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The Interaction of Human Serum Albumin with Dioctanoylphosphatidylcholine in Aqueous Solutions Pablo Martinez-Landeira,† Juan M Ruso,† Gerado Prieto,† Felix Sarmiento,† and Malcolm N Jones*,‡ Grupo de Biofisica e Interfases, Departamento de Fisica Aplicada, Facultad de Fisica, Universidade de Santiago de Compostela, E15706, Santiago de Compostela, Spain, and School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Received November 16, 2001. In Final Form: January 14, 2002 The interaction between human serum albumin (HSA) and the short-chain phospholipid dioctanoylphosphatidylcholine (diC8PC) in aqueous solutions at pH 3.2, 7.4, and 10.0 at high and low ionic strength has been investigated. Static and dynamic light scattering methods have shown that the HSA is partially associated in these aqueous solutions with an association number of 1.67 ( 0.02. On the addition of phospholipid up to a concentration equal to its critical micellar concentration (cmc) the association increases, and at high ionic strength (0.188 M) at pH 3.2 and 7.4 the HSA is largely dimeric with a small proportion of trimeric and possibly more associated species. The interaction can be followed using laser Do¨ppler velocimetry to measure the ζ potentials of HSA in the presence of diC8PC. At pH 3.2 where both HSA and diC8PC are positively charged, the interaction results in a marked decrease in ζ potential as the cmc of the PC is approached. At pH 10.0 where HSA is negatively charged and diC8PC is zwitterionic, the ζ potentials become less negative as the cmc is approached. Microcalorimetry measurements of the interactions at pH 3.2 and 7.4 suggest that after the initial interaction between dimeric HSA and diC8PC, further interaction involves the exothermic partial dissociation of dimeric HSA and the interaction between monomeric HSA with HSA-diC8PC complexes.
Introduction 1-3
Thermodynamic studies on the interactions of anionic, cationic,3 and nonionic4 surfactants and other small drug molecules5,6 with serum albumins have demonstrated that albumins have the capacity to bind a wide range of amphipathic molecules apart from their well-established role in vivo as fatty acid carriers.7 Human serum albumin (HSA) is the most abundant protein in blood plasma and is present at a concentration of the order of 5% w/v. It has a primary sequence of 609 amino acids,8 in a single polypeptide chain with 17 disulfide bridges and one free cysteine residue. The molecular mass is 69366.8 The molecule has a heart-shaped tertiary structure with a high R-helical content (67%)9,10 consisting of three domains, two of which (domains I and III) are connected by a flexible domain (II) that can bend apart when an amphipathic ligand binds.10 * To whom correspondence should be addressed. Present address: 77Broad Rd., Sale, Cheshire M33 2EU, U.K. E-mail:
[email protected]. † Universidade de Santiago de Compostela. ‡ University of Manchester. (1) Tipping, E.; Jones, M. N.; Skinner, H. A. J. Chem. Soc., Faraday Trans. 1 1974, 70, 1306. (2) Valstar, A.; Almgren, M.; Brown, W.; Vasilesu, M. Langmuir 2000, 16, 922. (3) Jones, M. N.; Skinner, H. A.; Tipping, E. Biochem. J. 1975, 147, 229. (4) Cordoba, J.; Reboiras, M. D.; Jones, M. N. Int. J. Biol. Macromol. 1988, 10, 270. (5) Taboada, P.; Mosquera, V.; Ruso, J. M.; Sarmiento, F.; Jones, M. N. Langmuir 2000, 16, 934. (6) Taboada, P.; Mosquera, V.; Ruso, J. M.; Sarmiento, F.; Jones, M. N. Langmuir 2000, 17, 6795. (7) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17. (8) Swiss-Prot Release 39.20, Accession no. P02768, June 2001. (9) Sugio, S.; Kashima, A.; Mochizuki, S.; Noda, M.; Kobayashi, K. Protein Eng. 1999, 12, 439. (10) Curry, S.; Mandelkow, H.; Brick, P.; Franks, N. Nat. Struct. Biol. 1998, 5, 827.
The crystal structure of the complex of HSA and five myristate ligands shows that the fatty acid binds in long hydrophobic pockets which are distributed asymmetrically throughout the molecule, three in domain III and two in domain I. In aqueous solution the protein can partially dimerize. Dimerization is reflected in the association number calculated from the measured molecular mass and the molecular mass of the monomeric molecule (69 366). Albumins have a tendency to partially dimerize in aqueous solution; Valstar et al.2 found bovine albumin had an association number of 1.34 and Matsumoto et al.11 found an association number of 1.75 for commercial preparations. Phospholipids are major components of cell membranes, and hence it is of some importance to consider their possible interaction with major serum proteins such as serum albumin. A major class of phospholipids is the phosphatidylcholines. In this study we have investigated the interaction of a short-chain phosphatidylcholine, dioctanoylphosphatidylcholine (diC8PC) with HSA in aqueous solutions (pH 3.2, 7.4, and 10.0) using a range of physical methods including static and dynamic light scattering, ζ potential measurements, and microcalorimetry. Experimental Section Materials. Human serum albumin (fraction V A 1653) and L-R-phosphatidylcholine, dioctanoyl (P-0958), were obtained from Sigma Chemical Co. and were used as supplied. All other reagents were of analytical grade and solutions were made up in doubledistilled deionized water. Buffers used were glycine (50 mM)HCl pH 3.2, ionic strength 0.0062 and 0.188 obtained by addition of sodium chloride; glycine (50 mM)-NaOH, pH 10.0, ionic strength 0.0312 and 0.188; and phosphate-buffered saline (PBS), pH 7.4, ionic strength 0.188 and one-tenth dilution PBS, ionic strength 0.0188.13 (11) Matsumoto, T.; Inoue, H. Chem. Phys. 1993, 178, 591. (12) Moreels, E., Ceuninck, W. D.; Finsy, R. J. Chem. Phys. 1987, 86, 618.
10.1021/la011681u CCC: $22.00 © 2002 American Chemical Society Published on Web 03/07/2002
Protein-Phospolipid Interactions
Langmuir, Vol. 18, No. 8, 2002 3301 microcalorimeter system.17 The instrument was used on the 30 µV range where the mean sensitivity of the detectors in the heat sinks of the two vessels was 14.66 ( 0.32 W V-1. The sample cell was charged with 2 g of buffered HSA, concentration 0.25% w/v, and 2 g of diC8PC of the required concentration in a range up to approximately 0.3 mM. The reference cell was charged with 2 g of diC8PC solution of identical concentration to that in the sample cell and 2 g of buffer solution. On mixing, the enthalpies of dilution of the phospholipid solution cancel and the enthalpy of dilution of the HSA was measured in a separate experiment and used to correct the data.
Results and Discussion
Figure 1. Static light scattering plots for HSA in aqueous solution at pH 3.2 based on eq 3: b, ionic strength 0.0062; O, ionic strength 0.188. Static Light Scattering (SLS). Measurements were made with a coherent DPSS 532 laser light scattering instrument equipped with a 0.5 W solid-state laser operating at 532 nm giving vertically polarized light. Solutions were clarified by ultrafiltration using 0.45 µm filters. The solutions had a dissymmetry not exceeding 1.10 when measured at 45° and 135°. The instrument was standardized using toluene at 532 nm, which has a Rayleigh factor of 31.6 × 10-6 cm-1.12 Refractive index increments of HSA at pH 3.2 and ionic strengths 0.0312 and 0.188 were measured at 298 ( 1 K using a Mettler Toledo RAS10M precision refractometer. Dynamic Light Scattering (DLS). Measurements of diffusion coefficients (D) were made at 298 ( 1 K using photon correlation spectroscopy (PCS) (Malvern Instruments Limited). The effective hydrodynamic radii of the complexes (Rh) were calculated from the Stokes-Einstein equation
D)
kT 6πηRh
(1)
where k is the Boltzmann constant, T the absolute temperature, and η the solvent viscosity (taken as 8.904 × 10-4 N m-2 s, the value for water at 25 °C). Electrophoretic Mobility Measurements. The electrophoretic mobilities (u) of the HSA plus diC8PC were measured using a Malvern Instruments Ltd., Zetasizer 3000. ζ potentials were calculated from the Henry equation14,15
ζ)
3ηu 20r f(κa)
(2)
where the permittivity of vacuum (o), relative permittivity (r), and viscosity of water (η) were taken as 8.854 × 10-12 J-1 C2 m-1, 78.5, and 8.904 × 10-4 N m-2 s, respectively. The Debye length (1/κ) and the measured complex radii (a) from DLS were used to calculate the Henry factors f(κa) for each ionic strength. A further condition for the use of the above equation is that the surface conductivity of the particles must be small relative to the bulk conductivity. As almost all of the measurements (apart from the low ionic strength (0.0062 M) system at pH 3.2) were made at electrolyte concentrations >10-2 M, the above condition is met and the ratio of particle to bulk conductivities (the Dukhin number16) is small. Microcalorimetry. Enthalpy measurements were made at 298.15 K using an LKB-Productkter 10700 twin-cell batch (13) Sanderson, N. M.; Guo, B.; Jacob, A. E.; Handley, P. S.; Cunniffe, J. G.; Jones, M. N. Biochim. Biophys. Acta 1996, 1283, 207. (14) Henry, D. C. Proc. R. Soc. London, Ser. A 1931, 133, 106. (15) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press: London, 1981; Chapter 3. (16) Dukhin, S. S.; Derjaguin, B. V. Surface and Colloid Science; Matijevic, E., Ed.; Wiley-Interscience: New York, 1974; Chapter 2, p 210.
Static Light Scattering of HSA and HSA-diC8PC Complexes. The sample of HSA was analyzed by SLS as a function of concentration at pH 3.2 and two ionic strengths. Figure 1 shows the light scattering plot based on the equation
Kc 1 ) + 2A2c R90 M
(3)
4π2n2 (dn/dc)2 4 Nλ
(4)
w
where
K)
and the wavelength λ ()532 nm), the refractive index n ) 1.33, and R90, A2, and Mw are the Rayleigh factor, second virial coefficient, and weight average molecular mass, respectively. The refractive index increments were found to be 0.1901 ( 0.0023 cm3 g-1 (pH 3.2, I ) 0.0062) and 0.1804 ( 0.0059 cm3 g-1 (pH 3.2, I ) 0.188). The intercepts of the plots in Figure 1 give a mean weight average mass of 115700 ( 1390, corresponding to an association number of 1.67 ( 0.02 taking the monomer molecular mass as 69 400. This compares with the values of 1.342 and 1.7511 reported for bovine serum albumin. The second vivial coefficients at low (0.0062) and high (0.188) ionic strength are 6.72 × 10-4 and 1.13 × 10-4cm3 g-2 mol, respectively. The decrease in A2 with increasing ionic strength is consistent with the suppression of ionic repulsion between the molecules. The product A2Mw is 13 cm3 g-1, which is considerably greater than the value of 3 cm3 g-1 expected for a spherical protein with a partial specific volume of 0.75 cm3 g-1.18 This may be because of lack of complete suppression of ionic interaction and/or the asymmetry of the dimers present. Figures 2, 3, and 4 show the scattering (S90, the ratio of the scattered light intensity at 90° relative to that of toluene, i.e., i90/i90(toluene)) of solutions of HSA at a fixed concentration of HSA (1.25 × 10-3 g cm-3) as the concentration of diC8PC is increased at pH 3.2, 7.4, and 10.0. The data largely relate to diC8PC concentrations below the critical micelle concentrations (cmc’s). The cmc’s of the diC8PC at the three pH values are 0.19 mM (pH 3.2) and 0.28 mM (pH 7.4 and pH 10.0).19 All the data in Figures 2-4 show an increase in S90 with increasing diC8PC concentration. The increases are greater at higher ionic strength. The increases may arise either from the addition of the phospholipid itself and/or from the increase in size of the HSA due to binding of the phospholipid. Measurements of the refractive index of diC8PC over the range of concentration up to 0.8 mM gave extremely small refrac(17) Wadso¨, I. Acta Chem. Scand. 1968, 22, 92. (18) Tanford, C. Physical Chemistry of Macromolecules; J. Wiley & Sons: New York, 1961; Chapter 4, p 234. (19) Martinez-Landeira, P.; Prieto, G.; Ruso, J. M.; Sarmiento, F. J. Colloid Interface Sci., in press.
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Figure 2. Light scattering relative to toluene from aqueous solutions of HSA on addition of dioctanoylphosphatidylcholine (diC8PC) at pH 3.2, [HSA] ) 0.125% w/v: b, ionic strength 0.0062 M; 9, ionic strength 0.188 M.
Martinez-Landeira et al.
Figure 4. Light scattering relative to toluene from aqueous solutions of HSA on addition of dioctanoylphosphatidylcholine (diC8PC) at pH 10.0, [HSA] ) 0.125% w/v: b, ionic strength 0.0312 M; 9, ionic strength 0.188 M. Table 2. Characterization Data for HSA + DiC8 PC Mixtures from DLS Measurements conditions
particle radius r (nm)
M
association no.
pH 3.2 (I ) 0.0062) pH 3.2 (I ) 0.188) pH 7.4 (I ) 0.0188) pH 7.4 (I ) 0.188) pH 10.0 (I ) 0.0312) pH 10.0 (I ) 0.188)
4.36 ( 0.33a 3.89 ( 0.18 3.61 ( 0.34 3.81 ( 0.40 3.62 ( 0.20 3.46 ( 0.19
224120 159170 127220 149550 128280 112010
3.23 2.29 1.83 2.15 1.85 1.61
a
Figure 3. Light scattering relative to toluene from aqueous solutions of HSA on addition of dioctanoylphosphatidylcholine (diC8PC) at pH 7.4, [HSA] ) 0.125% w/v: b, ionic strength 0.0188 M; 9, ionic strength 0.188 M.
tive index increments. The values were 0.0013 cm3 g-1 (r2 ) 0.9401) (pH 3.2, I ) 0.0063), 0.0015 cm3 g-1 (r2 ) 0.8107) (pH 3.2, I ) 0.188), 0.0014 cm3 g-1 (r2 ) 0.9615) (pH 10.0, I ) 0.0312), and 0.0012 cm3 g-1 (r2 ) 0.9507) (pH 10.0, I ) 0.188). It follows therefore that the increased scattering on addition of phospholipid could not arise from the phospholipid but from an increase in the apparent size of the HSA due to phospholipid-induced association. Extrapolation of the curves of Figures 2-4 to zero phospholipid concentration gave the weight average molecular masses shown in Table 1. These correspond to an association number of 1.65 ( 0.15, in good accord with the value given above from the light scattering data for HSA
Standard errors.
alone. The scattering data relates to a constant concentration of 0.125 × 10-2 g cm-3, so if the weight average molecular masses are corrected to zero HSA concentration using the second virial coefficient from the data in Figure 1, the molecular masses (see Table 1) give an average association of 1.86 ( 0.15. This suggests that the extrapolation of data for the systems HSA + diC8PC to zero diC8PC concentration does not lead to the true molecular masses as measured in the absence of phospholipid. Using the data in Figures 2-4 to calculate molecular masses of HSA at a phospholipid concentration equal to the critical micelle concentrations (as given above) gives the values in the final column in Table 1. The corresponding association numbers as a function of pH and ionic strength are shown in Table 3. These data suggest that phospholipid-induced association is greater at higher ionic strength and is inhibited by low pH at low ionic strength. At high ionic strength, association numbers for the HSA + diC8PC system suggest it consists of a mixture of largely dimeric HSA with some trimeric species. At low ionic strength the HSA is partially dimeric with the greatest population of dimers at pH 7.4. While the association numbers imply that the predominant species are dimers, it is not possible to deduce whether there are
Table 1. Weight Average Molecular Masses of HSA (concentration 1.25 × 10-3 g cm-3) from SLS Plots on HSA + DiC8PC Mixtures conditions pH 3.2 (I ) 0.0062) pH 3.2 (I ) 0.188) pH 7.4 (I ) 0.0188) pH 7.4 (I ) 0.188) pH 10.0 (I ) 0.0312) pH 10.0 (I ) 0.188) a
correlation coefficient r2(order)
(Mw)[PC])0
(Mw)b[PC])0
(Mw)[PC])cmc
0.8977(2) 0.9296(3) 0.9421(2) 0.9721(2) 0.9239(2) 0.9518(2)
96370 ( 116140 ( 12810 117140 ( 1650 125170 ( 2010 109210 ( 1935 123800 ( 3470
114980 120070 145830 129750 133750 128280
112970 174260 135730 162300 128070 161050
2660a
Standard errors. b Corrected to zero HSA concentration using second virial coefficients from Figure 1.
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Table 3. Association Numbers of Human Serum Albumin as a Function of pH and Ionic Strength in the Presence of Dioctanoylphosphatidylcholine at Concentration Equal to the Critical Micellar Concentration pH
low ionic strength (I)
high ionic strength (0.188 M)
3.2 7.4 10.0
1.63 ( 0.05a (0.0062) 1.96 ( 0.03 (0.0188) 1.85 ( 0.03 (0.0312)
2.51 ( 0.28 2.34 ( 0.04 2.32 ( 0.07
a
Standard errors.
Figure 6. Particle radii of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC) determined by dynamic light scattering: (, ), pH 7.4 ionic strength 0.0188 M; 2, 4, pH7.4 ionic strength 0.188 M. The open symbols are for the larger particles present in bimodal particle distributions. The arrow denotes the critical micelle concentration of diC8PC. The inset shows the particle size distribution for HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC) determined by dynamic light scattering: pH 7.4, ionic strength 0.188 M, [HSA] ) 0.125% w/v, [diC8PC] ) 0.297 mM.
Figure 5. Particle radii of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC) determine by dynamic light scattering: 9, O, pH 3.2, ionic strength 0.188 M. The open symbols are for the larger particles present in bimodal particle distributions. The arrow denotes the critical micelle concentration of diC8PC.
very small proportions of other aggregates with association numbers greater than 2. As ionic strength has a significant affect on association, the process is most likely largely a physical reversible process, although the possibility of association by disulfide bridging through the free sulfydryl group of HSA cannot be eliminated. Dynamic Light Scattering of HSA + diC8PC. Equivalent spherical particle radii for mixtures of HSA (0.125% w/v) and diC8PC at pH 3.2, 7.4, and 10.0 are shown in Figures 5-7, respectively. At each pH value, the data relating to phospholipid concentrations approaching the cmc give monodisperse distributions with an average particle radius in the range 3.3-4.4 nm. As the phospholipid concentration approaches the cmc, the particle size distributions become bimodal with the emergence of a small peak corresponding to a much larger species. The insert in Figure 6 shows an example of such a bimodal particle size distribution for the system HSA (0.125% w/v) plus diC8PC (0.297 mM) pH 7.4, ionic strength 0.188. The larger particles which arise as the cmc is approached are too large to be spherical micellar phospholipid, which would be expected to have radii of the order of the length of the C8 chain, approximately 2-3 nm, and hence must be large HSA aggregates induced by binding of possibly micellar-like phospholipid. Light scattering studies on diC8PC have given no evidence of vesicle formation, only large polydisperse micelles are formed.20 The data show that increasing the ionic strength not only leads to larger species but also causes them to form at lower phospholipid concentration. Considering the average particle sizes at phospholipid concentrations below the cmc (in Table 2) the molecular (20) Tausk, R. J. M.; Oudshoorn, C.; Overbeek, J. Th. G. Biophys. Chem. 1974, 2, 53.
Figure 7. Particle radii of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC) determined by dynamic light scattering: 2, pH 10.0, ionic strength 0.0312 M; 1, 3, pH 10.0, ionic strength 0.188 M. The open symbols are for the larger particles in bimodal particle distributions. The arrow denotes the critical micelle concentration of diC8PC.
masses (m) of the particles can be calculated assuming the particles are rigid from the equation for the hydrodynamic volume (Vh)
Vh )
4πr3 M ) (v2 + δ1v10) 3 N
(5)
where v2 is the partial specific volume, δ1 the hydration parameter, v10 the specific volume of the solvent, and N Avogadro’s constant .21 The partial specific volume of HSA is 0.73322 cm3 g-1, and taking δ1 as 0.2 g g-1 and v10 as 1 g cm-3, the molecular masses of HSA were calculated and are given in Table 2. The molecular masses correspond to (21) Tanford, C. Physical Chemistry of Macromolecules; J. Wiley & Sons: New York, 1961; Chapter 6, p 340. (22) Sober, H. A., Ed. Handbook of Biochemistry; The Chemical Rubber Co.: Cleveland, OH, 1968.
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Figure 8. ζ potentials of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (di-C8 PC): O, pH 3.2, ionic strength 0.0062 M; O, pH 3.2, ionic strength 0.188 M.
Figure 10. ζ potentials of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC): O, pH 10.0, ionic strength 0.0312 M; 0, pH 10.0, ionic strength 0.188 M.
Figure 9. ζ potentials of HSA in aqueous solution in the presence of dioctanoylphosphatidylcholine (diC8PC): O, pH 7.4, ionic strength 0.0188 M; 0, pH 7.4, ionic strength 0.188 M.
Figure 11. Enthalpy of interaction of HSA in aqueous solution with dioctanoylphosphatidylcholine (diC8PC) at 25 °C: b, pH 3.2, ionic strength 0.188 M; 9, pH 7.4, ionic strength 0.188 M.
associated numbers on average of 2.16 ( 0.60. As for the SLS data, these results suggest that dimeric HSA is the predominant species. ζ Potential Measurements. The ζ potentials of HSA plus diC8PC as a function of phospholipid concentration, pH, and ionic strength are shown in Figures 8-10. The ζ potentials are positive at pH 3.2 and negative at pH 7.4 and pH 10.0, which is consistent with previously reported isoelectric points of 4.2,6 4.9,23 and 4.7-4.924 for HSA. At pH 3.2 addition of phospholipid decreased the ζ potential of HSA at both low and high ionic strength suggesting a change in the structure of the double layer due to diC8PC interaction and a shift in the plane of shear. The ζ potentials become very small as the phospholipid cmc is approached (log[di-C8PC] ) -3.7). At pH 7.4 the ζ potentials become less negative with increasing phospholipid concentration, smoothly at low ionic strength and more sharply at high ionic strength. As at pH 3.2 the ζ potentials become very small as the cmc is approached (here log[diC8PC] ) -3.5). The data at pH 10.0 also show similar trends. The choline headgroups of diC8PC will be partly positively charged at pH 3.2 and zwitterionic at pH 7.4 and pH 10.0. At pH 7.4 and 10.0 the decrease in
negative ζ potential of HSA on addition of phospholipid must arise from hydrophobic interactions of the acyl chains of the phospholipid and a change in the structure of the double layer. At pH 3.2, charge interactions are probably also of minor importance since at this pH the protein and phospholipid have the same sign of charge. Microcalorimetry. Figure 11 shows the enthalpies of interaction between HSA and diC8PC at pH 3.2 and 7.4 as a function of the final diC8PC concentration after mixing. Note that these data predominantly relate to diC8PC concentrations below the cmc. The data have been corrected for the enthalpy of dilution of the HSA that was found to be the same at both pH values within the experimental error. The dilution enthalpy was exothermic, and for a final HSA concentration of 0.125% w/v was -1.04 ( 0.02 J g-1. The enthalpy of dilution of the diC8PC was canceled by mixing diC8PC and buffer in the reference cell. At pH 3.2 the interaction between HSA and diC8PC is endothermic but becomes less endothermic as the phospholipid concentration is increased. Such a result requires the presence of an exothermic effect arising when the diC8PC concentration is increased. Since the dilution enthalpies of both components have been corrected for, there must be some other source of the exothermic effect. One possibility is the dissociation of HSA dimers to monomers that become incorporated into the complexes. If we write the process occurring at low PC concentrations
(23) Houska, M,; Brynda, E. J. Colloid Interface Sci. 1997, 188, 243. (24) Bundschuh, I.; Jacklemeyer, I.; Luneberg, E.; Bentzel, C.; Petzodt, R.; Stotle, H. Eur. J. Clin. Biochem. 1992, 30, 651.
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as an interaction between HSA dimers and xPC molecules as
HSA2 + xPC T HSA2(PC)x
(6)
and at high PC concentration an interaction between HSA monomers and the dimer-PC complexes which requires the partial dissociation of n dimers (HSA2), thus
nHSA2 T (n - 1)HSA2 + 2HSA
(7)
(n - 1) HSA2PCx + 2HSA T (n - 1)HAS(2n/n-1)PCx (8) then at high PC concentrations the overall process may be written
nHSA2 + (n - 1)PCx T (n - 1)HAS(2n/n-1)PCx (9) If the dissociation of dimeric HSA to monomers is exothermic as the dilution data shows (see above), then the increasing exothermicity observed at higher PC concentrations arises from dissociation of dimers to monomers that are incorporated into the complexes. At pH 10 the interaction between diC8PC and HSA is exothermic, so on increasing the PC concentration the process becomes more exothermic.
Conclusions Both static and dynamic light scattering studies show that the interaction between partially associated HSA and diC8PC results in phospholipid-induced further association of HSA. The association is greater at higher ionic strength and is inhibited at low ionic strength and low pH. The interaction can be followed from measurements of the ζ potentials of HSA plus diC8PC. Very significant changes in ζ potentials of HSA occur as the cmc of the diC8PC is approached. The interactions are most likely hydrophobically driven, particularly at pH 3.2 where both species carry the same sign of charge. Microcalorimetry studies below the cmc of the phospholipid show that the enthalpies of interaction are endothermic at pH 3.2 and exothermic at pH 10. A possible explanation for the increasing exothermicity with increasing diC8PC concentrations at both pH values is the exothermic dissociation of HSA dimers to monomers that are incorporated into the HSAdiC8PC complexes at higher PC concentrations. The HSAdiC8PC interaction studied here is of interest in relation to the widespread occurrence of phosphatidylcholines in biological organisms and their short-chain precursors and breakdown products in anabolic and catabolic processes, respectively. LA011681U
