380
Biochimica et Biophysica Acta, 537 (1978) 380--385
© Elsevier/North-Holland Biomedical Press
BBA 3 8 0 4 0 THERMODYNAMICS OF THE ISOTHERMAL INTERACTION OF HUMAN IMMUNOGLOBULIN G WITH GUANIDINIUM CHLORIDE
SAVO LAPANJE, BRONISLAVA CRESNAR, VOJKO VLACHY and JOZE SKERJANC Department of Chemistry, University of Ljubljana, Ljubljana (Yugoslavia)
(Received March 15th, 1978)
Summary A t h e r m o d y n a m i c study of the isothermal interaction of human immunoglobulin G with guanidinium chloride, a strong denaturant, has been performed. Free energies of interaction were calculated using preferential binding data obtained by measuring densities at constant chemical potential and constant composition, respectively. Enthalpies of interaction were determined calorimetrically. The values of both t h e r m o d y n a m i c parameters as well as those of entropies of interaction have been found to depend crucially on the extent of denaturant binding.
Introduction In previous papers [1,2] the thermodynamics of the interaction of lysozyme and/3-1actoglobulin, respectively, with guanidinium chloride, a strong denaturant, has been studied. In this communication, we report on the interaction of another protein, h u m a n immunoglobulin G (IgG), with guanidinium chloride. Since in this case the preferential binding data for denaturant which are needed for calculation of the free energies of interaction were not available, it was necessary to perform separate experiments for determining the binding as a function of denaturant concentration. The m e t h o d chosen is based on density measurements [3,4]. The enthalpies of interaction were again determined calorimetrically. From them and the free energies of interaction the entropies of interaction can be calculated allowing a complete thermodynamic description of the protein-denaturant interaction. Materials and Methods Normal h u m a n serum was provided as a pool by the Institute of Blood Transfusion of Slovenia. IgG was isolated by (NH4)2SO4 precipitation, DEAE-
381 cellulose and G-200 chromatography [5]. The buffer used was 0.01 M TrisHC1/0.02 M NaC1 (pH 7.7). Protein concentrations were determined spectrophotometrically using the value 14.0 for the absorption coefficient {~lcm. 1% Guanidinium chloride was a product of Merck-Schuchardt and before use it was washed twice with acetone then dissolved in hot ethanol and precipitated with n-hexane. Density measurements were performed with a Precision Density Meter D M A 02 (A. Paar, Gratz). Solutions for measurements should not contain large particles; therefore, the solvent was passed through a sintered-glassfilterbefore dissolving the protein. To determine the extent of preferential binding, densities had to be measured at constant chemical potential and constant composition of solvent components (see Eqn. 2). Calorimetric experiments were carried out in an L K B Batch Microcalorimeter 10700-2. Initialprotein concentrations were around 1.5% (w/v). The procedure was identical to that described previously [2]. The essential feature is mixing of (2.00 +¢)ml protein solution with 4.00 ml appropriate denaturant solution in the reaction cell and of 2.00 ml buffer solution and 4.00 ml same denaturant solution in the reference cell.¢ is the protein displacement volume, i.e.,the product of protein mass x partial specificvolume. Results and Discussion
Determination of preferential binding and free energies o[ interaction. The preferential binding of a denaturant in three-compoent system, P3,pref, with components 1 (water), 2 (protein) and 3 (denaturant) is define by the following expression ,. M2(~g31 ~3.pref --'~~ \~"2/T,/./,1 j/, 3
(1)
where M2 is the molecular weight of the protein and M3 that of the denaturant, g is the concentration in g/g water, T is the temperature, and u the chemical potential. (Sg3/~g2)T,, ~,, 3 is obtained from de~usity..data at constant chemical potential and constant composition using the following equation [3,4]:
t gq 5p
'~2
where p is the densiW and m the molality. The values of (Sg3/~g2)r., 1., 3 for infinite dilution of protein are given in Table I which contains also the values of parameters used in their calculation. In Fig. 1 the values of (SP/~g2) are plotted as a function of protein concentration. Comparison ~ t h lysozyme [6] reveals that the preferential binding of guanidinium chloride to human IgG is, in general, smaller. Moreover, as with some other proteins, preferential binding reaches a maximum at about 5 M guanidinium chloride. Although no convincing interpretation for this, behavior is available, it could be due to changes in the structure of water at high denatur-
382 TABLE I PREFERENTIAL BINDING PARAMETERS OF HUMAN IMMUNOGLOBULIN G AT INFINITE DILUT I O N IN A Q U E O U S S O L U T I O N S O F G U A N I D I N I U M C H L O R I D E A T 2 5 ° C A N D p H 7.7
O6p) _
Concn. (M)
( 6p)o ~g2 T'P'm3
t~g2 T ' ~ l ' # 3
~P
(=---)T'P'm2og3
( c m -3 × 1 0 4 ) 1 2 3 4 5 6
2 . 3 7 2 _+0 . 0 0 7 2.201 1.954 1.665 1.471 1.022
2.0
(g/g p r o t e i n )
2.378 ± 0.007 2.188 1.945 1.623 1.414 0.996
2 . 2 7 5 _+ 0 . 0 0 1 1.888 1.549 1.243 0.983 0.754
0
0
3M
~g3 0 (~g2)T'gl'#3
0.00 0.01 0.025 0.035 0.06 0.035
0
± 0.01 ± 0.01 ± 0.01 ± 0.01 _+ 0.01 ± 0.02
O--
6Q ~
x
1.5
'E
u v
1.0 -
©
©
\-~g2 T,~I,F3
,(3--
6M
0.5
0
I
I
I
"5
10
15
conc.(g/I )
Fig. I . D e p e n d e n c e of p r e f e r e n t i a l b i n d i n g p a z a m e t e r s t i o n a t 25eC a n d p H 7.7.
(Op/~g2 ) o n
human immunoglobulin G concentra-
383
T A B L E II T H E R M O D Y N A M I C P A R A M E T E R S OF THE I N T E R A C T I O N OF H U M A N I M M U N O G L O B U L I N G W I T H G U A N I D I N I U M C H L O R I D E A T 25°C A N D p H 7.7 T h e v a l u e s o f A G i n t , A H i n t , a n d T,~Sin t r e f e r to t h e t r a n s f e r o f i m m u n o g l o b u l i n G at infinite d i l u t i o n from aqueous to d e n a t u r a n t solutions of designated molarity. Conen. (M)
--A Gin t
--AHint (kJ/mol)
--TASint
3.0 4.0 6.0
45 ± 30 95 ± 40 2 5 0 ± 80
1170 + 125 1590 ± 330 1 9 7 0 ± 500
1 1 2 5 -+ 1 5 5 1 4 9 5 -* 3 7 0 2720 ± 580
ant concentration affecting the solvation sheaths of the protein molecules. The free energies of transfer, i.e., overall interaction, AGint, can be calculated from preferential binding data using the equation [2,7] : (rn)
AGint = --R T (m=O) . (
P a,pref (m )dlna3
(3)
where m is the denaturant molality and a3 its activity. The activity of guanidinium chloride as a function of its molality is known [8]. Table II shows the values of AGint at 3, 4, and 6 M obtained by graphical integration of Eqn. 3. The actual integration encompasses the concentration range from 1 M to the upper limit. The contribution to the integral below 1 M is negligible, since at lower concentrations preferential binding is virtually non-existent (see Table I). All the values of AGint are negative, and the absolute values increase with increasing denaturant concentration. Moreover, as can be seen from Table II, the errors involved are quite large which is due to the large error accompanying the determination of preferential binding. Since in 3 M guanidinium chloride the protein is already completely unfolded [9], with constraints imposed by the disulfide bonds, the difference in AGint between this concentration and the higher ones may be attributed to positive preferential binding of denaturant with its increasing concentration. Thus, binding of denaturant is evidently the main factor in bringing about denaturation of human IgG, and it makes also the largest contribution to AVint, which is in agreement with previous findings [1,2]. Enthalpies of interaction. It has been shown previously [2] that the enthalpy changes on mixing (2.00 + ¢) ml protein solution and 4.00 ml denaturant solution, AH, in the reaction cell, and 2.00 ml buffer solution and 4.00 ml same denaturant solution, AH~f, in the reference cell are interrelated by the expression [2] AH= AHref + n2 (H231--H21 = AHref + n2AHin t
(4)
where n2 is the number of tool protein in the reaction cell and H23~ and g21 are the partial molar enthalpies of the Protein in the presence and absence of denaturant. The difference (t~23~--H21) is the enthalpy of interaction, AHint, resulting from transfer of 1 tool protein from buffer to the denaturant solution. Assumptions underlying the derivation of Eqn. 4 have been discussed previ-
384
3200
2800
2000
" •1 6 0 0 .c
-r
1200
I
800
400
0 L/ 0
I 1
1 2
t 3
I 4
l $
GdmCl
conc. ( m o l / I )
[ 6
7
Fig. 2. E n t h a l p i e s o f i n t e r a c t i o n of h u m a n i m m u n o g l o b u l i n G in a q u e o u s g u a n i d i n i u m c h l o r i d e s o l u t i o n s
at 25eC and pH 7.7.
ously [2]. Moreover, considering the relatively large experimental errors (see below) the contribution due to protein dilution has been neglected. The values of AHint calculated using Eqn. 4 are assembled in Table II, and in Fig. 2 they are plotted as a function of guanidinium chloride concentration. Examination of Table II and Fig. 2 shows that all the values of ~k/-/in t a r e negative and the errors involved relatively large. The curve displays an inflection point in the unfolding region. Thus, there is little doubt that the denaturant binding makes the largest contribution to AHint, whereas the contribution due to unfolding which is of opposite sign and smaller, is also clearly reflected. Similar behavior has been observed with ~-lactoglobulin in urea solutions [2]. Changes in protonation accompanying unfolding have not been ascertained. However, considering the presence of buffer the contribution to AHin t resulting from them cannot be significant [10]. Knowledge of A G i n t and ~ L / i n t allows the calculation of A S i n t. The values of T ~ i n t are included in Table II. Since the values of AGint are, in comparison with the values of AHlnt, relatively small, the values of TASint are not much smaller than the latter, This again suggests thermodynamic compensation [11].
385
In summary, it has been observed that human IgG in solutions of guanidinium chloride behaves similarly as other globular proteins studied so far. The largest contribution to the thermodynamic quantities of interaction stems from denaturant binding which is undoubtedly the necessary condition for protein unfolding so that any proposed mechanism of the unfolding by guanidinium chloride has to consider it. Acknowledgement This work was supported by the Research Council of Slovenia. References 1 2 3 4 5 6 7 8 9 10 11
Vlachy, V. and Lapanje, S. (1976) Biochim. Biophys. Acta 427, 387--391 Lapanje, S., Lunder, M., Vlachy, V. and ~kerjanc, J. (1977) Biochim. Biophys. Acta 491,482--490 Reisler, E. and Eisenberg, H. ( 1 9 6 9 ) Biochemistry 8, 4572--4578 Lee, J.C. and Timasheff, S.N. (1974) Biochemistry 13, 257--265 Stevenson, G.T. and Dorrington, K.J. (1970) Biochem. J. 118, 703--712 Span, J., Lenar~i~, S. and Lapanje, S. (1974) Bioehim. Biophys. Acta 359, 311--319 Vlachy, V. and Lapanje, S. (1978) Biopolymers 17, in the press Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4586--4590 Lapanje, S. and Dorrington, K.J. (1973) Biochim. Biophys. Acta 322, 45--52 Privalov, P.L. and Khechinashvili, N.N. (1974) J. Mol. Biol. 86, 665--684 Lumry, R. and Rajender, S. (1970) Biopolymers 9, 1125--1227
Biochimica et Biophysica Acta, 537 (1978) 380--385
© Elsevier/North-Holland Biomedical Press
BBA 3 8 0 4 0 THERMODYNAMICS OF THE ISOTHERMAL INTERACTION OF HUMAN IMMUNOGLOBULIN G WITH GUANIDINIUM CHLORIDE
SAVO LAPANJE, BRONISLAVA CRESNAR, VOJKO VLACHY and JOZE SKERJANC Department of Chemistry, University of Ljubljana, Ljubljana (Yugoslavia)
(Received March 15th, 1978)
Summary A t h e r m o d y n a m i c study of the isothermal interaction of human immunoglobulin G with guanidinium chloride, a strong denaturant, has been performed. Free energies of interaction were calculated using preferential binding data obtained by measuring densities at constant chemical potential and constant composition, respectively. Enthalpies of interaction were determined calorimetrically. The values of both t h e r m o d y n a m i c parameters as well as those of entropies of interaction have been found to depend crucially on the extent of denaturant binding.
Introduction In previous papers [1,2] the thermodynamics of the interaction of lysozyme and/3-1actoglobulin, respectively, with guanidinium chloride, a strong denaturant, has been studied. In this communication, we report on the interaction of another protein, h u m a n immunoglobulin G (IgG), with guanidinium chloride. Since in this case the preferential binding data for denaturant which are needed for calculation of the free energies of interaction were not available, it was necessary to perform separate experiments for determining the binding as a function of denaturant concentration. The m e t h o d chosen is based on density measurements [3,4]. The enthalpies of interaction were again determined calorimetrically. From them and the free energies of interaction the entropies of interaction can be calculated allowing a complete thermodynamic description of the protein-denaturant interaction. Materials and Methods Normal h u m a n serum was provided as a pool by the Institute of Blood Transfusion of Slovenia. IgG was isolated by (NH4)2SO4 precipitation, DEAE-
381 cellulose and G-200 chromatography [5]. The buffer used was 0.01 M TrisHC1/0.02 M NaC1 (pH 7.7). Protein concentrations were determined spectrophotometrically using the value 14.0 for the absorption coefficient {~lcm. 1% Guanidinium chloride was a product of Merck-Schuchardt and before use it was washed twice with acetone then dissolved in hot ethanol and precipitated with n-hexane. Density measurements were performed with a Precision Density Meter D M A 02 (A. Paar, Gratz). Solutions for measurements should not contain large particles; therefore, the solvent was passed through a sintered-glassfilterbefore dissolving the protein. To determine the extent of preferential binding, densities had to be measured at constant chemical potential and constant composition of solvent components (see Eqn. 2). Calorimetric experiments were carried out in an L K B Batch Microcalorimeter 10700-2. Initialprotein concentrations were around 1.5% (w/v). The procedure was identical to that described previously [2]. The essential feature is mixing of (2.00 +¢)ml protein solution with 4.00 ml appropriate denaturant solution in the reaction cell and of 2.00 ml buffer solution and 4.00 ml same denaturant solution in the reference cell.¢ is the protein displacement volume, i.e.,the product of protein mass x partial specificvolume. Results and Discussion
Determination of preferential binding and free energies o[ interaction. The preferential binding of a denaturant in three-compoent system, P3,pref, with components 1 (water), 2 (protein) and 3 (denaturant) is define by the following expression ,. M2(~g31 ~3.pref --'~~ \~"2/T,/./,1 j/, 3
(1)
where M2 is the molecular weight of the protein and M3 that of the denaturant, g is the concentration in g/g water, T is the temperature, and u the chemical potential. (Sg3/~g2)T,, ~,, 3 is obtained from de~usity..data at constant chemical potential and constant composition using the following equation [3,4]:
t gq 5p
'~2
where p is the densiW and m the molality. The values of (Sg3/~g2)r., 1., 3 for infinite dilution of protein are given in Table I which contains also the values of parameters used in their calculation. In Fig. 1 the values of (SP/~g2) are plotted as a function of protein concentration. Comparison ~ t h lysozyme [6] reveals that the preferential binding of guanidinium chloride to human IgG is, in general, smaller. Moreover, as with some other proteins, preferential binding reaches a maximum at about 5 M guanidinium chloride. Although no convincing interpretation for this, behavior is available, it could be due to changes in the structure of water at high denatur-
382 TABLE I PREFERENTIAL BINDING PARAMETERS OF HUMAN IMMUNOGLOBULIN G AT INFINITE DILUT I O N IN A Q U E O U S S O L U T I O N S O F G U A N I D I N I U M C H L O R I D E A T 2 5 ° C A N D p H 7.7
O6p) _
Concn. (M)
( 6p)o ~g2 T'P'm3
t~g2 T ' ~ l ' # 3
~P
(=---)T'P'm2og3
( c m -3 × 1 0 4 ) 1 2 3 4 5 6
2 . 3 7 2 _+0 . 0 0 7 2.201 1.954 1.665 1.471 1.022
2.0
(g/g p r o t e i n )
2.378 ± 0.007 2.188 1.945 1.623 1.414 0.996
2 . 2 7 5 _+ 0 . 0 0 1 1.888 1.549 1.243 0.983 0.754
0
0
3M
~g3 0 (~g2)T'gl'#3
0.00 0.01 0.025 0.035 0.06 0.035
0
± 0.01 ± 0.01 ± 0.01 ± 0.01 _+ 0.01 ± 0.02
O--
6Q ~
x
1.5
'E
u v
1.0 -
©
©
\-~g2 T,~I,F3
,(3--
6M
0.5
0
I
I
I
"5
10
15
conc.(g/I )
Fig. I . D e p e n d e n c e of p r e f e r e n t i a l b i n d i n g p a z a m e t e r s t i o n a t 25eC a n d p H 7.7.
(Op/~g2 ) o n
human immunoglobulin G concentra-
383
T A B L E II T H E R M O D Y N A M I C P A R A M E T E R S OF THE I N T E R A C T I O N OF H U M A N I M M U N O G L O B U L I N G W I T H G U A N I D I N I U M C H L O R I D E A T 25°C A N D p H 7.7 T h e v a l u e s o f A G i n t , A H i n t , a n d T,~Sin t r e f e r to t h e t r a n s f e r o f i m m u n o g l o b u l i n G at infinite d i l u t i o n from aqueous to d e n a t u r a n t solutions of designated molarity. Conen. (M)
--A Gin t
--AHint (kJ/mol)
--TASint
3.0 4.0 6.0
45 ± 30 95 ± 40 2 5 0 ± 80
1170 + 125 1590 ± 330 1 9 7 0 ± 500
1 1 2 5 -+ 1 5 5 1 4 9 5 -* 3 7 0 2720 ± 580
ant concentration affecting the solvation sheaths of the protein molecules. The free energies of transfer, i.e., overall interaction, AGint, can be calculated from preferential binding data using the equation [2,7] : (rn)
AGint = --R T (m=O) . (
P a,pref (m )dlna3
(3)
where m is the denaturant molality and a3 its activity. The activity of guanidinium chloride as a function of its molality is known [8]. Table II shows the values of AGint at 3, 4, and 6 M obtained by graphical integration of Eqn. 3. The actual integration encompasses the concentration range from 1 M to the upper limit. The contribution to the integral below 1 M is negligible, since at lower concentrations preferential binding is virtually non-existent (see Table I). All the values of AGint are negative, and the absolute values increase with increasing denaturant concentration. Moreover, as can be seen from Table II, the errors involved are quite large which is due to the large error accompanying the determination of preferential binding. Since in 3 M guanidinium chloride the protein is already completely unfolded [9], with constraints imposed by the disulfide bonds, the difference in AGint between this concentration and the higher ones may be attributed to positive preferential binding of denaturant with its increasing concentration. Thus, binding of denaturant is evidently the main factor in bringing about denaturation of human IgG, and it makes also the largest contribution to AVint, which is in agreement with previous findings [1,2]. Enthalpies of interaction. It has been shown previously [2] that the enthalpy changes on mixing (2.00 + ¢) ml protein solution and 4.00 ml denaturant solution, AH, in the reaction cell, and 2.00 ml buffer solution and 4.00 ml same denaturant solution, AH~f, in the reference cell are interrelated by the expression [2] AH= AHref + n2 (H231--H21 = AHref + n2AHin t
(4)
where n2 is the number of tool protein in the reaction cell and H23~ and g21 are the partial molar enthalpies of the Protein in the presence and absence of denaturant. The difference (t~23~--H21) is the enthalpy of interaction, AHint, resulting from transfer of 1 tool protein from buffer to the denaturant solution. Assumptions underlying the derivation of Eqn. 4 have been discussed previ-
384
3200
2800
2000
" •1 6 0 0 .c
-r
1200
I
800
400
0 L/ 0
I 1
1 2
t 3
I 4
l $
GdmCl
conc. ( m o l / I )
[ 6
7
Fig. 2. E n t h a l p i e s o f i n t e r a c t i o n of h u m a n i m m u n o g l o b u l i n G in a q u e o u s g u a n i d i n i u m c h l o r i d e s o l u t i o n s
at 25eC and pH 7.7.
ously [2]. Moreover, considering the relatively large experimental errors (see below) the contribution due to protein dilution has been neglected. The values of AHint calculated using Eqn. 4 are assembled in Table II, and in Fig. 2 they are plotted as a function of guanidinium chloride concentration. Examination of Table II and Fig. 2 shows that all the values of ~k/-/in t a r e negative and the errors involved relatively large. The curve displays an inflection point in the unfolding region. Thus, there is little doubt that the denaturant binding makes the largest contribution to AHint, whereas the contribution due to unfolding which is of opposite sign and smaller, is also clearly reflected. Similar behavior has been observed with ~-lactoglobulin in urea solutions [2]. Changes in protonation accompanying unfolding have not been ascertained. However, considering the presence of buffer the contribution to AHin t resulting from them cannot be significant [10]. Knowledge of A G i n t and ~ L / i n t allows the calculation of A S i n t. The values of T ~ i n t are included in Table II. Since the values of AGint are, in comparison with the values of AHlnt, relatively small, the values of TASint are not much smaller than the latter, This again suggests thermodynamic compensation [11].
385
In summary, it has been observed that human IgG in solutions of guanidinium chloride behaves similarly as other globular proteins studied so far. The largest contribution to the thermodynamic quantities of interaction stems from denaturant binding which is undoubtedly the necessary condition for protein unfolding so that any proposed mechanism of the unfolding by guanidinium chloride has to consider it. Acknowledgement This work was supported by the Research Council of Slovenia. References 1 2 3 4 5 6 7 8 9 10 11
Vlachy, V. and Lapanje, S. (1976) Biochim. Biophys. Acta 427, 387--391 Lapanje, S., Lunder, M., Vlachy, V. and ~kerjanc, J. (1977) Biochim. Biophys. Acta 491,482--490 Reisler, E. and Eisenberg, H. ( 1 9 6 9 ) Biochemistry 8, 4572--4578 Lee, J.C. and Timasheff, S.N. (1974) Biochemistry 13, 257--265 Stevenson, G.T. and Dorrington, K.J. (1970) Biochem. J. 118, 703--712 Span, J., Lenar~i~, S. and Lapanje, S. (1974) Bioehim. Biophys. Acta 359, 311--319 Vlachy, V. and Lapanje, S. (1978) Biopolymers 17, in the press Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4586--4590 Lapanje, S. and Dorrington, K.J. (1973) Biochim. Biophys. Acta 322, 45--52 Privalov, P.L. and Khechinashvili, N.N. (1974) J. Mol. Biol. 86, 665--684 Lumry, R. and Rajender, S. (1970) Biopolymers 9, 1125--1227
