234
PHOSPHOLIPASE
STRUCTURE-FUNCTION
TECHNIQUES
[21]
[21] T h e r m o d y n a m i c s o f P h o s p h o l i p a s e A2-Ligand Interactions
By RODNEY L. BILTONEN, BRIAN K. LATHROP, and JOHN D. BELL Introduction Phospholipases A2 can be activated on interaction with aggregated phospholipid substrates.l This activation is time-dependent and can thus be studied in considerable detail, m Specific methods for simultaneous study of various time-dependent properties of the enzyme and lipid substrate during the time course of hydrolysis have been described in a separate chapter in this volume. 4 Such investigations provide important clues regarding the mechanisms of the activation process. An important step in the development and testing of hypotheses of activation is the identification and quantitative thermodynamic characterization of the various equilibria involved in the process. Important equilibrium processes involved in phospholipase A 2 catalysis include protein-protein, protein-calcium, and protein-lipid interactions. In this chapter, we describe the use of spectroscopic and calorimetric techniques to obtain thermodynamic information relating to the latter two types of interactions. General Considerations For the simple reaction E + X ~-- EX the equilibrium constant is given by K = [EX] [E][X]
(1)
If some property (e.g., fluorescence intensity) of the enzyme (E) or ligand (X) is proportional to the amount of complex (EX) formed, the fractional degree of association of E as a function of the free concentration of X is given by f =
K[X] AA - - 1 + K[X] z~Amax
(2)
1 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 2 G. Romero, K. Thompson, and R. L. Biltonen, J. Biol. Chem. 262, 13476 (1987). 3 j. D. Bell and R. L. Biltonen, J. Biol. Chem. 264, 12194 (1989). 4 j. D. Bell and R. L. Biltonen, this volume [22].
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[21]
PHOSPHOLIPASE A2 THERMODYNAMICS
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where AA is the measured change in the observable at a given concentration of ligand and AAmax is the maximum change in the observable. ~Ama x and K can be estimated by nonlinear least-squares analysis of lk4 as a function of [X] or by linear regression analysis of the data in reciprocal form. The former method is preferred, but if linear regression of the reciprocal data is used, the data must be properly weighted. In any experiment, Z~ma x is the total change in the observable between the protein in the fully liganded state and the unliganded state. In a calorimetric experiment, z~tAmax is the total heat change for the reaction (AQmax), and the apparent enthalpy change per mole of enzyme is AH:pp = AQmax/ET
(3)
where ET is the total amount of enzyme used in the calorimetric experiment. The apparent Gibb's energy change for the association reaction is given by AGapp = - R T In K = A/~ap p O
--
O
TASapp
(4)
with AS:pp being the apparent entropy change for the reaction. If AH:pp is known, then AS:pp can be calculated. It must be noted that the values of AGapp and ASapp are dependent on the choice of reference state (i.e., concentration units of X used to calculate K). The authors prefer the use of a reference state of moles per liter or mole fraction. In any case, this reference condition should always be stated when reporting values of K, AG:pp and AS:pp. Although calorimetric experiments directly provide estimates for A/~aapp, A/a~app can also be estimated from the temperature dependence of K since O
O
A/'/:app -
d ( - R InK) d(1/T)
(5)
It is important to note that since estimates of the apparent thermodynamic quantities obtained as just described are highly dependent on the assumed model, comparison of the A/-'/~aapp obtained calorimetrically and that obtained by van't Hoff analysis [Eq. (5)] is an excellent test of the model. Generally, the two estimates will agree only if the assumed model used to calculate K is correct. It should be realized that the thermodynamic quantities derived as described above are apparent values. This is because it is generally unlikely that such an elementary representation of the interaction as given in Eq. (1) is correct; rather, the reaction may be coupled with other
236
PHOSPHOLIPASE STRUCTURE--FUNCTIONTECHNIQUES
[]21]
processes such as protein ionization. However, such coupling can be sorted out and in many cases used to advantage in experimental design. An example of this type of phenomenon is given in the discussion of calcium binding to porcine pancreatic phospholipase A 2 and is discussed in more general terms in a previous volume of this series. 5 Calorimetry has been used to study a variety of protein-ligand interactions but has seen limited application to phospholipase A2. All reported calorimetric studies of calcium and lipid binding to phospholipase A 2 have used LKB batch microcalorimeters equipped with twin gold cells, 6-8 and the procedure to be described applies specifically to such an instrument. Each of the cells consists of two compartments. On physical rotation of the calorimeter, the material in the two compartments of each cell is mixed, and the differential heat of mixing is measured. The reference cell contains buffer in both compartments, whereas the compartments of the sample cells contain the reactant solutions. The basic calorimetric experiment consists of mixing the two solutions which contain reactive components. The heat effect associated with the overall process is Qm "~- Qr + Qd + Qp
(6)
where Qm, Qr, Q0, and Qp are the measured heat, the heat of reaction, the heat of dilution of the components of the solutions, and the differential heat effect associated with the physical process of mixing. Qp is determined by measuring Qmwhen both compartments of the two cells contain buffer only. Qo is determined by measuring Qm = Qd + Qp when one compartment of the sample cell contains protein or ligand in buffer and the other contains only the buffer. Q0 should be measured at every concentration of protein and ligand used in the experiment. Qr is determined by measuring Qm = Qr + Qd + Qp when one compartment of the sample cell contains protein in buffer and the other contains the ligand in buffer. The use of buffers is helpful if the protein-ligand interaction results in the release or adsorption of protons, which is likely to be the case with phospholipase A 2 . On mixing, the heat generated or absorbed produces a temperature difference between the two cells which is measured by the potential difference across two thermoelectric devices. This temperature difference is proportional to the differential rate of heat flow into or out of the sample 5 R. L. Biltonen and N. Langerman, this series, Vol. 61, p. 287. 6 G. R. Hedwig and R. L. Biltonen, Biophys. Chem. 19, 1 (1984). 7 p. S. de Araujo, M. Y. Rosseneu, J. M. H. Kremer, E. J. J. van Zoelen, and G. H. de Haas, Biochemistry 18, 580 (1979). s D. Lichtenberg, G. Romero, M. Menashe, and R. L. Biltonen, J. Biol. Chem. 261, 5334 (1986).
[21]
PHOSPHOLIPASEA2 THERMODYNAMICS
237
compartment. The integrated signal over time is proportional to Qm, which can be calculated using a calibration constant determined with an electrical heater within the cell. The heat of reaction per mole of protein is given by
aapp
Or
(Om - Qd - Qp)
ET
ET
(7)
Qapp includes the apparent heat of binding plus the heat associated with proton release or adsorption by the buffer. That is, Qapp = Q + An AH°b
(8)
where Q is the heat associated with the protein-ligand interaction. An is the number of protons released or absorbed on binding of ligand. AH°b is the molar enthalpy change associated with the proton absorption by the buffer. Alternatively, titration microcalorimeters could be used for binding experiments. Modern titration calorimeters are capable of resolving heats in the range of about 10-100/xcal. In a titration calorimeter, one reactant is present in the calorimeter cell and the other is injected from a syringe in a stepwise fashion. An advantage of a titration system is that multiple concentrations of one of the reactants can be added to a single sample of the other. Thus, the time and material required to obtain a complete binding curve are much less with a titration instrument than with the batch calorimeter. Freire and co-workers have published a calorimetric study of the binding of the B subunit of cholera toxin to lipid vesicles using such a titration calorimeter.9 A detailed description of calorimetric instrumentation and its application to biological systems has been published in a previous volume in this series. 5,~°
Calcium B i n d i n g to Phospholipase A 2
Calorimetry There has been only one calorimetric study of calcium binding to phospholipase A2, and, thus, it must serve as the example for application of the technique. 6 The enthalpy of binding of calcium to phospholipase A2 was determined from the heats of mixing of solutions of enzyme in buffer with solutions of CaCI2 in buffer at the same pH and NaCI concentration. 9 A. Schrn and E. Freire, Biochemistry 211, 5019 (1989). l0 N. Langerman and R. L. Biltonen, this series, Vol. 61, p. 261.
238
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[9.1]
Heats of dilution of the enzyme and CaCI2 solutions were determined separately and subtracted from the measured heat of mixing to obtain the heat of reaction. The observed heats of reaction were in the range - 0.3 to - 1.5 mcal. To determine whether there is a concomitant proton uptake or release on calcium binding to phospholipase A2 at pH 8.0, the experiments were carried out in both Tris-HCl and HEPES-NaOH buffer systems (A/-Fb is different for each of the two buffers). In the mixing and dilution experiments, the calorimeter reference cell contained aliquots of buffer chosen to match closely the volumes in the reaction cell. The amount of solution added to each compartment of the calorimeter cell was determined gravimetrically. Prior to loading the calorimeter, the pH for both the enzyme and calcium solutions was adjusted to identical values, as necessary. After the experiment, the pH of the mixed solution was also determined to ascertain that the change in pH was never greater than 0.01 pH unit. Prior to mixing, the reactant solutions must be allowed to achieve thermal equilibrium with the calorimeter. The enzyme may adsorb to the walls of the gold reaction cell, and extreme care must be taken in the cleaning of the reaction cell at the end of each experiment. This adsorption is not peculiar to the gold cells but appears to be a property of phospholipase A2. As discussed elsewhere, phospholipase A2 has a tendency to adsorb also to glass surfaces. 3 The amount of protein adhering to the calorimetric cell surface in the calorimetric experiment described was small compared to the total amount of protein used in the experiment. The observed enthalpy change of reaction of calcium with phospholipase A2 in Tris buffer is shown as a function of the total calcium concentration in Fig. 1. A qualitatively similar response was obtained using HEPES buffer, but the values of AH were about 20% lower than with Tris buffer, indicating a net change in the degree of protonation of the enzyme on binding calcium. Since the heat of protonation of Tris and HEPES buffers are, respectively, - 11.33 and -4.93 kcal/mol, H,12 it follows that protons are released from the enzyme on binding calcium at pH 8.0. The AH data obtained in Tris buffer were analyzed assuming a l : l stoichiometry, yielding values for K (3507 M-l) and AH ( - 4.99 kcal/mol). 6
Spectroscopy Calcium binding to phospholipase A2 can also be monitored by measurement of any spectral parameter which changes on the interaction. Ultraviolet difference spectroscopy, circular dichroism spectroscopy, and " I. Grenthe, H. Ots, and O. Ginstrup, Acta Chem. Scand. 24, 1067 (1970). 12 L. Beres and J. M. Sturtevant, Biochemistry 10, 2120 (1971).
[21]
PHOSPHOLIPASE A 2 THERMODYNAMICS
239
a
I
t.3
"1-
1
PHOSPHOLIPASE
STRUCTURE-FUNCTION
TECHNIQUES
[21]
[21] T h e r m o d y n a m i c s o f P h o s p h o l i p a s e A2-Ligand Interactions
By RODNEY L. BILTONEN, BRIAN K. LATHROP, and JOHN D. BELL Introduction Phospholipases A2 can be activated on interaction with aggregated phospholipid substrates.l This activation is time-dependent and can thus be studied in considerable detail, m Specific methods for simultaneous study of various time-dependent properties of the enzyme and lipid substrate during the time course of hydrolysis have been described in a separate chapter in this volume. 4 Such investigations provide important clues regarding the mechanisms of the activation process. An important step in the development and testing of hypotheses of activation is the identification and quantitative thermodynamic characterization of the various equilibria involved in the process. Important equilibrium processes involved in phospholipase A 2 catalysis include protein-protein, protein-calcium, and protein-lipid interactions. In this chapter, we describe the use of spectroscopic and calorimetric techniques to obtain thermodynamic information relating to the latter two types of interactions. General Considerations For the simple reaction E + X ~-- EX the equilibrium constant is given by K = [EX] [E][X]
(1)
If some property (e.g., fluorescence intensity) of the enzyme (E) or ligand (X) is proportional to the amount of complex (EX) formed, the fractional degree of association of E as a function of the free concentration of X is given by f =
K[X] AA - - 1 + K[X] z~Amax
(2)
1 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 2 G. Romero, K. Thompson, and R. L. Biltonen, J. Biol. Chem. 262, 13476 (1987). 3 j. D. Bell and R. L. Biltonen, J. Biol. Chem. 264, 12194 (1989). 4 j. D. Bell and R. L. Biltonen, this volume [22].
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[21]
PHOSPHOLIPASE A2 THERMODYNAMICS
235
where AA is the measured change in the observable at a given concentration of ligand and AAmax is the maximum change in the observable. ~Ama x and K can be estimated by nonlinear least-squares analysis of lk4 as a function of [X] or by linear regression analysis of the data in reciprocal form. The former method is preferred, but if linear regression of the reciprocal data is used, the data must be properly weighted. In any experiment, Z~ma x is the total change in the observable between the protein in the fully liganded state and the unliganded state. In a calorimetric experiment, z~tAmax is the total heat change for the reaction (AQmax), and the apparent enthalpy change per mole of enzyme is AH:pp = AQmax/ET
(3)
where ET is the total amount of enzyme used in the calorimetric experiment. The apparent Gibb's energy change for the association reaction is given by AGapp = - R T In K = A/~ap p O
--
O
TASapp
(4)
with AS:pp being the apparent entropy change for the reaction. If AH:pp is known, then AS:pp can be calculated. It must be noted that the values of AGapp and ASapp are dependent on the choice of reference state (i.e., concentration units of X used to calculate K). The authors prefer the use of a reference state of moles per liter or mole fraction. In any case, this reference condition should always be stated when reporting values of K, AG:pp and AS:pp. Although calorimetric experiments directly provide estimates for A/~aapp, A/a~app can also be estimated from the temperature dependence of K since O
O
A/'/:app -
d ( - R InK) d(1/T)
(5)
It is important to note that since estimates of the apparent thermodynamic quantities obtained as just described are highly dependent on the assumed model, comparison of the A/-'/~aapp obtained calorimetrically and that obtained by van't Hoff analysis [Eq. (5)] is an excellent test of the model. Generally, the two estimates will agree only if the assumed model used to calculate K is correct. It should be realized that the thermodynamic quantities derived as described above are apparent values. This is because it is generally unlikely that such an elementary representation of the interaction as given in Eq. (1) is correct; rather, the reaction may be coupled with other
236
PHOSPHOLIPASE STRUCTURE--FUNCTIONTECHNIQUES
[]21]
processes such as protein ionization. However, such coupling can be sorted out and in many cases used to advantage in experimental design. An example of this type of phenomenon is given in the discussion of calcium binding to porcine pancreatic phospholipase A 2 and is discussed in more general terms in a previous volume of this series. 5 Calorimetry has been used to study a variety of protein-ligand interactions but has seen limited application to phospholipase A2. All reported calorimetric studies of calcium and lipid binding to phospholipase A 2 have used LKB batch microcalorimeters equipped with twin gold cells, 6-8 and the procedure to be described applies specifically to such an instrument. Each of the cells consists of two compartments. On physical rotation of the calorimeter, the material in the two compartments of each cell is mixed, and the differential heat of mixing is measured. The reference cell contains buffer in both compartments, whereas the compartments of the sample cells contain the reactant solutions. The basic calorimetric experiment consists of mixing the two solutions which contain reactive components. The heat effect associated with the overall process is Qm "~- Qr + Qd + Qp
(6)
where Qm, Qr, Q0, and Qp are the measured heat, the heat of reaction, the heat of dilution of the components of the solutions, and the differential heat effect associated with the physical process of mixing. Qp is determined by measuring Qmwhen both compartments of the two cells contain buffer only. Qo is determined by measuring Qm = Qd + Qp when one compartment of the sample cell contains protein or ligand in buffer and the other contains only the buffer. Q0 should be measured at every concentration of protein and ligand used in the experiment. Qr is determined by measuring Qm = Qr + Qd + Qp when one compartment of the sample cell contains protein in buffer and the other contains the ligand in buffer. The use of buffers is helpful if the protein-ligand interaction results in the release or adsorption of protons, which is likely to be the case with phospholipase A 2 . On mixing, the heat generated or absorbed produces a temperature difference between the two cells which is measured by the potential difference across two thermoelectric devices. This temperature difference is proportional to the differential rate of heat flow into or out of the sample 5 R. L. Biltonen and N. Langerman, this series, Vol. 61, p. 287. 6 G. R. Hedwig and R. L. Biltonen, Biophys. Chem. 19, 1 (1984). 7 p. S. de Araujo, M. Y. Rosseneu, J. M. H. Kremer, E. J. J. van Zoelen, and G. H. de Haas, Biochemistry 18, 580 (1979). s D. Lichtenberg, G. Romero, M. Menashe, and R. L. Biltonen, J. Biol. Chem. 261, 5334 (1986).
[21]
PHOSPHOLIPASEA2 THERMODYNAMICS
237
compartment. The integrated signal over time is proportional to Qm, which can be calculated using a calibration constant determined with an electrical heater within the cell. The heat of reaction per mole of protein is given by
aapp
Or
(Om - Qd - Qp)
ET
ET
(7)
Qapp includes the apparent heat of binding plus the heat associated with proton release or adsorption by the buffer. That is, Qapp = Q + An AH°b
(8)
where Q is the heat associated with the protein-ligand interaction. An is the number of protons released or absorbed on binding of ligand. AH°b is the molar enthalpy change associated with the proton absorption by the buffer. Alternatively, titration microcalorimeters could be used for binding experiments. Modern titration calorimeters are capable of resolving heats in the range of about 10-100/xcal. In a titration calorimeter, one reactant is present in the calorimeter cell and the other is injected from a syringe in a stepwise fashion. An advantage of a titration system is that multiple concentrations of one of the reactants can be added to a single sample of the other. Thus, the time and material required to obtain a complete binding curve are much less with a titration instrument than with the batch calorimeter. Freire and co-workers have published a calorimetric study of the binding of the B subunit of cholera toxin to lipid vesicles using such a titration calorimeter.9 A detailed description of calorimetric instrumentation and its application to biological systems has been published in a previous volume in this series. 5,~°
Calcium B i n d i n g to Phospholipase A 2
Calorimetry There has been only one calorimetric study of calcium binding to phospholipase A2, and, thus, it must serve as the example for application of the technique. 6 The enthalpy of binding of calcium to phospholipase A2 was determined from the heats of mixing of solutions of enzyme in buffer with solutions of CaCI2 in buffer at the same pH and NaCI concentration. 9 A. Schrn and E. Freire, Biochemistry 211, 5019 (1989). l0 N. Langerman and R. L. Biltonen, this series, Vol. 61, p. 261.
238
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
[9.1]
Heats of dilution of the enzyme and CaCI2 solutions were determined separately and subtracted from the measured heat of mixing to obtain the heat of reaction. The observed heats of reaction were in the range - 0.3 to - 1.5 mcal. To determine whether there is a concomitant proton uptake or release on calcium binding to phospholipase A2 at pH 8.0, the experiments were carried out in both Tris-HCl and HEPES-NaOH buffer systems (A/-Fb is different for each of the two buffers). In the mixing and dilution experiments, the calorimeter reference cell contained aliquots of buffer chosen to match closely the volumes in the reaction cell. The amount of solution added to each compartment of the calorimeter cell was determined gravimetrically. Prior to loading the calorimeter, the pH for both the enzyme and calcium solutions was adjusted to identical values, as necessary. After the experiment, the pH of the mixed solution was also determined to ascertain that the change in pH was never greater than 0.01 pH unit. Prior to mixing, the reactant solutions must be allowed to achieve thermal equilibrium with the calorimeter. The enzyme may adsorb to the walls of the gold reaction cell, and extreme care must be taken in the cleaning of the reaction cell at the end of each experiment. This adsorption is not peculiar to the gold cells but appears to be a property of phospholipase A2. As discussed elsewhere, phospholipase A2 has a tendency to adsorb also to glass surfaces. 3 The amount of protein adhering to the calorimetric cell surface in the calorimetric experiment described was small compared to the total amount of protein used in the experiment. The observed enthalpy change of reaction of calcium with phospholipase A2 in Tris buffer is shown as a function of the total calcium concentration in Fig. 1. A qualitatively similar response was obtained using HEPES buffer, but the values of AH were about 20% lower than with Tris buffer, indicating a net change in the degree of protonation of the enzyme on binding calcium. Since the heat of protonation of Tris and HEPES buffers are, respectively, - 11.33 and -4.93 kcal/mol, H,12 it follows that protons are released from the enzyme on binding calcium at pH 8.0. The AH data obtained in Tris buffer were analyzed assuming a l : l stoichiometry, yielding values for K (3507 M-l) and AH ( - 4.99 kcal/mol). 6
Spectroscopy Calcium binding to phospholipase A2 can also be monitored by measurement of any spectral parameter which changes on the interaction. Ultraviolet difference spectroscopy, circular dichroism spectroscopy, and " I. Grenthe, H. Ots, and O. Ginstrup, Acta Chem. Scand. 24, 1067 (1970). 12 L. Beres and J. M. Sturtevant, Biochemistry 10, 2120 (1971).
[21]
PHOSPHOLIPASE A 2 THERMODYNAMICS
239
a
I
t.3
"1-
1
