Albumin Binding of Long-Chain Fatty Acids: Thermodynamics and

Thermodynamics and kinetics of serum albumin binding of long-chain fatty acids (FA) with more than 14 carbon atoms are important in all chemical studi...
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J. Phys. Chem. 1996, 100, 17981-17985

17981

Albumin Binding of Long-Chain Fatty Acids: Thermodynamics and Kinetics Eigil Bojesen and Inge N. Bojesen*,† Department of Medical Biochemistry and Genetics, Laboratory of Medical Biochemistry B, UniVersity of Copenhagen, The Panum Institute, BlegdamsVej 3, DK-2200 Copenhagen N, Denmark ReceiVed: July 16, 1996X

Thermodynamics and kinetics of serum albumin binding of long-chain fatty acids (FA) with more than 14 carbon atoms are important in all chemical studies of FA in water. Furthermore, the energetics of this process should substantiate the molecular model (Brown, J. R.; Shockley, P. Lipid-Protein Interactions; John Wiley & Sons: New York, 1989; Vol. 1, pp 25-68). At pH 7.3, bovine serum albumin (BSA) has three equivalent high affinity binding sites for FA to BSA molar ratios below 2 at temperatures between 273 and 311 K. The corresponding Gibbs free energies (∆G°) of six FA transfers from buffer, ionic strength 0.173 M, to BSA are identical with those of transfers to heptane of FA excepting the head groups -CH2-COO- (Smith, R.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 289-293), suggesting similar binding free energy of the anionic head group in buffer and in BSA, corroborating NMR studies (Cistola, D. P.; Small, D. M.; Hamilton, J. A. J. Biol. Chem. 1989, 262, 10980-10985). From ∆H° and ∆G° of FA transfers from water to BSA and to aliphatic hydrocarbons, we calculate T∆S° and ∆H° of the FA hydrocarbon chains transfers from aliphatic hydrocarbons to BSA, with complete entropy/enthalpy compensation (∆G° ≈ 0). The values vary between FA but corroborate known conformational changes of BSA when binding FA. The kinetics of palmitate and oleate bindings reveal transition states of FA with low entropy as in water and higher enthalpies corresponding to carboxyl-group bindings by hydrogen bonds in water. Thus, FA are bound in BSA by combined electrostatic anionic head group bindings and van der Waals bindings of the hydrocarbon tails. The ∆G°’s of transfer from buffer are entirely accounted for by transfer of the hydrocarbon tail, making predictable the equilibrium constants of FA-BSA complexes in buffer at pH 7.3.

Introduction Our recent studies of bovine serum albumin, BSA, bindings of three long-chain fatty acids, FA, with more than 14 carbons revealed that BSA has three noncooperative equivalent sites.1 The thermodynamic analyses suggest predictions of hard-toestimate binding constants of very long FA, saturated as well as unsaturated, under the employed conditions: pH ) 7.3, KCl or NaCl, phosphate buffer, ionic strength 0.173 M, temperature 273-311 K. The presence of three equivalent, noncooperative FA binding sites is an interesting phenomenon to protein chemists but an understanding is outside the scope of the present paper. The aim of the present paper is to substantiate theoretically these predictions as they have important implications. In all studies of the poorly water soluble FA, well-defined concentrations of monomers are achieved only in equilibrium with FA bound to albumin in molar ratios, ν, below 3. Furthermore, the theory permits calculations of a spectrum of monomers corresponding to a spectrum of albumin-bound FA, such as found physiologically in the mammalian circulation, commuting as monomers with cells. FA binding to BSA is described by the three-domain model of Brown and Shockley2 which has got substantial support by NMR studies.3 The model suggests funnel-shaped binding sites with positively charged cups and narrow spouts lined by apolar groups. In water the spouts are probably closed by the hydrophobic effect. According to the NMR studies, the carboxyl groups of FA are bound somewhat differently in the three positively charged cups by electrostatic interactions in agreement with the anionic amphipath specificity of high affinity.4 The FA bindings in molar ratios below 3 induce conformational changes of albumin5 and our thermodynamic data should concur with this observation. † X

E-mail: [email protected]. Abstract published in AdVance ACS Abstracts, October 15, 1996.

S0022-3654(96)02141-7 CCC: $12.00

The phenomenon of enthalpy/entropy compensation is known for agonist and antagonist bindings to β-adrenergic receptors6 and is found in the transfer of FA from buffer or liquid hydrocarbons to BSA. Exemplified by new data on linoleic acid binding we will show that the thermodynamics of six saturated and unsaturated FA together with the kinetics of two FA bindings account quantitatively for the BSA properties of FA binding in that the free energies of bindings are the same as those of water/liquid hydrocarbon partitions of the carbon chains except for the -(CH2-COO-) group, which is bound by the same free energy in BSA and in water, solvated mainly by hydrogen bonds. Experimental Section Material and Methods. [14C(U)] Linoleic acid was purchased from DuPont NEN, Boston, MA. Unlabeled FA are from Sigma. Labeled as well as unlabeled FA were purified monthly by column chromatography, ascertaining the elution pattern of a single component.1 BSA (Behring Institute, Germany) was defatted according to the method of Chen.7 Scintillation Counting. The scintillation counter is calibrated to give dpm as output according to the spectral index of the external standard (SIE) and the efficiency was 67% for 3H and 95% for 14C in unquenched samples. Counting rates of samples were determined after the addition of 3.9 mL of Ultima Gold scintillation fluid to a probable error smaller than 1%. Preparation of Ghosts, the Reference Binder. Preparation of 0.05% BSA-filled pink ghosts from freshly drawn human blood1 was carried out by adding BSA to the hemolyzing solution. After resealing of the ghosts they were washed three times with 165 mM KCl, 2 mM phosphate buffer, and pH 7.3 and once with the same buffer containing 0.05% BSA (7.5 µM). Preparation of Buffer with FA-BSA Complex. Labeled FA and the calculated amount of unlabeled FA to give the molar © 1996 American Chemical Society

17982 J. Phys. Chem., Vol. 100, No. 45, 1996

Bojesen and Bojesen and binding capacity of the reference binder is so high that virtually no FA is released by quantitative removal of the buffer with the FA-BSA complex, using centrifugations and washings. The [FA] is in theory independent of the concentration of the suspended reference binder in contrast to that of a tiny amount of binding capacity released from the purified suspension. This latter is proportional to the concentration of the binder. Thus [FA] is in theory the FA concentration of an “infinitely” diluted suspension estimated by extrapolation from the four different dilutions of the same purified suspension. Accordingly, the relation is linear between the concentrations of supernatant FA and the binder (Figure 1, panel A) providing an estimate of [FA] as the ordinate intercept. As [FA] of the purified reference binder is the same as in the buffer with the FA-BSA complex we have got corresponding values of ν and [FA]. Results and Discussion

Figure 1. (A) Examples of plots to determine equilibrium water-phase monomer FA concentrations. Relations between supernatant linoleic acid concentrations and the four volume fractions of two washed ghost suspensions initially equilibrated at 311 K with solutions of FA-BSA complexes at ν ) 0.86, (0) and ν ) 0.11 (b). Constant dilution volume 3 mL. (B) Relationship between linoleic acid monomer water-phase concentrations ([FA]) and the molar ratio of FA to BSA (ν) presented according to the linearlized definition of the equilibrium constant Kd of N equivalent binding sites (Wilkinson8 at 283 K (b) and at 311 K (2). The regression lines are [FA]/ν ) 0.349 ((0.046) [FA] + 1.81 ((0.14), r ) 0.89 and [FA]/ν ) 0.329 ((0.035) [FA] + 5.62 ((0.20), r ) 0.92 respectively.

ratio of FA to BSA (ν) e 2 were deposited on 200 mg of glass beads (diameter 0.1 mm) with 3 × 50 µL benzene, which was sublimated after each addition at a pressure of about 16 mmHg. Finally, the glass beads were gently shaken for 15 min at room temperature with buffer containing 0.05% BSA. Such solutions contained 1 µCi [14C] linoleic acid per mL. Labeling of the Reference Binder. Ghosts were packed by centrifugation 7 min (at 36 400g) at the appropriate temperature. One volume of packed ghosts was equilibrated with 1.5 volumes of the solution containing labeled FA-BSA complexes, 50 min at 273 K, 40 min at 283 K, 20 min at 296 K, and 15 min at 311 K. After centrifugation as above, the ratioactivity of supernatants (Ca, dpm mL-1) was monitored in order to calculate final ν values as ν ) Ca/S × 7.5), where S is the specific activity of FA in dpm nmol-1 and 7.5 the concentration of BSA in nmol mL-1. Estimations of Water Phase Monomer Concentrations [FA] in Equilibrium with FA-BSA. After the reference binder was equilibrated with the FA-BSA complex at suitable final ν e 2, washings were carried out four times with 40 volumes of buffer without BSA at 277 K. The binder was now suspended in the same buffer to a total volume of 3 mL and this suspension was distributed into four tubes resulting in four different dilutions (Figure 1, panel A). After complete equilibration, ghosts were sedimented by centrifugation as described above and 2 × 200 µL of ghost-free supernatants were pipeted from the top of each tube for scintillation counting in order to determine FA concentration of supernatant. The FA affinity

Equilibrium Dissociation Constants Kd of N Number of BSA Binding Equivalents. When [FA] is the water phase monomer concentration, N is the number of equivalent BSA binding sites and ν is the molar ratio of FA to BSA; then Kd is defined by Kd ) [FA](N - ν)/ν, which linearized according to Wilkinson8 gives [FA]/ν ) [FA]/N + Kd/N. Previously, we showed for arachidonic acid (AR) and for oleic acid (OL) that corresponding values of [FA] and ν at four temperatures obey the linearized expressions of Kd with a value of N not different from 3.1 Figure 1, panel B, shows examples of such plots for linoleic acid (LI) at two temperatures, and Table 1 shows corresponding estimates of Kd and N for four FA. In calculations of Gibbs free energies of binding, we have used Kd values normalized to N ) 3. Comparison of Free Energies of FA Binding to BSA and Water/Liquid Hydrocarbon Phase Partition. The equilibrium constants, Kd, are now known at 296 K for three equivalent binding sites1,9 of six FA, from laurate, 12C, to oleate, 18C and arachidonate, 20 C. The thermodynamic state functions of water/liquid hydrocarbon partitions are expressed with unit mole fraction (X) as the standard state. When XBSA is the FA mole fraction in BSA, XBSA ) ν/(3 + ν) and Xw is the FA mol fraction in water and Kd ) Xw‚55.5(3 - ν)/ν, Xw/XBSA ) Kd(3 + ν)/(55.5(3 - ν)). Accordingly, the difference of standard potentials in BSA and water is µ°BSA - µ°W ) RT ln((3 + ν)/(55.5(3 - ν))) + RT ln (Kd). The free energy of binding ∆G° ) µ°BSA - µ°W depends therefore on ν but the effect is small. The second term of the expression above is about -45 kJ/mol and at 296 K the first term is -10 kJ/mol with ν , 3 and -8.3 kJ/mol with ν ) 1, the physiological upper limit of each one of the FA. The free energies, shown in Table 2, column 2, are calculated for ν , 3. The free energy of transfer from water decreases linearly with about 3.6 kJ/mol/(CH2)1 when an alkenyl group counts as a methylene group10 to compare with the values of columns 3 and 4 where the methylene equivalents are 3.510 and 3.8611 kJ/mol for water/ heptane partition of FA and water/aliphatic hydrocarbon partition of hydrocarbons, respectively. From phase partition of FA and correcting for an extrapolated hydrophilic effect of -(CH2COO-), 16-17 kJ/mol,10 the free energies of partition of the FA hydrocarbon chains get the values presented in Table 2, column 3. Using 3.86 kJ/mol11 as the methylene equivalent, ∆G° values are 6.5% more negative, Table 2, column 4. The results demonstrate that FA transfer from buffer to BSA may be entirely accounted for by “partition” of the hydrocarbon chains, i.e., most important, that the carboxyl and the R methylene groups may not contribute significantly to the free energy of FA transfer from buffer to BSA. This finding suggests

Albumin Binding of Long Chain Fatty Acids

J. Phys. Chem., Vol. 100, No. 45, 1996 17983

TABLE 1: Estimation by Regression Analyses According to Wilkinson8 of the Number of Binding Sites (N) on Bovine Serum Albumin (BSA) of Four Different Long-Chain Fatty Acids (FA) with Equilibrium Dissociation Constants (Kd, nM) of FA-BSA Complexes at Different Temperatures: Palmitic Acid (PA), Arachidonic Acid (AR), Linoleic Acid (LI), and Oleic Acid (OL) temperatures parameters PA N Kd AR N Kd LI N Kd OL N Kd

weighted mean N

0 °C

10 °C

23 °C

38 °C

2.27 ( 0.37 3.09 ( 0.59

2.63 ( 0.35 5.11 ( 0.84

4.34 ( 0.57 23.90 ( 3.30

3.13 ( 1.00 35.00 ( 4.00

2.79 ( 0.45

2.44 ( 0.46 3.70 ( 0.77

3.40 ( 0.90 8.94 ( 2.50

3.20 ( 0.90 17.60 ( 5.30

3.50 ( 0.60 29.27 ( 5.08

2.95 ( 0.24

2.94 ( 0.20 3.51 ( 0.49

2.87 ( 0.38 5.18 ( 0.80

3.05 ( 0.82 10.38 ( 2.95

3.04 ( 0.32 17.08 ( 1.90

2.96 ( 0.04

3.26 ( 0.50 1.40 ( 0.30

2.40 ( 0.60 1.60 ( 0.40

3.28 ( 0.57 3.19 ( 0.61

2.50 ( 0.70 3.90 ( 1.10

2.93 ( 0.24

TABLE 2: Free Energies ∆G° (kJ/mol) of Long-Chain Fatty Acid Transfer from Water (w) to Bovine Serum Albumin (BSA) with Occupancy ,3, Using Unit Mole Fraction as Standard State, from Water to Heptane, and Theoretical Free Energies of Equivalent Hydrocarbon Transfer from Water to Liquid Aliphatic Hydrocarbons (ah) fatty acid

w f BSA -∆G°(SE)

w f heptane -∆G°b

CH3(CH2)10COO- (laurate) CH3(CH2)12COO- (myristate) CH3(CH2)14COO- (palmitate) CH3(CH2)10(CHdCH)4COO- (arachidonate) CH3(CH2)12(CHdCH)2COO- (linoleate) CH3(CH2)14(CHdCH)COO- (oleate)

40a 48a 54.4(0.3) 54.5(0.4) 55.0(0.7) 58.0(0.5)

40 47 53.9 53.6 53.7 57.4

w f ah hydrocarbon 9CH2 + CH3 11CH2 + CH3 13CH2 + CH3 13CH2 + CH3 13CH2 + CH3 14CH2 + CH3

-∆G°c 43.0 50.6 58.3 58.3 58.3 62.0

a Values calculated on the basis of K values estimated by Spector;9 the others are based on 296 K K values measured by the authors and d d normalized to three binding sites. b Values taken from the work of Tanford.10 c Values taken from the work of Abraham.11

Figure 2. Van’t Hoff isochores. The temperature dependence of the equilibrium dissociation constants Kd calculated for three equivalent sites. (0) Oleic acid, (O) arachidonic acid, (4) palmitic acid, and (b) linoleic acid. The confidence limits (95%) are presented for oleic acid by (‚‚‚).

that the binding free energy of the head group, probably in the cups of the Brown and Shockley model, is the same as the solvation energy in buffer, probably mainly hydrogen and ionic bonds, in fact corroborating NMR studies with 13C-enriched carboxyl groups.3,12 In agreement with our binding studies,1 they suggest three equivalent high-affinity sites with chemical shifts of -COO- resembling that in water but somewhat different, probably indicating ion pair interactions even at pH < 6. The findings concur with preferential binding of anionic amphipaths.4 Enthalpy of FA Binding by BSA. Kd values of three equivalent binding sites of five FA are known for a reasonably large temperature range 273-311 K to provide estimates of the enthalpies according to the van’t Hoff isochore. Such plots from estimated Kd according to the described method are presented in Figure 2 and the binding enthalpies are presented in Table 3. The proportions of enthalpy and entropy contributions to free energies of FA transfer from water to BSA, Table 3, vary greatly between FA. In contrast, regarding transfer from water

to liquid hydrocarbon of the corresponding hydrocarbon chains, defined in Table 2, the proportions of the two state functions vary only a little, using the methyl and methylene partial values of Abraham11 lowered by 6.5%. This small correction arises from the different free energy values of Table 2, columns 3 and 4. The finding is important and for the following reasons it pertains to the effect on BSA conformation mentioned before. Figure 3 shows the linear relation between ∆H° and T∆S° of FA transfers from buffer to BSA. According to the discussion of ∆G°, the desolvation energy of the hydrophilic end group is offset by BSA binding and the thermodynamics therefore concern only transfer of the hydrocarbon tail from buffer to BSA. The regression shows enthalpy/entropy compensation which is partial, considering all the FA but of course complete for three FA’s with identical ∆G° as demonstrated for bindings of agonists and antagonists of β-adrenergic receptors.6 Thus, palmitic acid is in this sense analogous to agonists and linoleic acid analogous to antagonists. The differences of FA bindings can be illustrated by comparing the state functions ∆H° and T∆S° of transfer of the FA hydrocarbon equivalents of Table 2 from hydrocarbon to BSA, combining our data and the water to hydrocarbon transfer equivalents and thus eliminating water from considerations. The calculations, Table 3, show that ∆H° ≈ T∆S°, as ∆G° ≈ 0, and reflect the differences of the state functions of the hydrocarbon chains in BSA bindings and in liquid hydrocarbon solutions. The values are somewhat different if we compare with hexane or hexadecane but variations with FA show the same pattern. The BSA bindings of myristic (MY) and palmitic acid (PA) differ from solutions in liquid hydrocarbons by much smaller entropy contributions to the free energy of binding. This can probably not be assigned to the flexible hydrocarbon chain of FA3,12b but suggests a major contribution of the peptide chains, i.e., conformational changes of BSA are paid by the greater binding enthalpies. We believe this can be understood qualitatively from the nature of bindings as van der Waals-London dispersion interactions. Binding enthalpy is

17984 J. Phys. Chem., Vol. 100, No. 45, 1996

Bojesen and Bojesen

TABLE 3: Enthalpy and Entropy Contributions to Free Energies of Fatty Acid Transfers from Water (w) to Bovine Serum Albumin (BSA) and Hydrocarbon Equivalence to Hexadecane (Hexane) and from Hexadecane (Hexane) to BSA transfers of hydrocarbon (Table 2)

transfers of FA w f BSA

w f hexadecane

w f hexane

transfers hexadecane (hexane) f BSA

fatty acid

∆H°(SE) (kJ/mol)

T∆S° (kJ/mol)

∆H° (kJ/mol)

T∆S° (kJ/mol)

∆H° (kJ/mol)

T∆S° (kJ/mol)

∆H° (kJ/mol)

T∆S° (kJ/mol)

myristate palmitate arachidonate linoleate oleate

-47a -42.0(2.5) -35.0(1.5) -29.3(0.7) -30.7(2.4)

1.0 12.4 19.5 25.7 27.3

-16.0 -19.6 -19.6 -19.6 -21.5

31.5 35.0 35.0 35.0 36.7

-23.3 -28.5 -28.5 -28.5 -31.0

24.0 26.7 26.7 26.7 27.5

-31.0 (-23.7) -22.4 (-13.5) -15.4 (-6.5) - 9.7 (- 0.8) - 9.2 (+ 0.3)

-30.5 (-23.0) -22.6 (-14.3) -15.5 (- 7.2) - 9.3 (- 1.0) - 9.4 (- 0.2)

a This enthalpy value is taken from Pedersen et al.;19 the others are measured by the authors.1 T∆S° is calculated as -(∆G° - ∆H°). The values of columns 4, 5, 6, and 7 are calculated for the aliphatic hydrocarbon equivalents of Table 2 using the methylene and methyl partial values of Abraham11 corrected for 6.5% higher ∆G° corresponding to the measured w f BSA and w f hexadecane (hexane) values.

Figure 3. Enthalpy/entropy compensation in FA transfers from buffer to BSA (Table 3): ∆H° ) (0.688 ( 0.076)T∆S° - 48.6 ( 1.5R2 ) 0.965.

TABLE 4: Dissociation Rate Constants of FA-BSA Complexes (k-), Activation Energies of Dissociation (E1) and Association (E2), and Activation Entropies of Dissociation (T∆Sq°) FA

k(s-1)

E1 (kJ/mol)

T∆Sq°

T∆S° (kJ/mol)

OL 0.037a 52.3(1.3)a -30.7(1.3) -27.3 (2.4)d PA 0.043b 62.7c -20.0 -12.4 (2.5)d

E2 ) (E1 - ∆H°) (kJ/mol) 21.6(2.4) 20.7(2.5)

a The 296 K value of k- and E1 were calculated for the temperature interval 296-310 K as reported by Weisiger and Ma.14 b The 298 K value of k- reported by Svensson.15 c E1 calculated for the temperature interval 282-298 K.15 d Values from Table 3. For activation energies including 273 K, see the text.

favored by proximity of the molecules within critical ranges13 and counteracted by intra- and intermolecular motional energies, paying the binding with diminished degrees of freedom. In contrast to solvent hydrocarbon molecules, the apolar binding groups of BSA have only internal degrees of freedom favoring proximity. The strong binding between a highly flexible ligand and apolar sessile groups is then to be paid by decreased entropy of reactants, including the peptide chains. The entropies of OL bindings in BSA and liquid hydrocarbons are not much different, suggesting a relatively poor fit of the hydrocarbon chain of OL to the sessile apolar groups. Information on the Kinetics. BSA is a unique kind of water soluble “dispersed apolar phase”, enabling studies of the kinetics corresponding to the estimated equilibrium thermodynamics. The rate constants of dissociation and the Arrhenius activation energies (E1, Table 4) have been estimated directly for the small tempearture intervals 296-310 K for OL14 and 282-298 K for PA15 by measuring the rate of FA transfers from dissolved FA-

BSA complex to gel-bound albumin. At 273 K the dissociation rate constants are estimated indirectly by analyses of the exchange efflux kinetics from albumin containing ghosts.16 In spite of the small ∆T the precision of the estimated activation energy is good in the case of OL because the temperature has a large effect. The Arrhenius plot is apparently linear down to 273 K as the 273 K value for OL, estimated indirectly to 0.0063 ( 0.0003 s-1, is not different from the value predicted by the activation energy.14,16c This is not the case for PA as indirectly estimated 273 K rate constant for PA16a is considerably smaller, 0.0035 s-1, than predicted by the activation energy for the interval from 282 to 298 K obtained by the rate constants estimated directly (see Table 4). Using the thermodynamic formulation of the absolute rate theory17 using ∆Hq° ) E1 - RT, we calculate the activation entropy, T∆Sq°, which in theory is the difference of TS° in the bound and in the hypothetical transition state. Table 4 shows that the activation entropies are probably not different from the equilibrium values, T∆S°, and that the activation energies are considerably greater than ∆H° of transfer from BSA to water (Table 3). Applying the van’t Hoff isochore and the Arrhenius equation on Kd ) k-/k+ we get ∆H° ) E1 - E2, where E1 is the activation energy of dissociation and E2 that of association. E2 is the enthalpy required of FA in water to reach transition states. The high free energy of transition state involves insignificant change of the low entropy in water. This is remarkable because the low entropy in water is generally assigned to water structured more around the apolar chains than in the bulk. Thus the chain is apparently hydrated similarly in the transition state and in water solutions, suggesting similar additivity of the chain and head group contributions to the free energies of the transition state and of the equilibrium. The funnel model of the binding sites suggests that molecules in transition state must be near the cup where the head group is bound electrostatically by the same free energy as the solvation energy in water where the enthalpy, E2, is required to break hydrogen bonds. Thus, with the head group bound electrostatically in the cup the hydrocarbon chain is in a position to quit the water cage by entering the spout. Such a cooperation explains the preference for anionic amphipath binding.4 The interpretation is in agreement with similar E2 for OL and PA with a value a little higher than the estimated transfer free energy of the head group from water to hydrocarbon partitions, 1617 kJ/mol.10 In the past,18 association and dissociation kinetics suggested a two-step binding process. The dissociation kinetics of OL transfer from binding to human serum albumin, HSA, to BSA indicated activation enthalpy and entropy in striking agreement with Table 4. Furthermore, the activation enthalpy of laurate transfer was, unexpectedly, greater than that of OL, just as our E1 is greater for PA than for OL. On the other hand, contrary to our data, association of OL with HSA had a large

Albumin Binding of Long Chain Fatty Acids negative activation entropy, probably an artifact of adding OL to HSA in pure water. Conclusions Free energies of FA binding to BSA are identical with those of water to liquid hydrocarbon transfers of FA minus the carboxyl and R-methylene groups which is bound by the same free energy in water and in BSA. The theoretical entropy change of transfers of FA hydrocarbon tails from hexane or hexadecane to BSA vary with FA as expected of a multitute of sessile apolar binding groups and are compatible with conformational changes of BSA. The kinetics suggests a transition state with the low entropy of the hydrocarbon chain in water and an activation enthalpy of the head group bound similarly in water and in the cup of funnel-like high-affinity sites. The importance of the anionic head group binding in the cup is probably to ensure proximity and van der Waals binding of the hydrocarbon chain by apolar groups within the spout. Acknowledgment. We particularly wish to thank Prof. G. Bojesen, Institute of Chemistry, University of Copenhagen, for many helpful discussions. Aase Frederiksen provided skilful technical assistance, which is gratefully acknowledged. This work was supported by grants from The Danish Medical Research Council and from the NOVO-Foundation, Copenhagen, Denmark. References and Notes (1) (a) Bojesen, I. N.; Bojesen, E. J. Lipid Res. 1992, 33, 1327-1334. (b) Bojesen, I. N.; Bojesen, E. J. Lipid Res. 1994, 35, 770-778. (2) Brown, J. R.; Shockley, P. Lipid-Protein Interactions; John Wiley & Sons: New York, 1982; Vol. 1, pp 26-68.

J. Phys. Chem., Vol. 100, No. 45, 1996 17985 (3) (a) Cistola, D. P.; Small, D. M.; Hamilton, J. A. J. Biol. Chem. 1987, 262, 10971, 10979-10985. (b) Hamilton, J. A., Era, S., Bhamidipati, S. P., Reed, R. G. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2051-2054. (4) Reynolds, J. A.; Tanford, C. J. Biol. Chem. 1970, 245, 5161-5170. (5) (a) Gumpen, S.; Hegg, P. O.; Martens, H. Biochim. Biophys. Acta 1979, 574, 189-196. (b) Hvidt, Aa.; Wallevik, K. J. Biol. Chem. 1972, 247, 1530-1535. (c) Soeteway, F.; Rosseneu-Motreff, M.; Lamote, R.; Peeters, H. J. Biochem. 1972, 71, 705-710. (6) Searle, M. S.; Williams, D. H. J. Am. Chem. Soc. 1992, 114, 10690-10697. (7) Chen, R. F. J. Biol. Chem. 1967, 242, 173-181. (8) Wilkinson, G. N. Biochem. J. 1961, 80, 324-332. (9) Spector, A. A.; John, K.; Fletcher, J. E. J. Lipid Res. 1969, 10, 56-67. (10) (a) Tanford, C. The Hydrophobic Effect; John Wiley & Sons: New York, 1973; pp 4-11. (b) Smith, R.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 289-293. (11) Abraham, M. H. J. Am. Chem. Soc. 1982, 104, 2085-2094. (12) (a) Hamilton, J. A. N.I.P.S. 1992, 7, 264-270. (b) Hamilton, J. A.; Cistola, D. P.; Morrisett, J. D.; Sparrow, J. T.; Small, D. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 3718-3722. (13) Maitland, G. C.; Rigby, M.; Smith, E. B.; Wakeham, W. A. Intermolecular forces; Clarendon Press: Oxford, U.K., 1981. (14) Weisiger, R. A.; Ma, W-L. J. Clin. InVest. 1987, 79, 1070-1077. (15) Svensson, A.; Holmer, E.; Anderson, L. O. Biochim. Biophys. Acta 1974, 342, 54-59. (16) (a) Bojesen, I. N.; Bojesen, E. Biochim. Biophys. Acta 1991, 1064, 297-304. (b) Bojesen, I. N.; Bojesen, E. Acta Physiol. Scand. 1995, 154, 253-267. (c) Bojesen, I. N.; Bojesen, E. Acta Physiol. Scand. 1996, 156, 501-516. (17) Glasstone, S.; Laidler, K. J.; Eyring, H. The Theory of Rate Processes; McGraw-Hill Book Co.: New York, 1941; pp 1-27. (18) (a) Scheider, W. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 22832287. (b) Scheider, W. J. Phys. Chem. 1980, 84, 925-928. (19) Pedersen, A. O.; Honore´, B.; Brodersen, R. Eur. J. Biochem. 1990, 190, 497-502.

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