CHAPTER 7

THERMAL STABILITY OF PROTEINS IN AMINO

SULPHONIC ACID SALTS

126

INTRODUCTION

Certain organisms live under environmental stress like high osmotic pressure, freezing

or desiccation and accumulate low molecular weight compounds. These compounds are

known as osmolytes (Yancey et al., 1982; Somero, 1986). Compatible osmolytes, a

functional class of osmolytes, increase protein stability without having any effect on the

enzymatic activity. Most compounds used by organisms for this purpose are excluded

from the immediate vicinity of the protein molecules. They hav~ little or no propensity to

bind to proteins (Timasheff, 1992c ). Most osmolytic bacteria accumulate compounds

including sugars, polyols, amino acids and their derivatives, methyl amines, and amino

sulphonic acids like taurine. Some incompatible osmolytes like urea also get accumulated

to provide favourable cellular osmotic pressure (Timasheff, 1993) along with protein

structure protectants like trimethyl amine-N-oxide (TMAO), sarcosine, or betaine (Yancey

& Somero, 1980; Santoro et al., 1992). The interrelationship between protein structure

and dynamics plays an extremely important role in biological processes (Wang et al.,

1995).

This work was taken up to understand the mechanism of action of amino sulphnic

acids like taurine (aminoethane sulphonic acid), which is a compatible osmolyte also found

in milk of higher animals with a varied function in their physiology, in providing the

thermal stability to globular proteins. To understand the mechanism of action of sulphnic

acid . salts on the thermal stability of proteins and protein-solvent interactions, a

homologous series of these acids like sulfamic acid,· aminomethane sulphonic acid and

aminoethane sulphonic acid (APS) was taken and studied in detail. Except taurine, other

sulphonic acids have not been observed to function as natural osmolytes.

RESULtS

Thermal denaturation profiles of RNase A, Trp-Inh, lysozyme, and cyt c in the

presence of different amino sulphonic acids are shown in Fig. l(a-h). Amino methane

sulphonic acid could not be used due to it's limited solubility (~0.4M) and also because the

pH of its aqueous solution was not stabilizing even after adjusting several times. Taurine

also had low solubility (~.75M at pH 4.0 & l.OM at pH 7.0 and 9.2). The

thermodynamic parameters obtained by analyzing the thermal denaturation curves in the

presence of sulphonic acid salts have been summarized in Table 1.

Fig. l(a-c) shows the effect of sulphonic acids on the thermal stability of RNase A.

Sulfamic acid provided stability to the largest extent followed by APS and taurine.

Similar trends in stability provided by different compounds have been observed at all the

pH values and was found to be directly proportional to the concentration of the additives

used. APS decreased the thermal stability of cyt c at all the pH values.

127

Figure 1

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20 30 40 50 60 70 80 90

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Temperature (°C)

Fig. contd ...••.

128

g1 QO

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-0.2 ~.2

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Thermal denaturation profiles of various proteins at different pH values in the presence of amino sulphnic acid salts. RNase A at (a} pH 4.0, (b1,2) pH 7.0, (c1,2) pH 9.2; (d1,2) cyt cat pH 4.0; lysozyme at (e1,2) pH 7.0, (f1,2) pH 9.2, the up & down arrows indicate the onset of aggregation; and Trp-lnh at (g1,2) pH 7.0, & (h1,2) pH 9.2. Symbols used for cosolvents in panel (a) are used in all the following panels, and throughout this chapter to denote the same cosolvent system unless mentioned otherwise. All the experiments at pH 7.0 & 9.2 were carried out in the presence of 1.5M GdmCI.

Sulfamic acid was found to denature cyt c even at 20 °C as seen from the absence of

any thermal transition on heating the protein solution in its presence. Taurine was

observed to increase the thermal stability of cyt c only marginally (Fig.ld). Cyt c did not

show any cooperative thermal transition at pH 9.2 under any condition.

Fig.l(e,f) shows the thermal denaturation profile of lysozyme in the presence of

amino sulphonic acids at various pH values. Sulfamic acid aggregated the protein at all

the concentrations at pH 4.0 (data not shown), whereas at pH 7.0 and 9.2 aggregation was

observed at only higher concentrations. APS also aggregated lysozyme at pH 4.0 even at a

concentration of O.SM. All these compounds were found to increase the thermal stability

of lysozyme to varying extents. Thermal denaturation curves for Trp-Inh are shown in

Fig.l(g,h) and were possible only at pH 7.0 and 9.2. All the sulphonic acids increased the

thermal stability of Trp-Inh at both the pH values. Sulfamic acid on per molar basis

proved to be the most effective in providing thermal stability.

Surlace Tension Measurements

Surface tension measurements for aqueous sulphonic acid solutions were conducted at

25 °C. The results have been shown in Fig.2a. Surface tension values for all the

compounds in water increased linearly with an mcrease in the concentration of the

a~ditives and seems to be depending upon the -CH2 content in a particular compound, i.e.,

effectiveness of these compounds in raising the surface tension of water decreases with an

increase in their hydrophobicity.

129

Table 1

Thermodynamic parameters for the denaturation of several proteins in amino

sulphonic acid salts.

Cosolvent Tm ~Tm .Mim ASm MG(Tm)

(oC) (OC) (kcal. moJ-1) (e.u.) (kcal. mol-1)

RNase A in amino sulphonic acid salts.at pH 4.0

*Control 54.2 --- 94.0 287 0.00

Taurine (0.5M) 54.7 0.5 101.9 310 0.16

(0.75M) 55.0 0.8 96.26 293 0.23

APS (0.5M) 56.9 2.7 98.1 297 1.06

(I .2M) 64.1 9.9 102.0 302 2.67

Sulfarnic (0.5M) 59.2 5.0 98.6 297 1.40

acid (l.OM) 63.6 9.4 103.8 308 2.53

(2.0M) 69.4 15.2 107.8 315 4.00

RNase A in amino sulphonic acid salts and 1.5 M GdmCI.at pH 7.0.

*Control 46.0 --- 92.0 288 0.00

Taurine (0.5M) 48.0 2.0 84.3 262 0.50

(1.0M) 49.3 3.3 87.1 270 0.85

APS (0.5M) 48.0 2.0 90.8 283 0.55

(1.2M) 52.1 6.1 95.0 292 1.65

Sulfarnic(0.5M) 49.2 3.2 98.0 304 0.94

acid (1.0M) 52.7 6.7 97.2 298 1.85

(2.0M) 58.3 12.3 98.1 296 3.13

Table contd ....

130

Cosolvent Tm ATm Mlm ASm MG(Tm)

\C) (oC) (kcal. mol-l) (e.u.) (kcal. mol-1)

RNase A in amino sulphonic acid salts and 1.5M GdmCI at pH 9.2

*Control 45.2 --- 92.0 289. 0.00

Taurine (0.75M) 49.2 4.0 81.4 253 0.96

(l.OM) 49.9 4.7 96.0 297 1.32

APS (0.5M) 48.4 3.2 95.6 297 0.91

(I. 2M) 52.0 6.8 97.0 298 1.87

Sulfamic (0.5M) 49.0 3.8 95.9 298 1.08

acid · (l.OM) 52.1 6.9 95.3 293 1.86

(2.0M) 60.0 14.8 103.0 309 3.96

Cytochrome c in amino sulphonic acid salts at pH 4.0.

*Control 64.5 --- 61.5 177 0.00

Taurine (0.75M) 66.9 2.4 58.6 172 0.40

Cytochrome c in amino sulphonic acid salts and 1.5M GdmCI at pH 7.0

*Control 48.0 --- 38.4 128 0.00

Taurine (I. OM) 54.5 6.5 44.4 135 0.79

Lysozyme in amino sulphonic acid salts at pH 4.0.

*Control 73.1 --- 98.5 284 0.00

Taurine (0.75M) 74.4 1.3 94.3 271 0.35

Lysozyme in amino sulphonic acid salts and 1.5M GdmCI at pH 7.0

*Control 58.5 -- 94.9 286 0.00

APS (0.5M) 61.9 3.4 89.7 268 . 0.89

(I .2M) 68.7 10.2 84.0 246 2.29

Table contd ....

131

Cosolvent Tm .!1Tm Mlm .1Sm MG(Tm)

(oC) ('C) (kcal. moJ-1) (e.u;) (kcal. mol-1)

Lysozyme in amino sulphonic acid salts and l.SM GdmCI at pH 9.2

*Control 58.5 - 94.9 286 0.00

APS (0.5M) 60.7 2.2 81.0 243 0.52

Trypsin Inhibitor in amino sulphonic acid salts and l.5M GdmCl at pH 7.0

*Control 59.0 - 56.5 170 0.00

Taurine (0.5M) 62.2 3.2 61.3 183 0.56

(l.OM) 63.8 4.8 61.6 183 0.81

APS (0.5M) 62.6 3.6 62.0 185 0.63

(1.2M) 70.0 11.0 68.4 199 1.84

Sulfamic (0.5M) 63.1 4.1 55.2 164 0.62

acid (I. OM) 66.0 7.0 52.4 154 0.94

(2.0M) 70.3 11.3 54.9 160 1.43

Trypsin Inhibitor in amino sulphonic acid salts and in l.SM GdmCI at pH 9.2.

*Control 58.8 - 62.2 187 -Taurine (0.5M) 61.5 2.7 62.9 188 0.49

(l.OM) 64.6 5.8 62.9 186 0.98

APS (0.5M) 60.8 2.0 47.2 141 0.37

(1.2M) 68.3 9.5 68.0 199 1.63

Sulfamic (0.5M) 63.0 4.2 51.2 152 0.59

acid (2.0M) 73.2 14.4 57.5 166 1.79

*Control is the buffer solution in the presence or absence of GdmCI as indicated.

132

Figure 2

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Surface tension of water (a), and RNase A stability at pH 7.0 (b) as a function of amino sulphonic acid salt concentration. Symbols used in panel a are the same as that for panel b.

Fig.2b shows that stability of RNase A also increases linearly with an increase in the

cosolvent concentration, indicating the possible role of water-structuring in providing

thermal stabilty. However, it has been observed that the increase in the thermal stability of

the protein and the surface tension of water in the presence of these additives does not

follow similar trends (Fig.2a,b). For most of the proteins sulfamic acid was found to be

most effective in providing stabilization and was followed by APS and taurine. Surface

tension followed the trend directly related to the number of -CH2 groups present in the

compound, while a similar trend was not observed in the case of increase in the protein

stability (Fig. 2a,b ).

Fig.Ja Shows the relation between the increase in the thermal stability of RNase A

and the surface tension of water as a function of amino sulphonic acid concentration.

These two parameters increase linearly with the amino sulphonic acid concentration.

However, ATm for RNase A as a function of the number of CH2 groups in amino

sulphonic acids varies in a nonlinear way, which indicates that surface tension may not be

the sole factor responsible for the increase in the thermal stability ·of proteins in the

presence of amino sulphonic acids. Almost similar trend has been observed in the case of

Trp-lnh and lysozyme, wherein there was less difference between the stabilites provided

by APS and taurine at low concentrations although the difference became large at higher

concentrations. The results show very clearly that although the hydrophobicity of taurine

is in between that of sulfamic acid and APS, yet it has been observed to be the least

effective in providing thermal stability to most of the proteins, except cyt c.

133

Figure 3

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(a) Increase in thermal stabiliy, ~Tm of RNase A in the presence of increasing concentration of sulfamic acid salt at various pH values. The Y-axis on the right shows the increase in the surface tension, cr of water in the presence of sulfamic acid salt. (b) Influence of methylene, CH2 groups, present in cosolvent molecules,

on the thermal stability of RNase A at various pH values. The data points used were at 0.5M cosolvent concentration.

Figure 4

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Influence of net charge present on the surface of RNase A on its thermal stability provided by various amino sulphonic acids. Symbols used are same as in Fig. 1 a.

134

Fig.4 shows the effect of net charge on the thermal stability of RNase A in the

presence of different additives. It has been seen that sulfamic acid and APS follow the

same trend. There does not seem to be much effect of pH on RNase A stability between

pH 9.2 and 7.0. However, .1Tm is highly dependent on net charges at pH 4.0 where the

proteins have high net positive charge. The stability provided by both of these has a

minimum at pH 7.0 as can be seen in Fig.4. On the other hand, effectiveness of taurine

decreases almost linearly with an increase in the net charge on the protein surface. The

anomalous behavior of taurine has also been observed in relation to surface tension effect.

Fig.5 shows the relation of enthalpy of unfolding, .1Hm with the temperature of

denaturation for RNase A. It has been observed that the excess heat capacity of RNase A

depends upon the pH of the aqueous solution of the cosolvents. .1Hm for RNase A

denaturation is observed to increase in the presence of increasing concentration of sulfamic

acid at pH 4.0, while the increment was negligible at pH 7.0. Data on heat denaturation of

proteins in the presence of taurine and APS was not enough to evaluate their effect on the

heat capacity of proteins.

Figure 5

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Effect of pH on the enthalpy of denaturation of RNase A in the presence of sulfamic acid

135

Figure 6

25

~20 E 15

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~ 10. ~ -5

~ 0 .-5 RN•eA

Mlf' c- 43.3(~.1) + l8L0(~4)><M Rc0,996

n)11Rllrt1111Dr Mtf • ·204..6(:1:107) + 319.6(tt 19)xMS' Ro:0.9998

10~-r~~-r~~-r~~ -20 0 20 40 60 ll) 100 0 20 40 60 80 100

-~~so ( e.u) -~~so (e.l1)

Enthalpy-entropy compensation curves for (a) RNase A, and (b) Trp-lnh. The slope of the lines represents Tc, the temperature of compensation.

Fig. 6 presents the enthalpy-entropy compensation curves for RNase A and Trp­

Inh in the sulphonic acid studied at different pH values. Slopes of the plots represent

temperature of compenstaion, Tc which is 281 °K for RNase A and 320°K for Trp-Inh.

The strong correlation between the decrease in enthalpy and entropy has been suggested to

be originating from the changes in the state of solvent around the polar and nonpolar

groups. In the case of proteins such a behavior is not so well understood at the molecular

level. It has been suggested that the changes in the hydration of polar and nonpolar groups

upon protein denaturation may be responsible for the enthalpy-entropy compensation

(Gekko, 1981b).

Table 2

Surface tension values of aqueous solutions at different concentrations of amino sulphonic acid salts at 25°C

Cosolvent additive

Taurine

APS Sulfamic acid

0.3M

72.16±0.04

72.10±0.12

72.65±0.10 (l.OM)8

Surface Tension (dyne cm-1) 0.6M l.OM

72.45±0.20

72.23±0.11

73.94±0.02 (l.SM)

72.98±0.10

72.58±0.06

75.61±0.09 (2.0M)

a Cooncentrations in parenthesis is for sulfamic acid.

136

DISCUSSION

Unlike carboxylic acids and related compounds like amino acids, increase in the

thermal stability of proteins provided by amino sulphonic acids is not correlated linearly

with their ability to increase the surface tension of water. The linear increase in surface

tension is not reflected in the stability provided by a particular additive, especially APS

which is more effective at higher concentration.

The results indicate that sulfamic acid which does not contain any CH2 group

increases the stability of proteins and surface tension of water linearly as a function of

concentration and to the largest extent. Addition of the CH2 group, perhaps, alters the

nature of protein-solvent interactions in a very complex manner. Methylene groups due to

their hydrophobic nature may interact favorably with the exposed hydrophobic groups of

the protein upon unfolding. Therefore, the stability provided by sulfamic acid may be

offset to some extent by the methylene groups in the case of taurine and APS. Sulfarnic

acid has been observed to be the most effective compound in this series with an exception

for its effect on cyt c, where negatively charged sulfarnate anion may be affecting the

conformation of cyt c in a similar manner to that of carboxylic acids. Methylene groups

have previously been known to contribute negatively to the protein stability (Gerlsma,

1968, 1970) in the case of alcohols. However, in carboxylic acid salts CH2 group has been

observed to contribute positively or without having any appreciable effect (Busby &

Ingham, 1984). Malonate containing only one CH2 group was observed to be effective to

a lesser extent than succinate, which contains two CH2 groups, in providing thermal

stability to proteins and in raising the surface tension of water. Our results showing less

effectiveness of taurine than sulfamic acid and APS in raising the Tm of proteins indicate

that, although, methylene group does contribute in modulating the water-structure, yet it is

very difficult to relate its effect to the thermal stability of proteins.

Taurine has previously been known to be excluded from the surface of proteins

leading to their preferential hydration (Arakawa & Timasheff, 1985b ). This could

possibly be due to the effectiveness of taurine in raising the surface free energy of water.

Unfortunately, data on preferential interaction parameters for sulfamic acid and APS are

not available to rationalize the contribution of preferential interactions with their effect on

thermal s~bility of proteins. However, based on the Gibbs adsorption isotherm it can be

expected that these additive, due to their effectiveness in raising the surface free energy of

water, should also lead to the preferential hydration of proteins in the absence of any

specific binding interactions with proteins. Although the relation between surface tension

of the solvent and the thermal stability does not follow a linear trend, yet the positive role

of surface tension in leading to the increase in the thermal stability of proteins in the

presence of amino sui phonic acid salts seems quite obvious.

137

The thermal stability of proteins in aqueous solutions of cosolvents is highly related to

the effectiveness of the cosolvent in increasing the surface tension of water and hence the

degree of exclusion of the cosolvent from the vicinity of the protein in the absence of

specific cosolvent-protein interactions. Preferential binding or other attractive forces

between cosolvent and protein molecules may lead to the penetration of cosolvent

molecules in to the hydration shell of the protein. This will lead to a decrease in the

preferential hydration value of the protein and the effectiveness of the cosolvent in

increasing the thermal stability of proteins as well. A delicate balance between the

cosolvent exclusion, due to · the higher interfacial free energy, and the cosolvent

interactions with the protein due to the attractive coulombic or hydrophobic forces should

result in the net effect on the protein stability. The surface tension of water and the

thermal stability of proteins increases linearly with the sulphonic acid salt concentration

but the surface tension decreases with the increase in the hydrocarbon chain length in these

cosolvents. However, APS, containing propylene group ( -CH2CH2CH2-) is less effective

than sulfamic acid, containing no hydrophobic group, but more effective than taurine

which contains ethylene ( -CH2CH2-) group, in increasing the thermal stability of RNase A.

This anomaly could be arising due to differences in protein-cosolvent interactions in

the two end states of the protein. These cosolvents have similar and equal number of

ionizable groups, differing in their position by the presence of varying lengths of the

hydrocarbon chain, which may be the possible source of the anomaly. It is possible that

taurine containing two CH2 groups is attracted toward the hydrophobic patches on the

protein surface and decreases the water activity at the cosolvent-protein interface which

leads to a marked decrease in the preferential hydration as well as the effectiveness in

increasing the protein thermal stability in comparison to sulfamic acid. On the other hand,

APS having three CH2 groups might be excluded from the vicinity of the protein to a

greater extent due to strong repulsive forces between the highly hydrophobic propylene

group and the hydrophilic charged groups present on the protein surface. The additional

CH2 group in APS although may contribute to increase in the hydrophobic interactions

between protein and cosolvent molecules, yet they may not be strong enough to offset the

increased repulsive forces responsible for the exclusion of APS from the hydration shell.

In the case of taurine these repulsive forces may be counterbalanced by the attractive

forces to some extent leading to a decrease in its effectiveness in increasing the thermal

stability. Interestingly, taurine has been observed to be excluded weakly [(8g3/8g2h,111 ,,.u =

.-0.0331, at 0.667M taurine] from the vicinity of lysozyme and y-aminobutyric. acid

(GABA, NH2-CH2CH2CH2-COOH), an analogue of APS, strongly [(8g/8g2h,111,J13 = -0.0699, at 1M GABA], where (8g3/8g2h,111 ,J13 is the preferential interaction parameter

determined by equilibrium dialysis (Arakawa & Timasheff, 1985b ). A negative value of

the preferential interaction parameter indicates the preferential exclusion of cosolvent from

138

the hydration shell of the protein. The value of this parameter for taurine is very near to

that observed for ArgHCl [(ogiog2h-.~1 ,JL3 = -0.028, at 0.5M), which has been found to

penetrate the hydration shell and bind to proteins due to electrostatic attractive forces (Lin

& Timasheff, 1996). It seems quite possible that APS like GABA might be excluded

strongly as compared ~o taurine. Bhat and Timasheff (I 992) have observed that larger the

alkyl group, more negative will be the value of preferential interaction parameter.

However, a bulkier hydrophobic group may be excluded from the vicinity of the native

state, but may interact favorably with the larger exposed surface in the denatured state.

This situation may even lead to destabilization of the native state as has been observed in

the case of valine (Arakawa & Timasheff, 1985b ).

The less effectiveness of taurine at lower pH (Fig. 4) can be explained by considering

that at low pH the net charge present on the protein surface is more, but the total charge

decreases with a decrease in the pH due to the protonation of basic groups of the protein,

which should in tum result in a decrease in the repulsive forces. In this situation taurine

will be able to interact favorably with the exposed hydrophobic surface of the protein and

lead to a decrease in the thermal stability of proteins. By the same analogy APS should

also affect the stabilty in a similar manner. However, it has been observed to be more

effective at pH 4.0 than at pH 7.0 and 9.2. Similar trend is shown by sulfamic acid also.

As far as stability provided to RNase A is concerned, sulfamic acid and APS show similar

pattern. They seem to interact with proteins in similar ways. In the absence of preferential

interaction data it is very difficult to speculate on the nature of protein-cosolvent

interaction as a function of pH in the presence of these acid salts.

The polarity of a protein and the nature of charged cosolvent molecules are known to

affect the protein-cosolvent interactions. LysHCl and ArgHCl interact differently with

BSA (pl-5) and lysozyme or RNase A (both having pl-1 0) depending upon the pH (Kita

et al., 1994; Lin & Timasheff, 1996). Acidic amino acids like Asp, Glu or even LysGlu

and ArgGlu provide thermal stability due to the surface tension effect. ArgHCI and

LysHCl do not follow the same trend, since they have been observed to penetrate the

hydration shell of proteins due to attractive coulombic interactions and bind weakly to

proteins (Lin & Timasheff, 1996). The results obtained by us on sulfamic acid, taurine,

and APS clearly indicate the role of surface tension of the medium in providing stability to

proteins but their relative effect seems to be further governed by a balance between

attractive hydrophobic interactions between the cosolvent molecules and the proteins, and

repulsive forces between the non polar groups in the cosolvent and the charged protein

surface.

139