CHAPTER 3

THERMAL STABILITY OF PROTEINS IN AQUEOUS

POL VOL SOLUTIONS

43

INTRO.DUCTION

Sugars and polyols have been kno\vn to increase the stability of proteins against

thermal and chemical denaturation (Gerlsma, 1968, 1970; Gerlsma & Stuur, 1972, 1974;

Back et al., 1979; Uedaira & Uedaira, 1980; Lee & Timasheff, 1981; Gekko & Timasheff,

1981b; Gekko & Morikawa, 1981a,b; Arakawa & Timasheff, 1982a; Gekko & Koga,

1983; De Cordt et al., 1994a,b; Lozano et al., 1994; Gupta & Bhat, 1995). Polyols and

sugars have been known to be accumulated intracellularly in some organisms under

conditions of stress and have been found to be compatible osmolytes in living systems

· (Brown & Simpson, 1972; Yancey et al., 1982).

Several sugars and some polyols have been found to increase the thermal stability of

proteins by as much as 15°C at 2-3 M concentration. It has been proposed that these

compounds owe their effect to the hydroxyl groups. Uedaira and Uedaira (I 980) have

correlated the stabilizing effect with the number and position of hydroxyl groups in the

case of sugars. Sugar effect seems to. be non-specific, independent of the· nature of the

protein and mediated through water around the proteins. Extensive hydrogen bonding of

water in the hydration shell around proteins in the presence of sugars has been considered

to be a major driving force toward their stabilizing affect (Gerlsma, 1968). Also, in their

presence proteins have been found to be preferentially hydrated (Lee & Timasheff, 1981 ).

Since polyols have one additional hydroxyl group compared to their respective sugars

they are likely to impart a greater stabilizing effect as seen in some cases. Polyols have

also been shown to impart preferential hydration to bovine serum albumin (Gekko &

Morikawa, 1981 a). Although some studies on the effect of polyols on the thermal stability

of proteins are available in the literature, yet a comprehensive study is lacking and a~

general mechanism of their action for proteins of different characteristics is not yet clear.

It has been proposed that polyols and sugars work through their water structure making

effect. Addition of sugars increases the surface tension of water and hence increases the

energy requirement to create a cavity in such a medium upon protein denaturation (Lee &

Timasheff, 1981 ). However, protein stabilization due to the surface tension increment of

the medium has not been proposed as a mechanism for polyol action as glycerol decreases

the surface tension of water but still increases the protein stability (Gekko & Timasheff,

1981 b) and some higher polyols have also been reported to decrease the surface tension of

the solvent (Gekko, 1982a). · Preferential hydration, decrease in the hydrogen-bond

rupturing capacity of the medium (Gerlsma, .1968, 1970), and solvophobic effects

(Timasheff, 1993) have been proposed to be the operative forces for protein stabilization

in the presence of polyols. It is proposed that these forces are strong enough to offset the

effect of decreased surface tension in their presence. In this study ~e have determined the

surface tension of water in the presence of several polyols in order to establish its

44

contribution to protein stability. We have also attempted to find any relationship between

the surface hydrophilicity/hydrophobicity, net and total charges on the proteins with the

effectiveness of polyols in providing thermal stability by taking several globular proteins

varying in their surface characteristics in order to unravel the mechanism underlying their

stabilizing effect.

RESULTS

Fig. l(a-j) presents a set of thermal unfolding curves for RNase-A, cx.-CTgen,

lysozyme, cyt c and Trp-Inh in the presence of 1.0 M mannitol, 2.0 M sorbitol, 0.75 M

myo-inositol, 2.0 M adonitol and 2.0 M xylitol. The pH values selected were 2.5, 4.0 and

7.0. In the case of cyt c, studies at pH .2.5 were not possible as it is partially denatured at

this pH (Dyson & Beattie, 1982). Trp-inh studies were not possible at pH 2. 5 and 4. 0

because it gets aggregated when heated at these pH values. For all the proteins the thermal

denaturation experiments carried out at pH 7.0 included 1.5M GdmCI in the solutions.

This was necessary in order to avoid aggregation of proteins at higher temperatures and to

bring the transition temperature of stable proteins within the limit of experimental study.

Addition of GdmCI in this way has no effect on ~ Tm values as determined earlier for

lysozyme stability in the presence of osmolytes (Arakawa & Timasheff, 1985b ). The

concentration of the polyols selected were such as to have maximal effect on the thermal

stability of the proteins keeping in view their solubility in solution as well as their

tendency to precipitate the proteins at higher temperatures.

Figure 1

0.0 a -0.2 -0.2

• and

E -04 6. ~ E c -0.4 c . • Mn*j

lij lij 0 hll*j

~ -0.6 0~

~ -0.6 <il~

-RT -0.8 -0.8

-1.0 -1.0

20 30 40 50 60 70 3P 40 50 60 70 80

continued ....... .

45

c

E C-04 Iii .

~ -0.6

-0.8

-1.0 -1.0

25 35 45 55 65 20 30 40 50 60 70 80

1.0

0.8 0.8

~ ~0.6 C")

~ ~0.6 C")

~ 0.4 ~ 0.4

0.2 0.2

30 40 . 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90

Eo.6 ~ ~ 0.4

0.2

0.0

30 40 50 60 70 80 90

Eo.s ~ ~ 0.4

0.2

0.0

46

30 40 50 60 70 80 90 100

contd ..... .

0.0

-0.2

E c 0.6

~ ~ 0.4 -0.6

0.2 -0.8

0.0 -1.0

30 40 50 60 70 80 90 30 40 50 60 70 80 90 Temperature (°C)

Thermal denaturation profiles of proteins in the presence of polyols. RNase A at pH 2.5 (a), 4.0 (b), 7.0 (c); a-CTgen at pH 2.5 (d); Cyt cat pH 4.0 (e), 7.0 (f); Lysozyme at pH 2.5 (g), 4.0 (h), 7.0 (i) and Trypsin Inhibitor at pH 7.0 (j). All the experiments at pH 7.0 were carried out in the presence of 1.5M GdmCI. Symbols for polyols presented in panel 'a' have been used throughout this chapter.

The data presented in Fig.l(a-j) have been used to evaluate Tm, Mfm, ~Sm and

~ Tm, in the presence of polyols. Instead of calculating the free energy gain at 25°C we

have calculated the free energy change, ~G0, of proteins in the presence of polyols at the

Tm ofthe control, wherein ~G0(Tm) is zero. Hence the free energy change for proteins in

the presence of polyols will be the net free energy of stabilization, ~~G0, evaluated at the

Tm of the control. These data have been summarized in Table 1. The values presented

are the averages of at least three scans. The Tm values were found to be within ±0.5°C,

where as the uncertainties in~' ~Sin and ~~Go range from 3-7% depending on the

type of protein used. It is found that for lysozyme the uncertainties are on the upper side

of the scale, while for RNase A and cyt c it is usually 2-4 %. The thermodynamic

parameters for a-CTgen at pH 4.0 have not been calculated due to its irreversible

denaturation under these conditions. Tm and ~ Tm values are, however, presented for the

sake of comparison.

It can be seen from Fig. l(a-j) and Table 1 that the polyols studied increase the

thermal stability of all the proteins. On a molar basis it was found that inositol was the

best stabilizer with the highest value of ~ Tm followed by mannitol and sorbitol which are

epimers, whereas xylitol and adonitol which are also epimers, had the least stabilizing

effect. ~ Tm values per molar concentration of polyols were calculated by assuming a

linear dependence of Tm with the concentration of polyols as found earlier by Gekko &

Morikawa (I 981 b) and correlate well with the data for a-CTgen. It has been found that as

the number of hydroxyl groups increases, their stabilizing effect for protein also increases

47

Table 1 Thermodynamic parameters for several proteins in aqueous polyol solutions at various pH values.

RNase-A a.-CTgen Lysozyme Cyt-C

Cosolvent Tm t.Tm Mlm t.Sm MG(fm) Tm t.Tm Mlm t.Sm MG(fm) Tm t.Tm Mlm t.Sm MG(fm) Tm t.Tm Mlm t.Sm MG(fm_) 1 I

(OC) (OC) (kcal. (e.u.) (kcal.mot·l) (OC) (0C) (kcal. (e.u.) (kcal.mot·l) (OC) oq (kcal. (e.u.) (kcal.mot·l) (OC) (OC) (kcal. (e.u.) (kcal.mor ) mot· I) mot· I) mot· I) mol-l)

pH2.S

Control 38.3 - 81.1 260 0.00 44.9 -- 96.3 302 0.00 61.1 - 98.0 293 0.00 Mannitol (I M) 46.6 8.3 87.5 274 2.12 50.5 5.6 94.0 290 1.47 67.1 7.0 100.8 296 1.70 Inositol (0. 75M) 47.0 8.7 88.6 277 2.25 52.3 7.4 100.2 308 2.04 66.8 6.4 98.0 288 1.49 Xylitol (2.0M) . 48.5 10.2 88.8 276 2.60 52.6 7.7 101.9 313 2.10 68.7 8.0 103.7 303 2.18 Adonitol (2.0M) 50.2 11.9 89.0 275 2.98 52.5 7.6 99.5 305 1.97 69.1 9.1· 101.1 295 2.21 Sorbitol (2.0M) 51.5 13.2 91.6 282 3.38 54.7 9.8 98.2 300 2.45 71.0 9.9 103.1 300 2.78

pH4.0

Control 54.2 -- 94.0 287 0.00 59.0 -- 73.1 - 98.5 284 0.00 64.5 -- 61.5 182 0.00 Mannitol (I M) 59.0 4.8 97.2 293 1.40 63.0 4.0 77.9 4.8 100.8 287 1.32 69.8 5.4 66.5 190 0.94 Inositol (0. 75M) 59.8 5.6 98.1 295 1.64 62.0 3.0 77.7 4.6 101.0 288 1.32 70.9 6.5 54.2 158 0.90 Xylitol (2.0M) 61.4 7.2 98.8 296 2.11 64.5 5.5 80.1 7.0 101.0 286 1.89 72.5 8.1 62.1 180 1.30 Adonitol (2.0M) 63.3 9.1 103.3 307 2.66 64.0 5.0 79.6 6.5 102.0 289 1.78 72.6 8.2 67.4 195 1.42 Sorbitol (2.0M) 66.0 11.8 105.3 310 3.50 65.3 6.7 82.5 9.4 104.9 295 2.57 73.8 9.3 64.8 187 1.52

pH 7.0* Trypsin Inhibitor

Control 46.0 - 92.0 288 0.00 59.0 -- 56.5 170 0.00 58.5 - 94.9 286 0.00 48.0 - 38.4 120 0.00 Mannitol (I M) 50.2 3.9 97.6 302 1.26 64.8 5.8 71.3 211 1.08 63.5 5.0 96.6 287 1.37 52.7 4.7 38.4 118 0.50 lnoAitol (0. 75M) 50.7 4.5 98.5 304 1.44 64.5 5.5 71.2 211 1.06 64.5 6.0 96.1 285 1.63 54.0 6.0 40.2 123 0.65 Xylitol (2.0M) 52.9 6.5 97.7 300 2.14 67.3 8.3 71.2 209 1.53 66.5 8.0 100.9 297 2.25 56.11 11.11 40.0 121 0.1111 Adonitol (2.0M) 53.4 6.11 97.9 300 2.15 67.3 8.3 71.8 211 1.54 67.0 8.5 102.0 300 2.39 56.5 8.5 39.9 121 0.75 Sorbitol (2.0M) 56.1 10.3 99.5 302 2.50 71.8 12.8 73.0 212 2.22 70.1 11.6 105.5 307 3.29 '61.1 13.1 45.5 136 1.37

• Solutions contain 1.5M GdmCI.

(Gekko & Morikawa, 198lb). Our results are consistent with the observation for a wide

variety of proteins studied. AHm values, as reported in Table 1 increase in the presence of

polyols. Though the increase in AHm values in the presence of different polyols is within

the experimental and fitting errors, yet an overall gradual increasing trend is implied.

ASm values also show corresponding increase. The increase in AHm values with the

increase in Tm values has often been observed for several proteins studied using

differential scanning calorimetry (Gekko, 1982b; Privalov et al., 1989; Liu & Sturtevant,

1996). There is a very good correlation of the increase in AAG0 with the increase in ATm

for RNase A, lysozyme and cyt c. However, the slopes of the lines obtained vary with the

pH used (Fig. 2).

Figure 2

4.0

3.5 a 1.5 t:". -a 3.0 3.0

~ 2.5 2.5 '(; 1.0

u 2.0 2.0

~ 1.5 0

" 1.0 0.5 <I <I 0.5

0.0 0.0 1'-r-T"''"'T"''"'..,.......,....~I"'T"~ 0 2 4 6 8 10 12 ·14 0 2 4 6 8 10 12 14 2 4 6 8 10 12

.ATm (°C)

Dependence of free energy of stabilization, MG0 with the Tm of (a) RNase A, (b) Cyt c and (c) lysozyme. Symbols shown in panel 'a' are the same for 'b' and 'c'.

Effect of pH

Fig. 3 represents the plots of ll Tm per mole additive (ATm/M) versus the net charge

on RNase A. Since the data for mannitol and inositol is at lower concentration due to their

limited solubility compared to other polyols, the data needs to be normalized to a fixed

concentration for all these additives.

The net charges were calculated from the pH titration curves of the proteins, being

+15, +10 and +4 for RNase A (Tanford & Hauenstein, 1956) and +16, +12 and +9 for

lysozyme (Tanford & Roxby, 1972) at pH 2.5, 4.0 and 7.0 respectively.

49

Figure 3

b 0 - 8

~10 0

• 0 0 0 -a 6.

~ • 6.

~ 6 0

4 0 '\1 '\1

<l 4

2 + 6 +8 +10 +12 +14 +16 Net Charge

Effect of Net charge on RNase A (a) and Lysozyme (b) on the thermal stability in the presence of polyols. See Fig. 1a for description of symbols.

The data show that thermal stability increases linearly with the net charge on the

protein. However, in the case of lysozyme, variation of ~ Tm with the net charge is not

linear (Fig. 3b). Lysozyme melts at a very high temperature at pH 4.0 in the absence of

any denaturant. At high temperature polyols may not be effective enough to counter­

balance the high conformational entropy of denaturation.

Plots for other proteins have not been drawn due to availability of the ~ Tm values

only at two pH values for a-CTgen and cyt c, and at only one pH value in the case of

trypsin inhibitor.

Surface Tension Measurement and Protein Stability

Surface tension (cr) values of aqueous solution of polyols measured by drop method

have been shown in Table 2. Fig. 4 shows thermal stability of various proteins as a

function of surface tension of aqueous polyol solutions. There is an increase in the

thermal stability of proteins irrespective of the type of protein in the presence of all the

polyols used. Although thermal stability does not increase linearly with the surface

tension of the medium, yet a positive contribution of surface tension is obvious. It seems,

therefore, that both the nature of the cosolvent polyol as well as that of the protein govern

protein-cosolvent interactions and hence the thermal stability.

50

Figure 4

8.5 ,---..,---~-"""T"----r----,r---or---...,.--~---r---,

8.0

7.5

7.0

6.5 6 0 6.0 ._.

E 5.5

~ 5.0

4.5

4.0

3.5

3.0

0

0

. 1; •

6=0~Jj ..,...- R'.ase ~--'11 • -o-:- ottc

·--.~ -t:.-L~

r-'11- Trw.lm

8 .0 .2 .4 .6 a (dyne.cm· 1 )

Effect of surface tension of aqueous polyol solutions on the thermal stability of various proteins at pH 7.0.

Table 2

Partial molar heat capacities, volumes, and surface tension values of aqueous polyol solutions.

C0p (k.J mor') -Polyols yo (cm3 mor') O"/M (dyne cm-I morl)

a b a Xylitol 346.4 326 102.14 72.9±0.20

Adonitol 375.6 354 103.11 72.8±0.21

Inositol --- 369 --- 74.22±0.26

Mannitol 455.4 461 119.71 73.12±0.07

Sorbitol 423.1 424 119.16 73.15±0.19

column a From DiPaola & Belleau (1977). b From Jasra & Ahluwalia (1982a)

51

Excess Heat Capacity of Proteins in the Presence of Polyols

The change in the enthalpy of denaturation as a function oftransition temperature for

RNase A has been shown in Fig. 5. Enthalpy of denaturation increases linearly with the

. Tm, suggesting a positive increase in the heat capacity of RNase A upon denaturation. A

value of 0.89±0.05 kcal·moi-1.°K-1 has been obtained in the presence of polyols. Pace &

Laurents (1992) have reported a value of2.2±0.3 kcal.moi-1.°K-1 evaluated by varying the

Tm of RNase A by adding urea. A calorimetric value of 1.4 kcal.mol-1.°K-1 has been

reported by Privalov et al. (1973). Recently Liu and Sturtevant (1996) have very

rigorously investigated the effect of various cosolvents on the calorimetric heat capacity

value ofRNase A and lysozyme. They have reported a value of 1.74±0.07 kcal.moi·1.0K-1

for RNase A in buffer which differs from their earlier reported value of 2.06 kcal.

mol-1.°K~1 (Tsong et al., 1970). Liu & Sturtevant (1996) have pointed out that the origin

of such a variation could be lying in the method and conditions employed in the evaluation

of heat capacity of proteins.

Polyols seem to have a depressing effect on the heat capacity of denaturation of

RNase A. The positive heat capacity of denaturation of proteins has its origin in the

unfavorable interactions of solvent with hydrophobic groups which get exposed upon

denaturation. The increase in the ordering of water molecules around the denatured

protein molecules, which present a large hydrophobic surface to the hydrophilic solvents,

leads to an increase in the heat capacity of the system. Since ~Cp values are strongly

increased by the hydrophobic hydration effects which in tum would depend on the

chemical nature of the solvent molecules, hydrophilic groups like hydroxyls present in

polyols are expected to increase the excess heat capacity of proteins. This has been further

confirmed by calorimetric studies on the effect of sucrose on the excess heat capacity of

RNase A (Liu & Sturtevant, 1996). A decrease in heat capacity of denaturation for several

other proteins has also been observed in the presence of polyols. The data in Table 1

clearly show that there is only a marginal increase in the enthalpy of denaturaion, LUim as

a function of Tm in the presence of polyols than in water. A lower value of heat capacity

obtained by us for the studied proteins could either be due to lesser exposure of

hydrophobic groups upon unfolding (Griko et al., 1994c) or due to the inherent problem in

the evaluation of such a sensitive thermodynamic parameter by indirect techniques like

spectroscopy which is based on the two-state assumption of protein denaturaion.

However, Gekko (1982a) predicted the decrease in heat capacity ofBSA by 15-20% in IM

sorbitol solution than in water.

52

Figure 5

-~ 105

~100 . ~ 95 -~90 <l

85

45

~ oo ~

L\Cp= 0.89±0.051caLrrot10f<"1

R=0.988

50 55 .§9 rmeG)

65 70

Enthalpy of denaturation, ~Hm as a function melting temperature, Tm. Data at pH 2.5 & 4.0 have been used to evaluate the value of heat capacity. Data at pH 7.0 were having a larger scatter in values and hence could not be used.

Role of Protein in Polyoi-Protein Interactions

As evident from Fig. l(a-j) and Table 1, the nature of the protein plays a significant

role in modulating the stabilizing effect due to the polyols. Is there a physico-chemical

property associated with the protein which could be governing polyol-protein interactions

mediated via the solvent water? To answer this question we examined in detail the

correlation of several properties of proteins like average hydrophobicity and

hydrophilicity, using the scale of Hopp and Woods (1981 ), and net charge with the thermal

stability ofproteins. Plots of L1 Tm!M additive versus these properties at several pH values

did not give any direct correlation and uniform dependence.

This shows that none of these properties is a predominant factor as far as the proteins

are concerned. It therefore appears that a delicate balance of' the interactions between the

surface hydrophobic groups, hydrophilic groups and the charged residues with water and

the ability of the polyols to alter the physico-chemical properties of water especially the

interfacial tension at protein-water interface plays a very significant role in the stabilizing

action of polyols.

. The L1Tm values have been found to correlate rather well especially at pH 7.0 wherein

an increase in L1 Tm was observed as a function of increasing net buried hydrophobic

surface area. MSAl'-p-AA.SAp, where MSANP is the buried nonpolar accessible surface

53

area (ASA) which gets exposed upon denaturation of the native state, and MSAp is the

buried polar ASA getting exposed upon unfolding of the protein (Fig. 6a). Interaction of

.1ASANP with hydrophilic solvent should drive the protein toward the native state. On the

other hand the favorable interactions of the polar groups, which get exposed upon

denaturation, with the solvent should destabilize the native conformation. The balance

between these two opposing interactions should decide the role of particular additives in

providing the stability to proteins. The difference of the .1ASANP and .1ASAp can be taken

as the force responsible to drive the protein to fold and to avoid· the unfavorable

interactions with the solvent system. The positive· correlation between the thermal stability

provided by different polyols with the net hydrophobic surface (.1ASANp-.1ASAp) reflect

the origin of conformational stability of proteins. ~ASANP and aASAp for RNase A,

lysozyme, a-CTgen and cyt c were taken from Myers et al. (1995). .1ASA for Trp-Inh

was not available and hence was calculated from the equation given below taking the

number of total amino acid residues present in it to be 186 (Kato et al., 1986) and the

presumption that about 30% of the buried ~ASA is contributed by the polar groups,

especially buried peptide bonds .

.1ASA = -907 + 93x(# residues)

The origin of excess heat capacity of proteins upon denaturation, .1Cp has also been

known to depend on the extent of aASA upon denaturation (Carra et al., 1994;

Makhatadze & Privalov, 1995; Myers et al., 1995). A direct correlation has been

observed between .1Cp and ~ASA as follows:

~CPcal = -119+0.2x(MSA) and

dCPobs = -96 + 0. 72 x,1CPcal

Wherein .1CPobs is the .1Cp determined experimentally and. ~CPcai is the calculated

heat capacity based on ~ASA. The ilCPobs for Trp-Inh evaluated by using these equations

comes out to be 2.2 kcal.mol-I.°K-I. A good correlation of ~Tm with net hydrophobicity

and charges on the surface of the proteins was observed at only pH 7.0, whereas at pH 4.0

(Fig. 6b) and 2.5 no appreciable correlation was obtained. This could possibly be due to

change in the hydrophobicity of different proteins as the charges disappear or appear on

their surface. At lower pH, groups like -Coo- get protonated leading to an increase in the

hydrophobicity (Kuhn et al., 1995). . Different proteins have different compositions and

distribution of charged groups making the situation very complex. This is likely to result

in a varied correlation of properties like net hydrophobicity with the thermal stability at

different pH values as observed by us.

54

Figure 6

10 I I I I . I I

9+ b

. - 9t- a . 0

i 0

81- . 8 t- 0 0 .

0 0 7o,..

0 7 - t- .

i A A 6t- A

0 . 6 0 A

• • ~

A 5

St- • . • ft A

• 41- lJ <:] 0 B .

4 t- • 0 • <;J

8 {} 3o,.. -

3 ! 4 5 6 3~2 3~6 4~0 3 7 4.4

(MSANp·MSAp)x10·3 (A2)

I I I I I I I I I . I

-a c 0 d

i t- 0 0 0

8- . 0 p A - 0 A

i6 ... A • 6- A 0

• • A ft

~ • • A

• 4- lJ 0

<:]4 <;J 0 <;J

.,_ . 0 • 8 A

{} 0 8 2

~ ~5 ~ ~7 ~8 ~9 +1o +11 +12 +h Net Charge

Dependence of the thermal stability of proteins on their physicochemical characteristics. Contribution of Net hydrophobicity at pH 7.0 {a), pH 4.0 {b), and Net charge at pH 7.0 {c) and pH 4.0 (d). !!J.AS~p-MSAp (in

A2) values for various proteins are as follows RNase A: 3181, lysozyme: 3542, cyt c: 4148, Trp-lnh: 6556. Net charge on Trp-lnh is - 9 at pH 7.0, but th,l:! data have been shown against +9 net charge.

Fig. 6c,d shows the effect of the net charges on the protein surface irrespective of the

type of protein at a given pH. At pH 7.0, ~Tm increases with an increase in the net

charge, whereas at pH 4.0 the relationship gets reversed. This clearly indicates that

although net charge. is important, yet in addition some other property of the protein also

plays a role iii modulating the protein-cosolvent interactions. Different properties of the

proteins seem to vary in a non parallel way leading to a varied net effect on the protein

55

stabilty. pH 7.0 is a physiological pH for which most of the proteins· have evolved to

function optimally. Deviation from natural conditions may cause a significant departure

from their normal behavior. Going away from neutral pH may bring a change in various

properties especially the hydrophilicity of the protein, which can always modulate the

protein-cosolvent interactions.

Figure 7

0 a 0 b

~ -5

i-5 -10

~-10 -15

~ ·20 -J: -15

-25

-al Tc = 272.6±4.5 oK -30

-35 -00 -6) -40 -al 0 -100 -80 -60 -40 -20 0

_o 4 ... d e - 2 0 E -2 0 .

~ -4 ·2 ~ - -4

-6

·8

-10

-40 -30 -20 -10 0 -40 -30 -20 -10 0 10

MSo (e.u.)

Enthalpy-Entropy compensation curves for (a) RNase A, (b) a-CTgen, (c) Cyt c, (d) Lysozyme, and (e) Trp­lnh.

Enthalpy-Entropy Compensation

Fig. 7 illustrates the trends in changes in enthalpy and entropy of denaturation to

compensate each other in response to the effect of polyols on the protein-water

interactions. In all the cases the relationship has been observed to be linear with varying

slopes. The slope of the compensation curve is the compensating temperature, Tc. A

knowledge of Tc can provide us the insight into the mechanism of action underlying the

56

stability provided by the cosolvent additives. A change in the protein-cosolvent

interactions at the molecular level is well reflected in a change in thermodynamic

parameters. The value of Tc calculated from the slopes of the compensation curves has

been given iri the figures, representing several proteins as indicated. Several previous

studies also show the compensating trend in enthalpy and entropy in the polyol mediated

stabilization. The origin of the enthalpy-entropy compensation phenomenon has been

proposed to be related closely to the solvation effects around nonpolar and polar peptide

groups (Gekko, 1981 b). The variation in Tc values for different proteins may be due to

differences in the content of polar and nonpolar groups in them, since the extent of

preferential hydration also depends upon the polarity and nature of the protein (Gekko &

Timasheff, 1981 a)

DISCUSSION

The data presented in Fig. 1 (a-j) and Table 1 clearly indicate that the polyols used

in the study stabilize a protein to varying extents. The polyols selected were such that we

could compare the effect of sorbitol and mannitol having six carbons in the chain with

xylitol and adonitol having five carbons in their chain. Also inositol which is a cyclic

polyol with six carbon atoms could be compared with the analogous acyclic polyols

sorbitol and mannitol.

In a three component system of protein, water, and polyol, the interactions that could

decide the stabilizing ability of a polyol arise from those between polyol and water,

between polyol and protein, and between water and protein. Since polyols are known to

induce preferential hydration for proteins ( Gekko, 1981 a) they would not bind and interact

with the protein at room temperature appreciably. Recently in the case of RNase A in the

presence of sorbitol it has been observed that under denaturing conditions the protein has

even much higher preferential hydration than in the native state (Timasheff, 1993).

Considerable information is available on polyol-water interactions both from spectroscopic

(Carpenter et al., 1993) and thermodynamic (DiPaola & Belleau, 1977; Jasra & Ahluwalia,

1982a) point of view. The studies indicate an increase in the hydrogen bonded network of

water in the presence of polyols. This would mean that the increase in the hydrogen

bonding in the hydration shell around proteins shouid lead to the increase in the thermal

stability of proteins. This also explains the higher stabilizing power of mannitol and

sorbitol as compared to adonitol and xylitol having less number of hydroxyl groups. It is

known that sugars and polyols are highly compatible with water structure and that OH

distances in these additives correspond to the nearest neighbour oxygen distance in water

(Warner, 1962). The studies on inositol clearly indicate that inositol is highly compatible

with water structure as has be~n found in the case of glucose, both being cyclic in nature.

Therefore, it appears that cyclic sugars and inositol would structure water to better extent

57

compared to acyclic polyols with similar numbers of C atoms in their Chain. It has also

been observed that our values of ATm on a molar basis for inositol correspond well with

that of glucose reported by Back et.al. (1979) and are higher than those for other polyols.

Role of surface tension

Recently surface tension of water and its change by the addition of cosolvents has

been thought of playing a major role in the stabilization of proteins in their presence (Kita

et al., 1994; Lin & Timasheff, 1996). Using rigorous thermodynamic analysis, the affect

of sugars like sucrose (Lee & Timasheff, 1981 ), glucose and lactose (Arakawa &

Timasheff, 1982a) and trehalose (Kita et.al., 1994) on the surface tension of water has

been found to be the main force behind the stabilization of proteins and their preferential

hydration. However, polyols including glycerol have been reported to reduce the surface

tension of water and their stabilizing action has been considered to be one of 'solvophobic'

in nature (Gekko, 1982a, Timasheff, 1995). To our surprise, the polyols studied by us lead

to a considerable increase in the surface tension of water. More so, it was found that on a

molar basis inositol leads to the largest increase in the surface tension of water followed by

sorbitol and mannitol which gave nearly equal values. Xylitol and adonitol increased the

surface tension of water to similar extents but to lesser magnitudes than sorbitol and

mannitol (Table 2). This is very much consistent with our observation of the increase in

the stability of proteins. in their presence.

Compounds which increase the surface tension of water are known to be excluded

from the protein surface leading to their preferential hydration. This should lead to an

excess of water molecules at the protein-solvent interface. According to equation (1)

(chapter 1) any compound leading to a positive increase in cr with increasing concentration

of cosolvent should give a negative value of preferential interaction parameter, (arn3/arn2)

and hence lead to preferential exclusion of the cosolvent molecules from the protein

surface (Timasheff, 1993). The preferential hydration induced by the polyols in the case

ofBSA (Gekko & Morikawa, 198Ia) has been found to be in the. order inositol >sorbitol­

mannitol > xylitol. Interestingly, it correlates very well with the order of increase in the

surface tension of water in the presence of these polyols as observed by us (Table 2). We,

therefore, consider that the increase in the surface tension of water by polyols is the main

driving force for imparting thermal stability to proteins. This is further supported by the

observation of Sinanoglu and Abdulnur (1964, 1965), Breslow and Guo (1990), and

recently Timasheff and coworkers (Kita et al., 1994; Lin & Timasheff, 1996) who have

proposed that the free energy of cavity formation in water to accommodate the exposed

groups of proteins upon denaturation is proportional to the increase in surface tension of

water by various solvent additives. However, it may not hold good for all the cosolvents

which leads to the stabilization of proteins against denaturation, e.g., as observed in the

58

case of amino-acid salts (Kita et al., 1994). Urea and GdmCI have been found to increase

the surface· free energy of water (Breslow & Guo, I 990) and at the same time are

preferentially bound to proteins at high concentration (Prakash et al., 1981; Arakawa &

Timasheff, 1984b ).

On the other hand, glycerol, which reduces the surface free energy has been found to

lead to the preferential hydration of proteins (Gekko & Timasheff, 1981a). These

observations point to a much more complex situation for the mode of action of these

cosolvents. In general surface tension increase leads to the preferential hydration· and the

magnitude of preferential interaction parameter (8mi8m2) calculated from equation (1)

(Chapter!) has been shown to be very close to that determined experimentally by

equilibrium dialysis (Lin & Timasheff, 1996). We have observed a good correlation of the

surface tension increments by polyols with the increase in thermal stability of several

globular proteins, varying in their several physico-chemical properties (Fig. 4). These

observations suggest the dominant role of surface tension of the medium in providing

stability to the globular proteins, given that polyols are inert toward the protein surface.

It has been observed that glycerol decreases the specific volume (Gekko & Timasheff,

1981 a) and adiabatic compressibility of proteins. Glycerol has been found to squeeze out

water molecules from the cavities in the protein structures and it decreases the flexibility

of the polypeptide chain to minimize the surface area exposed to the solvent. This effect is

similar to that exerted by pressure on the protein structure. In the same way higher polyols

can also exert pressure on the protein structure due to increased surface tension and

viscosity of their aqueous solution to minimize the surface area of the protein and its

flexibility. An entropic loss by a decrease in the intrinsic motions may be offset by an

increase in the intramolecular interactions due to compaction of the structure. However,

glycerol is known to decrease the surface tension of the medium and its action has been

proposed to be mediated chiefly through preferential exclusion from the surface of the

protein.

To substantiate the role of polyols in water-structuring we also tried to look at the

other thermodynamic properties like partial m·olar heat capacity, Cp0 and volume, yo for

polyol-water mixtures. It has been observed that the partial molar heat capacities of

polyols in water are highly positive (Jasra & Ahluwalia, 1982a) and have also been related

to their relative degree of fitting to water structure. A large heat capacity of water in the

presence of polyols and sugars has been attributed to stronger or more extensive hydrogen

bonding between solute hydroxyl groups and water molecules (Jasra & Ahluwalia, 1982a)

as has been suggested from various spectroscopic (Symon et al., 1980) and thermodynamic

studies (Taylor & Rowlinson, 1955). An increase in yo also indicates the better

structuring of water molecules in the presence of polyols. Gerlsma & Stuur (1972, 1974)

have suggested extensive hydrogen bonding between polyhydroxy compounds and water

59

explained in terms of a specific hydration model (Tait et al., 1972). The values o(Cp0 and

yo for the aqueous polyols solutions studied are presented in Table 2. It can be seen that

sorbitol and mannitol have largest Cp0 and yo values compared to adonitol and xylitol.

However, inositol has a lower value of ~p0 than sorbitol and mannitol which can not be

explained considering that inositol is supposed to fit better with water structure. The

distance between the hydroxyl oxygen atoms on the same side of the inositol ring has been

found to correspond to the second nearest neighbor oxygen distance in water (Warner,

1962). The same has been observed for D-glucose which als~ has a lower Cp0 and yo value than other acyclic analogues in solutions but has a greater surface. tension effect on

water and hence larger stabilizing effect (Lee & Timasheff, 1981 ). The lower values of

Cp0 and yo for inositol or glucose can be ascribed to its cyclic nature. From the

thermodynamic studies on model compounds it has also been shown that cyclization leads

to a lowering of Cp 0 and yo values (Gill, 1988). However, it remains to be explained how

inositol having lower Cp0 than sorbitol and mannitol increases the surface tension of water

to larger extents.

pH dependence of thermal stabilities

The results on RNase A clearly indicate that the protein is stabilized to varying

extents in the presence of polyols at different pH values (Table 1). Interestingly it has

been observed that, lower the Tm of the Protein in the buffer, higher the ability of the

polyol to stabilize it. ~G(H20) for RNase as a function of pH evaluated by Pace et al.

(1990) also indicates a lower stability at pH 2.5 compared to that at pH 4.0 and 7.0,

although circular dichroism (CD) studies show no apparent differences in the secondary

structure of the protein at these values (Privalov et al., 1989).

Polyols and sugars have been known to decrease the dielectric constant of water

(Akerlof, 1932) leading to the strengthening of inter- and intramolecular hydrogen

bonding in the water structure and the peptide chain, respectively. Increased strength of

hydrogen bonding among water molecules should lead to a greater energy requirement for

cavity formation in water to accommodate the unfolded peptide chain. In addition,

stronger intra-peptide hydrogen bonding should lead to the stabilization of the native state

of protein molecules. The increased network of hydrogen bonding in the solvent system

should decrease the solvent-protein interactions leading to the strengthening of protein

intra-molecular hydrogen bonding (Schiffer et al., 1995). A decrease in the dielectric

constant should also lead to the strengthening of other electrostatic interactions

(Gerlsma, 1968, 1970; Gerlsma & Stuur, 1972, 1974; Back et al., 1979).

According to equation (1) (chapter 1 ), cosolvents increasing the surface tension of

water should be depleted from the surface. It has been found that the zone of depletion

could be 2-3 water molecules from the interface (Lonsdale, 1958). It has been proposed

60

that the ftrst layer at the interface could be making less extensive hydrogen bonding with

its neighbours than the 3.45 typical for the bulk at 30°C (Karplus & Rossky, I980,I981).

Collins and Washabaugh (I985) proposed that the highly inobile water molecules in the

ftrst layer will show a slight preference for orientations in which they can be net hydrogen

donors (Lewis acid centres) to the bulk solution. This leads to the less extensively

hydrogen bonded oxygen atoms of the surface water molecules carrying a slight negative

charge, while the extensively hydrogen bonded oxygen atoms pf the bulk have a slightly

positive charge. But due to the high electronegativity of oxygen the negative charge is

more localized on the surface atom while the slight positive charge in the bulk is spread

over neighbours also. This leads to a slight negative charge on the surface of the pure

water-air interface. Neutral kosmotropes have been found to further increase the surface

potential difference (Collins & Washabaugh, I 985). It is possible that the negative surface

provided by water at the water-protein interface may be effective in reducing the repulsion

among the positive charges on the surface of the proteins, especially at the acidic pH

values. This should lead to a decrease in the effective positive charges on the surface of

the protein and hence the repulsion among them. The same forces may also be operative

even at high pH values. However, due to the presence of negative charges on the surface

of the protein at those pH values, the negative surface provided by water in the presence of

kosmotropes may not be so much effective.

Neutral kosmotropes which increase the surface potential difference should provide a

more negative surface at the water-protein interface leading to a more effective screening

of the repelling positive charges on the protein surface. This should result in a greater

hydration of the protein. The data for an entirely different class of additives viz., salts like

MgS04 and MgC12 (Arakawa et al., I 990a) also indicate an increase in the preferential

hydration of RNase A in the order of pH 1.5>2.0>2.8>5.5 and correlate well with the

higher thermal stability at lower pH values. It is therefore likely that in the present case

RNase A is preferentially hydrated to larger extents at low pH in the presence of polyols

and stabilized to larger extent. However, the results of Gekko and Timasheff (198 I a) for

RNase A in 30% glycerol at pH 2.0, 2.8, 4.3 and 5.8 indicate a decreased preferential

hydration of the protein at pH 2.0. Glycerol has been known to decrease the surface

tension of water and is, therefore, behaving much different from the higher order polyol

like sorbitol. The net effect of a cosolvent on the stability of a protein should be governed

by the relative preferential hydration of the two end states of the protein molecule. The

unpublished results of Xie and Timasheff (as quoted in Timasheff, I 993) indicate an

increase in the preferential hydration of RNase in the presence of 30% sorbitol in the

destabilized state at 48°C relative to the native state at 20°C. The low pH state of RNase

A in our studies can be considered to be identical to the high temperature state ( 48°C)

where larger preferential hydration in the presence of sorbitol is observed. It is, therefore,

61

likely that the low pH state of the protein which has lower thermal stability but higher

preferential hydration is expected. to be stabilized to larger extents in the presence of

polyols ..

Another alternative reason for a higher stability provided by polyols could be because

of the fact that with the decrease in pH the hydrophilic negative charges on the polypeptide

chain disappear (Carra et al., 1994), due to the protonation of carboxylate ions which are

more hydrophilic than their protonated form (Kuhn et al., 1995). Decrease of repulsive

electrostatic interactions and reduced solubilty of groups due to their less hydrophilic

nature at low pH could result in strengthening of the hydrophobic interactions (Privalov,

1996). The presence of the hydrophilic groups like polyols around the protein molecules

should, therefore, strengthen the solvophobic effects to a greater extent at these conditions.

Similar trend has been observed for a-CTgen at pH 2.5 and 4.0, lysozyme at pH 2.5,

4.0 and 7.0 and for cyt c at pH 4.0 and 7.0, except for some exceptions at pH 7.0 in the

case of lysozyme and cyt c. For the sake of comparison of pH 7.0 results containing

GdmCl with that of pH 4.0 and 2.5, around 15°( has to be added to the Tm values as 1.5

M GdmCl has been found to decrease the Tm of RNase A and lysozyme by this amount.

However, the assumption is that the ~ Tm values would remain unaltered as observed

earlier for amino acid solutions (Arakawa & Timasheff, 1985b ). The anomaly in the case

of lysozyme, wherein there is a minimum at pH 4.0 in its thermal stability plotted against

pH or net charge (Fig. 3) could be arising from the high Tm of this protein even in the

absence of any additive. Tm for lysozyme is around 73°C at pH 4.0 in the absence of any

denaturant and stabilizer. At high temperature polyols may not be effective enough to

counter-balance the high conformational entropy of denaturation. · Moreover, the

effectiveness of these cosolvents may be decreasing due to the decrease in the surface

tension of their aqueous solutions at higher temperature.

From the thermal denaturation curves in the presence of polyols, the mo and ~so

values evaluated at a temperature equal to the Tm in the control experiment (without

polyol) revealed that the stabilizing effect of polyols for the protein is entropically

controlled, i.e., ~so term is decreasing to larger extent in the presence of polyols relative

to control which compensates for the decrease in mo to give a small increase in ~Go

tabulated as Ll~Go in Table 1. Similar observations have been made earlier by Gekko and

Morikawa (1981b) also. In Fig. 2a,b,c it can be seen that for RNase A, lysozym~ and cyt

c, ~~Go is a linear function of ~ Tm. However, the slopes of the lines vary as a function

of pH. This can be interpreted in terms of the variation in t}le compensation of mo and

~so at different pH values, although at all the pH values the two terms are decreasing in

magnitude in the presence of polyols (Fig. 7). The variation in slopes is also dependent on

the nature of the protein.

62

In nutshell, both the nature of the polyol and that of the protein is important in

governing the stability of proteins in the presence of polyols. Surface tension of the

medium and the structuring of water in the presence of polyols appears to be a dominant

factor though not the sole factor. Given that several physico-chemical properties of

proteins did not show any direct correlation with their thermal stability in the presence of

polyols indicates that a delicate balance of the interactions between the surface

hydrophobic, hydrophilic and charged residues with water an.d the ability of polyols to

alter the physico-chemical properties of water especially ·the interfacial tension at the

protein-water interface plays a very significant role in the stabilizing action of polyols.

63