcosine (0.0, 0.5, 1.0 and 2.0 mol kg-') at 298.15, 313.15 and 313.15 K are illustrated in Fig. 4. AGO values for the thermal unfolding of RNase A in aqueous solutions of TMAO in temperature range 298.15-373.15 K are illustrated in Fig. 5. These functions are obtained using the following equations with the assumption that the A, C, is independent of temperature,

AHo(T) = Ad H - (G - T ) Ad C,

Aso( T ) = A, H/Td - Ad c, In (&/T)

(1)

(2) and

Heat capacity of denaturation A,C, for each solvent com- position determined experimentally has high degree of uncer- tainty. The AdCp values of RNase A and lysozyme in methylamines and urea solutions were assumed to be 4.6 and 6.1 kJ K-' mol-', respectively, which are the values obtained for the proteins in aqueous solutions at pH 6.00.

Discussion Thermal denaturation of lysozyme and RNase A in aqueous solution of urea and methylamines is a two-state transition with a stoichiometry of one, as the mean value of q(=

0 n 0.3 0

365 -ftT

& 225 d

4 I

155 -!<y 120

0 0.4 0.8 1.2 1.6 2.0 rn/mol kg-'

Fig. 4 Variation of standard thermodynamic parameters of denaturation of lysozyme at 298 (O), 313 (A) and 333 (0) K with sarcosine concentration

60

40

c 20

E 3 0

8

-

2-

-20

-40

I L

-60 1 280 300 320 340 360 380

T/K Fig. 5 Variation of AGO of ribonuclease A with temperature, varying TMAO concentration. m/mol kg-': 0, 0.0; V, 0.5; 0, 1.0; 0, 2.0.

Ad H/AHvH), the cooperativity index, determined are 0.994 & 0.036 and 1.018 0.038, respectively, for RNase A and lysozyme. The transition temperatures of RNase A and lysozyme at pH 6.00 are 335.1 and 349.2 K, respectively. These values are close to the values reported in the liter- a t ~ r e . ~ ~ ~ ~ ~ * ~ ~ A, H at pH 6.00 for RNase A and lysozyme are 441 and 480 kJ mol-', respectively, which are close to the values obtained by Privalov.22 The G of RNase A and lysozyme decrease in the presence of urea as expected (Tables 1 and 2). A significant decrease of 73 and 45 kJ mol-1 in A, H of lysozyme and RNase A is observed in 2.0 mol kg- aqueous solution of urea. A depression of the denaturation temperature of proteins by urea has been- reported e a r l i e r . " ~ ~ ~ - ~ ~ This lowering of G increases with urea con- centration. The disruption of protein structure by the addi- tion of urea is said2' to be due to the direct interaction of urea with protein and its effect on the structure of the water in contact with the non-polar residues. There is ample proof that urea acts as a structure breaker.30

In the presence of methylamines, both proteins are ther- mally more stable (Table 1 and 2). The order of increase in G by methylamines is TMAO > sarcosine > betaine (Fig. 2 and 3). The enhancement of & is more for RNase A than for lysozyme. In the case of lysozyme, precipitation occurs in TMAO solutions, hence results could not be compared, though in betaine and sarcosine a similar increase in the G of lysozyme is observed. While A d H of both RNase A and lysozyme in urea solutions decreases, it increases in the pres- ence of methylamines. The recent DSC studies of Santoro et aL3' on the effect of glycine-based osmolytes (glycine, sarco- sine, dimethylglycine and betaine) on the thermal stability of RNase A and lysozyme also indicate that thermal foldings are reversible, two-state transitions even in very high concen- trations of osmolytes. They also observed that these osmolytes increased the thermal stability of lysozyme and RNase A, sarcosine being the maximum stabilizer. Our values of A% and Ad H in the presence of 2 mol kg - ' sarcosine and betaine are of the same order of magnitude as reported by Santoro et aL3'

AGO, AHo and AS" for the thermal unfolding of lysozyme in aqueous solutions of sarcosine (Fig. 4) and AGO of RNase A in aqueous solutions of TMAO (Fig. 5) are more positive

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online

J. CHEM. SOC. FARADAY TRANS., 1993, VOL. 89 2773

TMAO >

u-ahnine >

glyune > palanine

c0,-

betaine > proline

Scheme 1

than in water, indicating enhanced stability of the proteins in aqueous methylamine solutions. With increase in tem- perature AGO decreases, as expected, while AHo and ASo increase.

At all concentrations, the q of RNase A and lysozyme in sarcosine solution is more than in betaine. The probable explanation is that the betaine molecule is more hydrophobic because of the presence of two additional bulky CH, groups. From our studies,' on the effect of various amino acids (glycine, a-alanine, /3-alanine, y-aminobutyric acid, valine and leucine) on the stability of RNase A, it has been observed that the increase in the hydrophobicity of amino acids results in a decrease in the stability of RNase A. Santoro et aL3' have also shown that at low concentration of osmolytes, the greater the degree of glycine methylation the less effective the osmolyte tends to be in increasing &. Extending the co- sphere to betaine and sarcosine it can be said that there exist hydrophobic hydration cospheres for the alkyl sidechains and thehydrophilic hydration cospheres for the charged zwitterionic headgroups. The overlap of cospheres of these solutes with those of the exposed non-polar residues (of the unfolded protein) would squeeze out water from their hydrophobic hydration cospheres. This effect is greater in betaine due to the presence of two additional methyl groups. The enhanced hydrophobic interactions between the betaine and the non-polar groups of the unfolded protein reduces its stabilizing effect.

Although the structure of TMAO cannot be compared with betaine or sarcosine or amino acids, owing to the pres- ence of three methyl groups one would expect it to be hydro- phobic in nature and thus less effective in stabilizing proteins. However, TMAO, which is essentially an uncharged mol- ecule, is found to be the most stabilizing of the methylamines and amino acids. Possibly, it interacts favourably with water by entering into the water lattice, strengthening the solvent structure and increasing the hydrophobicity of the protein. Thus the non-polar groups on the protein surface would be expected to react unfavourably to contact with this mixed solvent (TMO-water). The polar or ionic groups on the protein surface may 'bind' TMAO through the interaction of TMAO with the water of hydration around polar groups of the proteins. This overall effect might be responsible for the most stabilizing effect of TMAO among all the osmolytes. Lee and Timasheff3' attributed the stabilizing effect of sucrose on proteins to the positive Gibbs energy required to form a cavity in the solvent needed for accommodating the protein molecule due to the increase in the solvent cohesive force when sucrose is added to water in the solvent system. Bull and Bree~e ,~ suggested that substances which stabilize proteins do so by enhancing the water structure in the imme- diate neighbourhood of the protein.

The q of RNase A in presence of mixture of urea and methylamines in a molal concentration ratio of 2 : 1 is found to be close to that of RNase A in water. The structure- stabilizing influence of methylamines and structure- destabilizing influence of urea on the thermal transition of RNase A are very nearly additive. They do not appear to interfere with each other's individual actions on protein. Owing to their opposite actions on the protein, at a particu- lar connection of urea and methylamine their individual effects become nullified, i.e. the methylamines counteract the effect of urea at a molal concentration ratio of cu. 2 : 1 (Fig. 2). For urea and betaine as the solvent system, at a molal concentration ratio of 2 :1, the effect is not fully counter- acted, as compared to that of TMAO and sarcosine, as the stabilizing effect of betaine is much less than that of TMAO or sarcosine.

Comparing methylamines with amino acids32 we find the order of their thermal stabilizing propensities to be as shown in Scheme 1.

At 1.0 mol kg-' of glycine and sarcosine A% for RNase A is 4.2 and 3.4 K, respectively. This indicates that the replace- ment of H by CH, in glycine reduces the magnitude of the thermal stability of RNase A. Similarly, because of the pres- ence of two additional methyl groups, betaine is less effective in stabilizing RNase A than a-alanine. The change in the rela- tive position of NH; and CO, groups could alter the elec- trostriction of water, which could also modify the overall hydrophobic hydration propensity of solutes indirectly, as in the case of sarcosine and a-alanir~e.~' The methyl group is attached to the electron-accepting ammonium group in sar- cosine, while in a-alanine, the methyl group is comparatively free to partake in the hydrophobic interaction. This effect gives more stabilizing ability to sarcosine than a-alanine.

References 1 G. Nemethy, W. J. Peer and H. A. Scheraga, Annu. Rev.

Biophys., 1981,10,459. 2 P. H. Yancey, M. E. Clark, S. C. Hand, R. D. Bowlus and G. N.

Somero, Science, 1982,217, 1214. 3 R. D. Bowlus and G. N. Somero, J . Exp. Zool., 1979,208, 137. 4 P. H. Yancey and G. N. Somero, Biochem. J. , 1979,183,137. 5 P. H. Yancey and G. N. Somero, J . Exp. Zool., 1980,212,205. 6 S. C. Hand and G. N. Somero, J . Biol. Chem., 1982,257,734. 7 C. Tanford, Ado. Protein Chem., 1970,24,1. 8 J. F. Brandts, in Structure and Stability of Biological Macro-

molecules, ed. s. N. Timasheff and G. D. Fasman, Marcel Dekker, New York, 1969, p. 213.

9 E. G. Finer, F. Franks and M. J. Tait, J . Am. Chem. Soc., 1972, 94,4424.

10 D. B. Wetlaufer, S. K. Malik, L. Stoller and R. L. Coffin, J . Am. Chem. SOC., 1964,86,508.

11 F. Franks and D. Eagland, CRC Crit. Rev. Biochem., 1975, 3, 165.

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online

2774 J. CHEM. SOC. FARADAY TRANS., 1993, VOL. 89

12

13 14 15

16 17 18

19

20 21

22

23

H. Edelhoch and J. C. Osborne, Adv. Protein Chem., 1976, 30, 183. M. Roseman and W. P. Jencks, J. Am. Chem. SOC., 1975, W, 631. J. H. Stem and J. D. Kulluk, J. Phys. Chem., 1969,73, 2795. G. C. Kresheck and H. A. Scheraga, J. Phys. Chem., 1965, 69, 1704. T. S. Sarma and J. C. Ahluwalia, J. Phys. Chem., 1972,76,1366. K. P. Prasad and J. C. Ahluwalia, Biopolymers, 1980,19,273. P. K. Nandi and D. R. Robinson, J. Am. Chem. SOC., 1972, 94, 1299. F. Franks, in Water: A Comprehensioe Treatise, ed. F. Franks, Plenum Press, New York, 1972, vol. 2. J. A. Gorden and W. P. Jencks, Biochemistry, 1963,2,47. P. H. von Hippel and T. Schleich, in Structure and Stability of Biological Macromolecules, ed. S. N. Timasheff and G. D. Fasman, Marcel Dekker, New York, 1969, p. 417. P. L. Privalov and N. N. Khechinashvili, J . Mol. Biol., 1974, 86, 665. G. Velicelebi and J. M. Sturtevant, Biochemistry, 1979, 18, 1180.

24 25

26 27 28

29 30 31

32 33

34 35 36 37

F. P. Schwarz, Thermochim. Acta, 1989,147,71. F. P. Schwarz and W. H. Kirchhoff, Thermochim. Acta, 1988, 128,267. D. 0. V. Alonso and K. A. Dill, Biochemistry, 1991,30,5974. J. F. Brandts and L. Hunt, J. Am. Chem. SOC., 1967,89,4826. C. N. Pace, D. V. Laurents and J. A. Thomson, Biochemistry, 1990,29,2564. C. Tanford, J. Am. Chem. SOC., 1962,84,4240. L. L. Bright and J. R. Jezorek, J. Phys. Chem., 1975,79,800. M. M. Santoro, Y. Liu, S. M. A. Khan, L. Hou and D. W. Bolen, Biochemistry, 1992,31, 5278. S . Gopal and J. C. Ahluwalia, Pure Appl. Chem., in the press. R. W. Gurney, in Ionic Processes in Solution, McGraw Hill, New York, 1953. H. S. Frank and M. W. Evans, J. Chem. Phys., 1945,13,507. J. C. Lee and S. N. Timasheff, J . Biol. Chem., 1981,256,7193. H. B. Bull and K. Bresse, Biopolymers, 1978, 17,212. K. P. Prasad and J. C. Ahluwalia, J. Solution Chem., 1976, 5, 491.

Paper 3/01 103H; Received 24th February, 1993

Publ

ishe

d on

01

Janu

ary

1993

. Dow

nloa

ded

by T

he U

nive

rsity

of

Man

ches

ter

Lib

rary

on

22/1

0/20

14 2

2:45

:31.

View Article Online