- Email: [email protected]
Thermodynamics of hapten-antibody interaction(s)
72
TIBS - March 1980
5 Lee, S. G. and Lipmann, F. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2343-2347 6 Mukerjee, A. K. and Singh, A. K. (1978) Tetrahedron Lett. 34, 1711-1763 7 Katz, E. and Demain, A. L. (1977)Bacteriol. Rev. 41,449-474 8 Demain, A. L. (1973)Adv. Appl. Microbiol. 16, 177-202 9 Malik, V. S. (1979)Adv. Genet. 20, 37-126 10 Miller, J. H. and Reznikoff, W. S. (1978) The Operon, Cold Spring Harbor Laboratory
11 Floss, H. G., Robbers, J. E. and Heinstein, P. F. (1974) in Recent Advances in Phytochem. 8, 141-178, Appleton, New York 12 Drew, S. and Demain, A. L. (1977) Annu. Rev. Microbiol. 31,343-356 13 Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46, 263-293 14 Jones, A. and Westlake, D. W. S. (1974) Can. J. MicrobioL 20, 1599--1603 15 Luckner, M., Nover, L. and Bohm, U. (1977) Sec-
Thermodynamics of hapten-antibody interaction(s) T. K. S. Mukkur Recent thermodynamic investigations o f hapten-antibody interaction(s), conducted over a wide range o f temperatures, have revealed that the formation o f hapten-antibody complexes at lower temperatures is predominantly enthalpy-driven, while at higher temperatures these interactions are predominantly entropy-driven, thus permitting one to rationalize the mechanism o f hapten--antibody interactions(s) as enthalpy--entropy compensation. A combination of thermodynamic and structural information is necessary to gain a complete understanding of the nature of antigen-antibody interaction. While the results of some earlier investigations suggested a possible role of entropy in hapten-antibody interaction(s) [1--4], others indicated contribution by both enthalpic and entropic terms [5,6]. In the past, thermodynamic functions for hapten-antibody interaction(s) were generally calculated from the binding data obtained by the use of techniques such as equilibrium dialysis, fluorescence quenching or light scattering. Relatively recently, however, microcalorimetry was used to obtain direct measurement of binding enthalpy for the formation of hapten-antibody complexes [7-9]. In almost all investigations, the range of temperatures used for the analysis of various hapten-antibody interaction(s) was quite narrow and revealed the standard enthalpy change, AH°, to be quite large, the latter being interpreted to the driving force for the formation of hapten-antibody complexes [7-10]. Recently, thermodynamic functions for the interaction of ¢-DNP4-1ysine with heterogeneous specific rabbit and bovine antidinitrophenyl (DNP) IgG and their Fab fragments calculated from binding data obtained using equilibrium dialysis over a wide range of temperatures ( - 3 to +67°C), revealed that the mechanism of hapten-antibody interaction(s) could be described by enthalpy-entropy compensation [11-13]. Since the bioenergetics of T. K. S. Mukkur is at the CSIRO, Division of Animal Health, McMaster Laboratory, Glebe, N.S. W. 2037, Australia.
hapten-antibody interaction(s) has not been reviewed for well over the past decade and a half [14], it appears quite appropriate to attempt to detail current developments in this field of research. In initial studies, Barisas et al. [7] reported discrepancies between the van't Hoff enthalpy values deduced from fluorescence titrations and calorimetric values. In contrast, a good agreement between the binding enthalpies obtained from equilibrium dialysis measurements was reported [9]. Therefore, it was suggested that possibly the Sip's distribution function used for calculating the average association constant, K0, did not give a proper representation of the heterogeneity in the binding data [15,16]. Therefore, Johnston et al. [9] investigated the thermodynamics of hapten binding to mouse myeloma proteins which exhibited a substantial affinity for a broad spectrum of polynitrophenyl and related haptens. Haptens used included ~-DNP4-1ysine, menadione, dinitronaphthol and ~-tdnitriphenyl-Llysine, DNP-glycine and DNP-aminocaproic acid. Binding data were obtained at 4 and 25°C by fluorescence queiching, equilibrium dialysis and calorimetry. The binding enthalpies (Z~LIb)were found to be large (-12.1 to -20.2 kcal) and were therefore considered to be the driving force for ligand-protein interaction(s). Furthermore, the change in heat capacity on binding (ACp) was negative which was rationalized in terms of possible charge transfer association of ligands with the tryptophan in the antibody combining site. Halsey and Biltonen [6] studied the thermodynamics of the interaction of
16 17 18 19 20
ondary Metabolism and Cell Differentiation, Springer-Verlag Hitchcock, J. M. and Katz, E. (1978) Antimicrob. Agents Cheraother. 13, 104-114 Tyler, B. (1978) Annu. Rev. Microbiol. 47, 1127-1162 Raper, K. B. (1978)ASMNews 44, 645--653 Vining, L. C. (1979) Adv. Appl. Microbiol. 25, 147-168 Robbers, J. E., Eggert, W. W. and Floss, H. G. (1978) Lloydia 41,120--129
c-DNP- l-lysine with heterogeneous rabbit IgG fractions with different binding affinities by the use of a calorimeter. An average AH of - 1 3 . 9 kcal/mol was measured. Although there was no significant correlation between the enthalpy and free energy changes, a statistically significant correlation was observed between the free energy changes and unitary entropy changes which varied from -5.1 to -10.6cal/mol deg. Since hydrophobic interactions are thought to be involved in the formation of hapten-antibody complexes, one would have predicted a positive entropy contribution. Based on their observations of a negative heat capacity change for the transfer of DNP-lysine from water to ethanol, these authors attempted rationalization by suggesting that perhaps this large negative entropic contribution was associated with other aspects of the hapten-antibody interaction. Similarly, Epstein et al. [2] who studied the interaction of haptens containing the phenylarsonic acid group with rabbit anti-arsanilic antibody using a light scattering method at temperatures ranging from 15 to 37°C reported a positive entropy change (AS°) of 2 2 _ 9 e.u./mol bonds at 25°C. These results were explained in terms of the liberation of a number of water molecules on combination of hapten with the antibody molecules. Clearly, the calorimetric experiments need to be performed over a wider range of temperatures before any definite conclusions can be drawn. Binding enthalpy values for the interaction of Lac dye with rabbit antilactose antibody fractions, calculated from binding data obtained by equilibrium dialysis, ranged from - 9 . 5 kcal to -11.3 kcal per mol of hapten bound [17]. This narrow range of values of AH were interpreted to suggest that the antibody-ligand interaction utilized almost the full capacity of the lactosyl moiety for hydrogen bond formation in an antibody site. It was, therefore, concluded that the AH term was primarily due to multiple hydrogen bonding and that there was no significant change in the heat capacity associated with the formation of hapten-antibody complex. The contribu© Elsevier/North-HollandBiomedicalPrc,~ |gNI)
TIBS - March 1980
73
temperatures appeared to be compensated by the negative enthalpy factor resulting in a favourable free energy change for each of the anti-DNP antibody preparations. Therefore it would appear that the Temperature at which the enthalpy factor was a primary contributor enthalpy factor equals the entropy factor to the driving force for the formation of Before correction (*C) After correction (*C) Antibody preparation AC~ hapten-antibody complex at lower temperatures as suggested previously [7-10] but Rabbit anti-DNP IgG + 472.2 17.5 16 Bovine anti-DNP IgGl +332.0 41.5 39 the entropy factor assumed greater imporBovine anti-DNP Fab' of IgG~ + 362.0 26 22 tance at increased temperatures, thus perBovine anti-DNP IgGs + 280.1 25 19.5 mitting one to rationalize a mechanism for hapten-antibody interaction(s) in terms of enthalpy--entropy compensation effect tion of hydrophobic bonding in this system [11,12], and bovine anti-DNP IgG2 [18] which has previously been used to explain was not assigned any significant :role. over a wide range of temperatures ( - 3 to various enzymatic catalytic reactions [ 19]. The predominance of enthalpy factor at One difficulty associated with practically +67°C), non-linear van't Hoff plots were all the previous thermodynamic investiga- obtained. This forbade the use of the classi- low temperatures could be rationalized by tions is that the binding experiments have cal van't Hoff equation [ 18] for the calcula- suggesting the predominance of hydrogen been carried out over a narrow range of tion of various thermodynamic functions bonding possibly formed for each bound temperatures. Further difficulty encoun- for which purpose appropriate equations hapten [10,20]. However, it does not rule tered with some investigations is the lack of were therefore derived [11,12]. The unit- out the participation of hydrophobic clarification as to whether any corrections ary free energy change (AG,) and unitary interactions, the latter being expected to be for non-specific binding of the hapten to entropy change (AS,) could then be calcu- predominant if the entropy contributions the non-antibody immunoglobulins have lated according to previously described are positive instead of unfavourable negabeen made [9]. In another instance, the equations [7,14]. A typical van't Hoff plot tive unitary entropy changes [7,8,10]. That non-antibody immunoglobulin used for is shown in Fig. 1. The extent of ctirvatures hydrophobic bonding possibly participates making non-specific corrections was dif- was found to be indicative of large positive in formation of hapten-antibody comferent from the one used for specific bind- heat capacity changes, ACg (Table I), which plexes in spite of a negative heat capacity ing studies [6]. This factor could be quite were indicative of the unfolding of an anti- change has already been suggested because significant, particularly if the immuno- body combining site as a result of combina- of the observed negative heat capacity globulins under investigation undergo con- tion with the hapten. Various ther- change for the transfer of DNP-lysine from formational changes on addition of the modynamic functions, when plotted versus water to ethanol [6]. Our experiments hapten, and exhibit negative cooperativity temperature, revealed an enthalpy- [11-13], however, revealed a positive heat in a manner similar to the one reported for entropy compensation for the interaction capacity change (AC~,)for the interaction of of c-DNPd-lysine with all the anti-DNP c-DNP- l-lysine and anti-DNP antibodies bovine IgGl [16]. In recent investigations of the thermo- antibody preparations used in the investi- of rabbit and bovine origin (Table I). If one dynamics for the interaction of ~-DNP- gation. A typical enthalpy-entropy com- assumed that the negative values of AHtot~ at low temperatures were predominantly l-lysine with rabbit anti-DNP IgG, bovine pensation plot is shown in Fig. 2. anti-DNP IgG1 and its fragments, Fab' The unfavourable entropy factor at low due to AHbinding of ~-DNP4-1ysine with specific antibody, the trend towards increasing positwe values with increasing 6temperatures would be compensation by AH~oivent, thus suggesting a possible mode t for the participation of solvent-protein interactions in the formation of hapten-antibody complexes. The entropy effects could also be 5.6rationalized in terms of a compensation mechanism. The observed trend in AS, could be considered to represent a compensating effect(s) of ASinternaland AS~olvent, the former being more important at lower and the latter at higher temperatures. If 5.2t ASt°,,~ (AS~) was corrected for the entropy of mixing to obtain AS,, the unitary entropy change, and utilized for the compensation plot, enthalpy-entropy compensation was observed again, with the exception that the temperature at which the enthalpy factor 4.a I I I I 2.9 3.1 3.3 3.5 3.7 equalled the entropy factor was slightly lowered (Table I), thus suggesting an in103fr significant contribution of the entropy of Fig. 1. A van' t Hoff plot for the interactic.n o f c-DNP-l-lysine with rabbit anti-DNP lgG. K r denotes total affinity mixing to the entropy due to specific hapconstant, T denotes absolute temperature.
TABLE I Standard heat capacity changes (ACp) and the temperature (°C) at which the enthalpy factor equals the entropy factor, before and after correction for the entropy of mixing, for the interaction of ~-DNPd-lysine with anti-DNP antibodies of rabbit and bovine origin.
I
74
TIBS - March 1980
TABLE II Thermodynamic functions for the interaction of E-DNP4-1ysine, and rabbit and bovine anti-DNP antibodies Temperature (*C)
Rabbit anti-DNP lgG AGu AH° ASu
Bovine anti-DNP IgG, AGu AH ° ASu
-3 4 12 15 20 22 25 36 37 52 57 60 67
-9.27
-9.65
-18.20
-31.90
-9.31
-13.29
-13.96
-9.83
-14.22
-3.35
-18.34
+20.32 -9.23
-9.87
+4.69
+46.99
-11.11
+14.15
+78.55
-11.95
+18.88
+90.69
=9.32 -9.65
-10.19
-9.97
-0.50 -0.02
+0.50
Bovine anti-DNP Fab' IgGl AGu AH ° ASu -8.72
-13.96
Bovine anti-DNP IgG, AGu AH* ASu
-19.38
-8.63
-5.27
+11.45
-8.95
+0.16
+29.46
-7.06
-8.05
-3.64
-7.07
-4.97
+7.27
-7.18
-2.17
+16.75
-7.49
+1.19
+27.89
-7.99 -8.32
+5.40 +7.65
+41.13 +47.94
-2.51
+13.96 +29.64
+44.62
-9.81
+7.76
+53.27
-10.29
+11.38
+64.17
While AGu and AlP are expressed in kcal/moi, ASu is expressed in entropy units (e.u.). The data presented in this table have been calculated from results published previously [ 11-131.
ten-antibody interaction(s). Further, the AS, increased to positive values with increasing temperature (Table II), thus suggesting a greater contribution of hydrophobic bonding in the formation of hapten-antibody complexes. Information regarding the interaction of multivalent antigens with specific antibodies is scanty. The first such report to appear in the literature [3] dealt with the interaction of bovine serum albumin (BSA) with anti-BSA antibody. The heat
of reaction determined calorimetrically was negative but the entropy change was calculated to be positive. However, recent studies which also involved the interaction of BSA with specific anti-BSA antibody [6] reported negative unitary entropy changes accompanying the above reaction. Clearly, the results are inconclusive at present and represent an area of research which needs further investigation. Future investigations need be aimed at the determination of
20
--TZS? 10
i
0
t~
-10
/ -20
-
-30
-
--40 240
260
280 300 Temperature (°K)
320
340
Fig.2. Compensation plot for the interaction of e DNP-l-lysine with rabbuann • • DNP lgG. AG oand AS~denotethe o standard free energy and entropy changes, respectively. AH ° denotes the standard enthalpy change.
(a) the thermodynamic parameters for the interaction of multivalent antigens with specific antibodies, (b) influence of antibody polyvalency on various thermodynamic functions and (c) the relative contribution of various types of forces participating in the formation of hapten/antigen-antibody complexes. References 1 Berson, S. A. and Yalow, R. S. (1959) J. Clin. Invest. 38, 1996-2016 2 Epstein, S. I., Doty, R. and Boyd, W. C. (1956) Z Am. Chem. Soc. 78, 3306-3315 3 Steiner, R. F. and Kitzinger, C. (1956) J. Biol. Chem. 222, 271-284 4 Velick, S. F., Parker, C. W. and Eisen, H.N. (1960) Proc. Natl. Acad. Sci. U.S.A. 46, 1470-1482 5 Karush, F. ( 1 9 5 6 ) J . Am. Chem. Soc. 78, 5519--5526 6 Halsey, J. F. and Biltonen, R.L. (1975) Biochemistry 14, 800-804 7 Barisas, B. G., Sturtevant, J. M. and Singer, S. J. (1971) Biochemistry 10, 2816-2821 8 Barisas, B. G., Singer, S. J. and Sturtevant, J. M. (1972) Biochemistry 11, 2741-2744 9 Johnston, M. F. M., Barisas, B. G. and Sturtevant, J. M. (1974) Biochemistry 13,390-396 10 Ghose, A. C. and Karush, F. (1974) Biochemistry 13, 1959-1964 11 Szewczuk, M. R. and Mukkur, T. K. S. (1977) Immunology 32, 111-119 12 Szewczuk, M. R. and Mukkur, T. K. S. (1977) Immunology 33, 11-16 13 Mukkur, T. K. S. (1978) Biochem. J. 172, 39--44 14 Karush, F. (1962)Adv. Immut~l. 2, 1--40 15 Werblin, T. P. and Siskind, G. W. (1972) lmmunochemistry 9, 987-1011 16 Mukkur, T. K. S. and Fang, W. D. (1976) Anal Biochem. 75, 183-191 17 Medof, M. E. and Aladjem, F. (1971) Fedn. Proc. 30, 657-(abstract 2581) 18 Mahan, B. H. (1963) Elementary Chemical Thermodynamics, W. A. Benjamin, Inc., California, U.S.A. 19 Hammes, G, (1964) Nature (London) 204, 342-343 20 Eisen, H. N. and Siskind, G. W. (1964) Biochemistry 3, 996-1008
TIBS - March 1980
5 Lee, S. G. and Lipmann, F. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2343-2347 6 Mukerjee, A. K. and Singh, A. K. (1978) Tetrahedron Lett. 34, 1711-1763 7 Katz, E. and Demain, A. L. (1977)Bacteriol. Rev. 41,449-474 8 Demain, A. L. (1973)Adv. Appl. Microbiol. 16, 177-202 9 Malik, V. S. (1979)Adv. Genet. 20, 37-126 10 Miller, J. H. and Reznikoff, W. S. (1978) The Operon, Cold Spring Harbor Laboratory
11 Floss, H. G., Robbers, J. E. and Heinstein, P. F. (1974) in Recent Advances in Phytochem. 8, 141-178, Appleton, New York 12 Drew, S. and Demain, A. L. (1977) Annu. Rev. Microbiol. 31,343-356 13 Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46, 263-293 14 Jones, A. and Westlake, D. W. S. (1974) Can. J. MicrobioL 20, 1599--1603 15 Luckner, M., Nover, L. and Bohm, U. (1977) Sec-
Thermodynamics of hapten-antibody interaction(s) T. K. S. Mukkur Recent thermodynamic investigations o f hapten-antibody interaction(s), conducted over a wide range o f temperatures, have revealed that the formation o f hapten-antibody complexes at lower temperatures is predominantly enthalpy-driven, while at higher temperatures these interactions are predominantly entropy-driven, thus permitting one to rationalize the mechanism o f hapten--antibody interactions(s) as enthalpy--entropy compensation. A combination of thermodynamic and structural information is necessary to gain a complete understanding of the nature of antigen-antibody interaction. While the results of some earlier investigations suggested a possible role of entropy in hapten-antibody interaction(s) [1--4], others indicated contribution by both enthalpic and entropic terms [5,6]. In the past, thermodynamic functions for hapten-antibody interaction(s) were generally calculated from the binding data obtained by the use of techniques such as equilibrium dialysis, fluorescence quenching or light scattering. Relatively recently, however, microcalorimetry was used to obtain direct measurement of binding enthalpy for the formation of hapten-antibody complexes [7-9]. In almost all investigations, the range of temperatures used for the analysis of various hapten-antibody interaction(s) was quite narrow and revealed the standard enthalpy change, AH°, to be quite large, the latter being interpreted to the driving force for the formation of hapten-antibody complexes [7-10]. Recently, thermodynamic functions for the interaction of ¢-DNP4-1ysine with heterogeneous specific rabbit and bovine antidinitrophenyl (DNP) IgG and their Fab fragments calculated from binding data obtained using equilibrium dialysis over a wide range of temperatures ( - 3 to +67°C), revealed that the mechanism of hapten-antibody interaction(s) could be described by enthalpy-entropy compensation [11-13]. Since the bioenergetics of T. K. S. Mukkur is at the CSIRO, Division of Animal Health, McMaster Laboratory, Glebe, N.S. W. 2037, Australia.
hapten-antibody interaction(s) has not been reviewed for well over the past decade and a half [14], it appears quite appropriate to attempt to detail current developments in this field of research. In initial studies, Barisas et al. [7] reported discrepancies between the van't Hoff enthalpy values deduced from fluorescence titrations and calorimetric values. In contrast, a good agreement between the binding enthalpies obtained from equilibrium dialysis measurements was reported [9]. Therefore, it was suggested that possibly the Sip's distribution function used for calculating the average association constant, K0, did not give a proper representation of the heterogeneity in the binding data [15,16]. Therefore, Johnston et al. [9] investigated the thermodynamics of hapten binding to mouse myeloma proteins which exhibited a substantial affinity for a broad spectrum of polynitrophenyl and related haptens. Haptens used included ~-DNP4-1ysine, menadione, dinitronaphthol and ~-tdnitriphenyl-Llysine, DNP-glycine and DNP-aminocaproic acid. Binding data were obtained at 4 and 25°C by fluorescence queiching, equilibrium dialysis and calorimetry. The binding enthalpies (Z~LIb)were found to be large (-12.1 to -20.2 kcal) and were therefore considered to be the driving force for ligand-protein interaction(s). Furthermore, the change in heat capacity on binding (ACp) was negative which was rationalized in terms of possible charge transfer association of ligands with the tryptophan in the antibody combining site. Halsey and Biltonen [6] studied the thermodynamics of the interaction of
16 17 18 19 20
ondary Metabolism and Cell Differentiation, Springer-Verlag Hitchcock, J. M. and Katz, E. (1978) Antimicrob. Agents Cheraother. 13, 104-114 Tyler, B. (1978) Annu. Rev. Microbiol. 47, 1127-1162 Raper, K. B. (1978)ASMNews 44, 645--653 Vining, L. C. (1979) Adv. Appl. Microbiol. 25, 147-168 Robbers, J. E., Eggert, W. W. and Floss, H. G. (1978) Lloydia 41,120--129
c-DNP- l-lysine with heterogeneous rabbit IgG fractions with different binding affinities by the use of a calorimeter. An average AH of - 1 3 . 9 kcal/mol was measured. Although there was no significant correlation between the enthalpy and free energy changes, a statistically significant correlation was observed between the free energy changes and unitary entropy changes which varied from -5.1 to -10.6cal/mol deg. Since hydrophobic interactions are thought to be involved in the formation of hapten-antibody complexes, one would have predicted a positive entropy contribution. Based on their observations of a negative heat capacity change for the transfer of DNP-lysine from water to ethanol, these authors attempted rationalization by suggesting that perhaps this large negative entropic contribution was associated with other aspects of the hapten-antibody interaction. Similarly, Epstein et al. [2] who studied the interaction of haptens containing the phenylarsonic acid group with rabbit anti-arsanilic antibody using a light scattering method at temperatures ranging from 15 to 37°C reported a positive entropy change (AS°) of 2 2 _ 9 e.u./mol bonds at 25°C. These results were explained in terms of the liberation of a number of water molecules on combination of hapten with the antibody molecules. Clearly, the calorimetric experiments need to be performed over a wider range of temperatures before any definite conclusions can be drawn. Binding enthalpy values for the interaction of Lac dye with rabbit antilactose antibody fractions, calculated from binding data obtained by equilibrium dialysis, ranged from - 9 . 5 kcal to -11.3 kcal per mol of hapten bound [17]. This narrow range of values of AH were interpreted to suggest that the antibody-ligand interaction utilized almost the full capacity of the lactosyl moiety for hydrogen bond formation in an antibody site. It was, therefore, concluded that the AH term was primarily due to multiple hydrogen bonding and that there was no significant change in the heat capacity associated with the formation of hapten-antibody complex. The contribu© Elsevier/North-HollandBiomedicalPrc,~ |gNI)
TIBS - March 1980
73
temperatures appeared to be compensated by the negative enthalpy factor resulting in a favourable free energy change for each of the anti-DNP antibody preparations. Therefore it would appear that the Temperature at which the enthalpy factor was a primary contributor enthalpy factor equals the entropy factor to the driving force for the formation of Before correction (*C) After correction (*C) Antibody preparation AC~ hapten-antibody complex at lower temperatures as suggested previously [7-10] but Rabbit anti-DNP IgG + 472.2 17.5 16 Bovine anti-DNP IgGl +332.0 41.5 39 the entropy factor assumed greater imporBovine anti-DNP Fab' of IgG~ + 362.0 26 22 tance at increased temperatures, thus perBovine anti-DNP IgGs + 280.1 25 19.5 mitting one to rationalize a mechanism for hapten-antibody interaction(s) in terms of enthalpy--entropy compensation effect tion of hydrophobic bonding in this system [11,12], and bovine anti-DNP IgG2 [18] which has previously been used to explain was not assigned any significant :role. over a wide range of temperatures ( - 3 to various enzymatic catalytic reactions [ 19]. The predominance of enthalpy factor at One difficulty associated with practically +67°C), non-linear van't Hoff plots were all the previous thermodynamic investiga- obtained. This forbade the use of the classi- low temperatures could be rationalized by tions is that the binding experiments have cal van't Hoff equation [ 18] for the calcula- suggesting the predominance of hydrogen been carried out over a narrow range of tion of various thermodynamic functions bonding possibly formed for each bound temperatures. Further difficulty encoun- for which purpose appropriate equations hapten [10,20]. However, it does not rule tered with some investigations is the lack of were therefore derived [11,12]. The unit- out the participation of hydrophobic clarification as to whether any corrections ary free energy change (AG,) and unitary interactions, the latter being expected to be for non-specific binding of the hapten to entropy change (AS,) could then be calcu- predominant if the entropy contributions the non-antibody immunoglobulins have lated according to previously described are positive instead of unfavourable negabeen made [9]. In another instance, the equations [7,14]. A typical van't Hoff plot tive unitary entropy changes [7,8,10]. That non-antibody immunoglobulin used for is shown in Fig. 1. The extent of ctirvatures hydrophobic bonding possibly participates making non-specific corrections was dif- was found to be indicative of large positive in formation of hapten-antibody comferent from the one used for specific bind- heat capacity changes, ACg (Table I), which plexes in spite of a negative heat capacity ing studies [6]. This factor could be quite were indicative of the unfolding of an anti- change has already been suggested because significant, particularly if the immuno- body combining site as a result of combina- of the observed negative heat capacity globulins under investigation undergo con- tion with the hapten. Various ther- change for the transfer of DNP-lysine from formational changes on addition of the modynamic functions, when plotted versus water to ethanol [6]. Our experiments hapten, and exhibit negative cooperativity temperature, revealed an enthalpy- [11-13], however, revealed a positive heat in a manner similar to the one reported for entropy compensation for the interaction capacity change (AC~,)for the interaction of of c-DNPd-lysine with all the anti-DNP c-DNP- l-lysine and anti-DNP antibodies bovine IgGl [16]. In recent investigations of the thermo- antibody preparations used in the investi- of rabbit and bovine origin (Table I). If one dynamics for the interaction of ~-DNP- gation. A typical enthalpy-entropy com- assumed that the negative values of AHtot~ at low temperatures were predominantly l-lysine with rabbit anti-DNP IgG, bovine pensation plot is shown in Fig. 2. anti-DNP IgG1 and its fragments, Fab' The unfavourable entropy factor at low due to AHbinding of ~-DNP4-1ysine with specific antibody, the trend towards increasing positwe values with increasing 6temperatures would be compensation by AH~oivent, thus suggesting a possible mode t for the participation of solvent-protein interactions in the formation of hapten-antibody complexes. The entropy effects could also be 5.6rationalized in terms of a compensation mechanism. The observed trend in AS, could be considered to represent a compensating effect(s) of ASinternaland AS~olvent, the former being more important at lower and the latter at higher temperatures. If 5.2t ASt°,,~ (AS~) was corrected for the entropy of mixing to obtain AS,, the unitary entropy change, and utilized for the compensation plot, enthalpy-entropy compensation was observed again, with the exception that the temperature at which the enthalpy factor 4.a I I I I 2.9 3.1 3.3 3.5 3.7 equalled the entropy factor was slightly lowered (Table I), thus suggesting an in103fr significant contribution of the entropy of Fig. 1. A van' t Hoff plot for the interactic.n o f c-DNP-l-lysine with rabbit anti-DNP lgG. K r denotes total affinity mixing to the entropy due to specific hapconstant, T denotes absolute temperature.
TABLE I Standard heat capacity changes (ACp) and the temperature (°C) at which the enthalpy factor equals the entropy factor, before and after correction for the entropy of mixing, for the interaction of ~-DNPd-lysine with anti-DNP antibodies of rabbit and bovine origin.
I
74
TIBS - March 1980
TABLE II Thermodynamic functions for the interaction of E-DNP4-1ysine, and rabbit and bovine anti-DNP antibodies Temperature (*C)
Rabbit anti-DNP lgG AGu AH° ASu
Bovine anti-DNP IgG, AGu AH ° ASu
-3 4 12 15 20 22 25 36 37 52 57 60 67
-9.27
-9.65
-18.20
-31.90
-9.31
-13.29
-13.96
-9.83
-14.22
-3.35
-18.34
+20.32 -9.23
-9.87
+4.69
+46.99
-11.11
+14.15
+78.55
-11.95
+18.88
+90.69
=9.32 -9.65
-10.19
-9.97
-0.50 -0.02
+0.50
Bovine anti-DNP Fab' IgGl AGu AH ° ASu -8.72
-13.96
Bovine anti-DNP IgG, AGu AH* ASu
-19.38
-8.63
-5.27
+11.45
-8.95
+0.16
+29.46
-7.06
-8.05
-3.64
-7.07
-4.97
+7.27
-7.18
-2.17
+16.75
-7.49
+1.19
+27.89
-7.99 -8.32
+5.40 +7.65
+41.13 +47.94
-2.51
+13.96 +29.64
+44.62
-9.81
+7.76
+53.27
-10.29
+11.38
+64.17
While AGu and AlP are expressed in kcal/moi, ASu is expressed in entropy units (e.u.). The data presented in this table have been calculated from results published previously [ 11-131.
ten-antibody interaction(s). Further, the AS, increased to positive values with increasing temperature (Table II), thus suggesting a greater contribution of hydrophobic bonding in the formation of hapten-antibody complexes. Information regarding the interaction of multivalent antigens with specific antibodies is scanty. The first such report to appear in the literature [3] dealt with the interaction of bovine serum albumin (BSA) with anti-BSA antibody. The heat
of reaction determined calorimetrically was negative but the entropy change was calculated to be positive. However, recent studies which also involved the interaction of BSA with specific anti-BSA antibody [6] reported negative unitary entropy changes accompanying the above reaction. Clearly, the results are inconclusive at present and represent an area of research which needs further investigation. Future investigations need be aimed at the determination of
20
--TZS? 10
i
0
t~
-10
/ -20
-
-30
-
--40 240
260
280 300 Temperature (°K)
320
340
Fig.2. Compensation plot for the interaction of e DNP-l-lysine with rabbuann • • DNP lgG. AG oand AS~denotethe o standard free energy and entropy changes, respectively. AH ° denotes the standard enthalpy change.
(a) the thermodynamic parameters for the interaction of multivalent antigens with specific antibodies, (b) influence of antibody polyvalency on various thermodynamic functions and (c) the relative contribution of various types of forces participating in the formation of hapten/antigen-antibody complexes. References 1 Berson, S. A. and Yalow, R. S. (1959) J. Clin. Invest. 38, 1996-2016 2 Epstein, S. I., Doty, R. and Boyd, W. C. (1956) Z Am. Chem. Soc. 78, 3306-3315 3 Steiner, R. F. and Kitzinger, C. (1956) J. Biol. Chem. 222, 271-284 4 Velick, S. F., Parker, C. W. and Eisen, H.N. (1960) Proc. Natl. Acad. Sci. U.S.A. 46, 1470-1482 5 Karush, F. ( 1 9 5 6 ) J . Am. Chem. Soc. 78, 5519--5526 6 Halsey, J. F. and Biltonen, R.L. (1975) Biochemistry 14, 800-804 7 Barisas, B. G., Sturtevant, J. M. and Singer, S. J. (1971) Biochemistry 10, 2816-2821 8 Barisas, B. G., Singer, S. J. and Sturtevant, J. M. (1972) Biochemistry 11, 2741-2744 9 Johnston, M. F. M., Barisas, B. G. and Sturtevant, J. M. (1974) Biochemistry 13,390-396 10 Ghose, A. C. and Karush, F. (1974) Biochemistry 13, 1959-1964 11 Szewczuk, M. R. and Mukkur, T. K. S. (1977) Immunology 32, 111-119 12 Szewczuk, M. R. and Mukkur, T. K. S. (1977) Immunology 33, 11-16 13 Mukkur, T. K. S. (1978) Biochem. J. 172, 39--44 14 Karush, F. (1962)Adv. Immut~l. 2, 1--40 15 Werblin, T. P. and Siskind, G. W. (1972) lmmunochemistry 9, 987-1011 16 Mukkur, T. K. S. and Fang, W. D. (1976) Anal Biochem. 75, 183-191 17 Medof, M. E. and Aladjem, F. (1971) Fedn. Proc. 30, 657-(abstract 2581) 18 Mahan, B. H. (1963) Elementary Chemical Thermodynamics, W. A. Benjamin, Inc., California, U.S.A. 19 Hammes, G, (1964) Nature (London) 204, 342-343 20 Eisen, H. N. and Siskind, G. W. (1964) Biochemistry 3, 996-1008