Langmuir 2000, 16, 10449-10455
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Interaction of Amphiphilic Propranolol Hydrochloride with Haemoglobin and Albumin in Aqueous Solution J. M. Ruso, D. Attwood,† M. Garcı´a, G. Prieto, F. Sarmiento, P. Taboada, L. M. Varela, and V. Mosquera* Grupo de Fı´sica de Coloides y Polı´meros, Departamento de Fı´sica Aplicada y Departamento de Fı´sica de la Materia Condensada, Facultad de Fı´sica, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain, and School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, U.K. Received July 10, 2000. In Final Form: September 12, 2000 ζ-potential and UV difference spectroscopy techniques have been utilized to study the interaction of the amphiphilic drug propranolol hydrochloride with human haemoglobin (HH) and human serum albumin (HSA) in aqueous solution at pHs below, above, and at the isoelectric points of both proteins at 25 °C. The number of adsorption sites on both proteins was determined from the observed increases of the ζ-potential as a function of drug concentration in the regions of positive ζ-potential, where the adsorption was a consequence of the hydrophobic effect. The Gibbs energies of adsorption of the drug onto the proteins showed an exponential decrease with increase of drug concentration. The interactions between HSApropranolol complexes in the presence of propranolol hydrochloride were interpreted from dynamic lightscattering data using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The concentrations of the amphiphilic drug were maintained below its critical concentration, so its behavior could be considered to be that of a 1:1 electrolyte. This treatment has been widely used to obtain interactions between micelles in electrolyte solution but to our knowledge has not previously been applied to systems of the type considered here.
I. Introduction The self-association characteristics and surface activity of the amphiphilic drug propranolol hydrochloride have been reported previously.1-3 Many of the β-adrenoceptor blocking agents including propranolol exhibit a range of pharmacological effects that are independent of their β-blocking activity and which arise as a result of modification of the cell membrane. These effects, collectively referred to as the membrane stabilizing activity, include nonspecific cardiac depression and depression of myocardial conduction velocity and local anaesthetic activity. The magnitude of the membrane effects has been correlated with the hydrophobicity of the drug molecules,4,5 as indicated by oil/water partitioning characteristics and surface activity. The hydrophobicity of the propranolol molecule (see Chart 1) also has an influence on its potential interaction with relatively hydrophobic molecules such as haemoglobin and albumin, which will be explored in this paper. Haemoglobin is a tetrameric molecule whose quaternary structure is composed of two R- and two β-peptide chains (R2β2) with eight R-helical segments and 75% R-helix. The four subunits which correspond to the myoglobin molecule in their chain conformation are held together at their contact sites by hydrophobic bonds and salt bridges.6 * To whom correspondence should be addressed. Telephone: +34 981 563-100. Fax: +34 981 520 676. E-mail:
[email protected]. † University of Manchester. (1) Attwood, D.; Agarwal, S. P. J. Pharm. Pharmacol. 1979, 31, 392. (2) Mosquera, V.; Ruso, J. M.; Attwood, D.; Jones, M. N.; Prieto, G.; Sarmiento, F. J. Colloid Interface Sci. 1999, 210, 97. (3) Ruso, J. M.; Attwood, D.; Rey, C.; Taboada, P.; Mosquera, V.; Sarmiento, F. J. Phys. Chem. B 1999, 103, 7092. (4) Hellenbrecht, D.; Lemmer, B.; Wiethold, G.; Grobecker, H. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1973, 277, 211. (5) Levy, J. V. J. Pharm. Pharmacol. 1968, 20, 813. (6) Takeda, K.; Sasa, K.; Kawamoto, K.; Wada, A.; Aoki, K. J. Colloid Interface Sci. 1988, 124, 284.
Chart 1
Human serum albumin plays an important role in the transport and deposition of a variety of endogenous and exogenous substances in blood. This protein has three domains, each consisting of a large double loop, a short connecting segment, a small double loop, a long connecting segment, another large double loop, and a connecting segment to the next domain.7 Protein-amphiphilic molecule interactions have been extensively studied by a variety of experimental methods8 including quantitative equilibrium dialysis, calorimetry, viscometry, static and dynamic light scattering, and UV difference spectroscopy. In general, the amphiphilic molecules chosen for these studies are ionic surfactants in view of their application in the area of membrane studies.9,10 There is evidence that the initial interaction between ionic surfactants and proteins is predominantly ionic.11,12 These initial interactions can cause the protein (7) Winzor, D. J.; Sawyer, W. H. Quantitative Characterization of Ligand Binding; Wiley-Liss Inc.: New York, 1995. (8) Jones, M. N. In Biological Thermodynamics; Jones, M. N., Ed.; Elsevier: Amsterdam, 1988. (9) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29. (10) Jones, O. T.; Earnest, J. P.; McNamee, M. E. Solubilization and Reconstitution of Membrane Proteins. In Biological Membranes; Findlay, J. B. C., Evans, W. H., Eds. IRL Press: Oxford, 1987; Chapter 5, pp 101-134. (11) Oakes, J. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200. (12) Tipping, E.; Jones, M. N.; Skinner, H. A. J. Chem. Soc., Faraday Trans. 1 1974, 70, 1306.
10.1021/la000965w CCC: $19.00 © 2000 American Chemical Society Published on Web 11/15/2000
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to unfold, so exposing more binding sites. The existence of nonpolar amino acid side chains in protein molecules suggests the possibility of interaction between proteins and small molecules containing hydrocarbon chains, such as hydrocarbons themselves, simple amphiphiles, or biological lipids.13 This hydrophobic interaction between proteins and ionic surfactants can give rise to a conformational change even when the concentration of the ligands is remarkably low.14 Saturation of the binding sites generally occurs below the critical micelle concentration of the surfactant.15 In this study, the interaction of propranolol hydrochloride with haemoglobin and albumin has been initially investigated by measurements of the ζ-potential of the complex formed. We have used this approach previously in the analysis of the interactions of alkylsulfates16 and alkyltrimethylammonium bromides17 with lysozyme. The data were interpreted in terms of the theory of Ottewill and Watanabe,18 which describes the adsorption of counterions to colloidal particles by the Langmuir isotherm and is applicable to independent and identical adsorption sites. The ζ-potential data have been used to calculate the Gibbs energies of adsorption. The study has been made at pHs above, below, and at the isoelectric point of both proteins, defined as the pH at which the ζ-potential is zero. The adsorption of propranolol onto these proteins may induce conformational changes in the proteins that affect the amino acid residues located on the surface of the proteins. We have used the technique of UV difference spectroscopy19 to detect any such induced conformational changes. Finally, we have used dynamic light scattering to determine diffusion coefficients of the albumin in the presence of propranolol to evaluate the stability of this colloidal system. The concentrations of propranolol were kept below its critical concentration, so its behavior could be considered that of a 1:1electrolyte. These data were interpreted using the Derjaguin-Landau-VerweyOverbeek (DLVO) theory of colloid stability, facilitating quantification of the interactions between these complexes by means of the interaction potential. This treatment has been widely used to obtain interactions between micelles in electrolyte solution but to our knowledge has not previously been applied to systems of the type considered here. II. Experimental Section Materials. Human haemoglobin, HH (9008-02-0, 98% purity), human serum albumin, HSA (70024-90-7, 98% purity), and propranolol (1-[isopropylamino]-3-[1-naphthyloxy]-2-propanol) hydrochloride were obtained from Sigma Chemical Co. The buffers used were glycine (50 mmol dm-3)-hydrochloric acid pH 3.20, glycine (50 × 10-3 mol dm-3)-hydrochloric acid pH 4.90, glycine (50 × 10-3 mol dm-3)-hydrochloric acid pH 6.50, and glycine (50 × 10-3 mol dm-3)-sodium hydroxide pH 10.0. The ionic strength corresponding to these concentrations was 0.0312. All other materials were of analytical grade, and solutions were made using doubly distilled and degassed water. All measurements were below the literature values of the critical micelle concentration of propranolol hydrochloride,2 0.128 mol dm-3. (13) Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; Wiley: New York, 1980. (14) Steinhardt, J.; Reynolds, J. A. Multiple Equilibria in Proteins; Academic Press: New York, 1969. (15) Jones, M. N. Biological Interfaces; Elsevier: Amsterdam, 1975. (16) Mosquera, V.; Ruso, J. M.; Prieto, G.; Sarmiento, F. J. Phys. Chem. 1996, 100, 16749. (17) Sarmiento, F.; Ruso, J. M.; Prieto, G.; Mosquera, V. Langmuir 1998, 14, 5725. (18) Ottewill, R. H.; Watanabe, A. Kolloid-Z. 1960, 170, 132. (19) Jones, M. N.; Skinner, H. A.; Tipping, E.; Wilkinson, A. E. Biochem J. 1972, 135, 231.
Ruso et al. Zeta Potential Measurements. The ζ-potentials of the proteins and protein/drug solutions were measured at 25.0 °C by a Zetamaster 5002 (Malvern Instruments Ltd) using a 5 mm × 2 mm rectangular quartz capillary cell and taking the average of five measurements at a stationary level. The ζ-potentials were calculated from the electrophoretic mobilities, uE, assuming a radius of 3 nm for both proteins estimated from molecular masses for haemoglobin21 and albumin;22 this condition was satisfied at all the pHs examined. The product κa is 4.04 (where κ is the reciprocal Debye length and a the particle radius), corresponding to a Henry factor, f(κa), of 1.1420
ζ)
3uEη 1 20r f(κa)
(1)
η, 0, and r are the viscosity, the permittivity of vacuum, and the relative permittivity of the medium, taken as 8.904 × 10-4 N m-2 s, 8.854 × 10-4 J-1 C2 m-1, and 78.5, respectively. To obtain measurements of the ζ-potential at different pHs, the zetamaster was connected to a Mettler DL21 Titrator, which is a complete analysis station for titrimetric analysis. Spectroscopy. Difference spectra were measured using a Beckman spectrophotometer (model DU 640), with six microcuvettes, operating in the UV-visible region, with a full scale expansion of 0.2 absorbance units. For absorbance difference spectra, five of the six microcuvettes were filled with protein/ drug solutions; the remaining microcuvette contained only protein in the corresponding medium and was used as a blank reference. The microcuvettes were filled and placed in the same orientation for all the tests. Absorbance was measured at the temperature 25 °C using a temperature controller (Beckman DU Series), based on the Peltier effect. Adsorption of Propranolol Hydrochloride by Haemoglobin and Albumin. Aliquots of 2.5 cm3 of the protein solution of concentration 0.125% w/v were equilibrated at 25 °C with 2.5 cm3 of drug solutions covering the required range of concentration. Equilibrium adsorption was achieved using equilibration times in excess of 3 h. Dynamic Light Scattering. Measurements were made at 25.0 ( 0.1 °C and at a scattering angle of 90° with a BI-200SM Brookhaven laser light scattering instrument equipped with a 4 W argon ion laser (Coherent Innova 90) operating at 488 nm with vertically polarized light. This was combined with a Brookhaven BI 9000AT digital correlator with a sampling time range of 25 ns to 40 ms. Solutions were clarified by ultrafiltration through 0.1 µm filters, until the ratio of light scattering at angles of 45° and 135° did not exceed 1.10. Diffusion coefficients were determined from a single-exponential fit to the correlation curve. Hydrodynamic radii were calculated from measured diffusion coefficients by means of the Stokes-Einstein equation.
III. Results and Discussion A. Complex Characterization and Adsorption. Figure 1 shows the ζ-potential of HH and HSA as a function of pH. In calculating the ζ-potential of a small particle (as here), the deformation of the applied field by the presence of the particle in its neighborhood can be neglected. Moreover, it may also be assumed that the electrophoretic retardation does not affect the particle to any great extent, and the only retarding force on the particle is the viscous drag from the water. As the protein charge is usually low, and electrophoresis is carried out at normal ionic strengths, the ζ-potential of such a particle can be expected to be low. Consequently, although the relaxation effect is relatively unimportant, the electrophoretic retardation (20) Hunter, R. J. Zeta Potential in Colloid Science. Principles and Applications; Academic Press: New York, 1981. (21) Beretta, S.; Lunelli, L.; Chirico, G.; Baldini, G. Appl. Opt. 1996, 35, 3763. (22) Frank, M.; Mears, A.; Labov, S. E.; Benner, W. H.; Horn, D.; Jaklevic, J. M.; Barknecht, A. T. Rapid Commun. Mass Spectrom. 1996, 10, 1946.
Interaction of Propranolol Hydrochloride with HH and HSA
Figure 1. ζ-potential of human haemoglobin (9) and human serum albumin (b) (0.125% w/v) in aqueous glycine-saline solution (ionic strength 0.0312) as a function of pH at 25 °C.
Figure 2. ζ-potential of human haemoglobin (0.125% w/v) in aqueous glycine-saline solution (ionic strength 0.0312) as a function of logarithm of propranolol hydrochloride concentration at pH 2.5 (9), pH 6.8 (b), and pH 8.0 (2) at 25 °C.
should not be neglected.23 ζ-potential-pH data were fitted by a fourth order polynomial with r2 ) 0.9977 and 0.9964 for HH and HSA, respectively. These expressions give a zero ζ-potential at pHs of 6.83 and 5.10 for HH and HSA, respectively, values in close agreement with the isoelectric points from the literature of 6.824 and 4.9.25 The ζ-potentials of HH and HSA as a function of propranolol concentration c at different pHs are shown in Figures 2 and 3, respectively. At pH 8.0, both systems show a gradual increase of the ζ-potential from the negative values at very low propranolol concentrations (see also Figure 1 in the absence of drug) to positive values with increase of propranolol concentration, reflecting changes in the surface charge. The propranolol concentration at which the point of zero ζ-potential was reached was very close for both proteins, being 0.027 and 0.026 mol kg-1 for haemoglobin and albumin, respectively. There is, however, a difference in behavior of the HH and HSA systems in the presence of drug at pH 2.5. Whereas the ζ versus c plot for the HSA/propranolol system at pH 2.5 is similar to that at its isoelectric point (pH 4.9), that of (23) Bier, M. Electrophoresis. Theory, Methods and Applications; Academic Press: New York, 1967. (24) Hayashi, K.; Kugimiya, M.; Imoto, T.; Funatsu, M.; Bigelow, C. C. Biochemistry 1968, 7, 1461. (25) Houska, M.; Brynda, E. J. Colloid Interface Sci. 1997, 188, 243.
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Figure 3. ζ-potential of human serum albumin (0.125% w/v) in aqueous glycine-saline solution (ionic strength 0.0312) as a function of logarithm of propranolol hydrochloride concentration at pH 2.5 (9), pH 4.9 (b), and pH 8.0 (2) at 25 °C.
Figure 4. Difference spectra of human haemoglobin (0.125% w/v) in the presence of propranolol hydrochloride of concentration (1) 0.03, (2) 0.045, (3) 0.05, (4) 0.06, and (5) 0.12 mol kg-1, relative to native haemoglobin at pH 8.0.
the HH/propranolol system at pH 2.5 shows an abrupt change of potential at a propranolol concentration of approximately 0.05 mol kg-1. At this pH the albumin is protonated and hence there is no expected electrostatic interaction between the protein and the positively charged propranolol. The sigmoidal shape of the curve suggests an induced conformational transition of haemoglobin at this propranolol concentration, which as discussed below may be associated with a tetramer-dimer transition. The beginning of this denaturation can also be observed at pH 6.8 at a similar propranolol concentration. Figure 4 shows the difference spectra in the wavelength range 380-440 nm at pH 8.0 between a HH solution of concentration 0.125% w/v and a solution of HH of the same concentration containing propranolol hydrochloride, at a range of drug concentrations. Addition of propranolol results in the development of an absorption band in the region of 410 nm that is positive with respect to native HH, arising from the extended π-electron system of the porphyrin ring.26 Similar spectra to those of Figure 4 were obtained for the other pHs, with peaks at 406 and 411 nm for pH 2.5 and 6.8, respectively. For the HSA/propranolol (26) Neubacher, H.; Lohmann, W. In Biophysics; Hoppe, W., Lohmann, W., Markl, H., Ziegler, H., Eds.; Springer-Verlag: Berlin, 1983.
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Ruso et al. Table 1. Number of Adsorption Sites, N1, and Standard Gibbs Energy of Adsorption, - ∆G°ads, of Human Haemoglobin (HH) and Human Serum Albumin (HSA) as a Function of the pH of the Medium HH
pH 2.5
(m-2)
1.25 × 14.3
N1 -∆G°ads (kJ mol-1) HSA (m-2)
pH 2.5
N1 -∆G°ads (kJ mol-1)
Figure 5. Absorbance at 25 °C of human haemoglobin (0.125% w/v) at (9) pH 2.5, (b) pH 6.8, and (2) pH 8.0 as a function of logarithm of propranolol hydrochloride concentration measured at the wavelengths of peak absorption.
1017
6.7 × 14.1
1016
pH 6.8 2.13 × 15.3
1017
pH 4.9 1.3 × 14.1
(
(27) Jones, M. N. In Surface Activity of Proteins; Magdassi, S., Ed.; Marcel Dekker: New York, 1996. (28) Bhattacharyya, J.; Bhattacharyya, M.; Charaborti, A. S.; Chaudhuri, U.; Poddar, R. K. Int. J. Biol. Macromol. 1998, 23, 11.
pH 8.0 1.5 × 1017 14.2
1016
4.606kBT dζ ) d log c ze
systems the main peaks were observed at 410, 413, and 417 nm for pHs 2.5, 4.9, and 8.0, respectively. Figures 5 and 6 show the absorbance changes of HH and HSA at the peak wavelength of the difference spectra, as a function of propranolol concentration, for the three pHs studied. Absorbance increases gradually with concentration for all HSA/propranolol systems and for HH/ propranolol systems at pH 6.8 and 8.0; the greatest increase of absorbance being at pH 8.0 (near the physiological pH of 7.4). The curve obtained at pH 2.5 for HH is the typical sinusoidal curve accompanying a change of conformation of globular proteins,28 suggesting, as in the case of ζ-potential data, that the HH at pH 2.5 undergoes a significant change in conformation. The analysis of data from ζ-potential measurements and difference spectroscopy seems to indicate the same pathway for the conformational changes induced by propranolol in HSA at the three pHs under study. For HH these changes are similar at pHs 6.8 and 8.0, but they are more abrupt at pH 2.5. It is well-known that at low electrolyte concentrations HH exists mostly as a tetramer while at high electrolyte concentrations it exists pre-
1.32 × 1017 14.6
dominantly as a dimer.28 The difference in behavior of HH over the pH range can be attributed to the high electrolyte concentration at low pH, the protons (H+) and anions (Cl-, the counterion of propranolol hydrochloride) playing the role of electrolyte. The increases of the ζ-potential as a function of drug concentration in the regions of positive ζ-potential are a consequence of adsorption due to the hydrophobic effect, since in such regions the protein and drug ion have the same sign of charge. Consequently, the following equation can be used to calculate the number of adsorption sites N1.29,30
( )
Figure 6. Absorbance at 25 °C of human serum albumin (0.125% w/v) at (9) pH 2.5, (b) pH 6.8, and (2) pH 8.0 as a function of logarithm of propranolol hydrochloride concentration measured at the wavelengths of peak absorption.
pH 8.0
(
)
sinh(zeζ1/2kBT) - sinh(zeζ2/2kBT) cosh(zeζ2/2kBT)
x8n0kBT[sinh(zeζ1/2kBT) - sinh(zeζ2/2kBT)] - 1 zeN1
)
(2)
ζ1 and ζ2 are selected ζ-potentials on the line, kB the Boltzmann constant, c the concentration, T the absolute temperature, n0 the ionic concentration, e the proton charge, z the valence of the ion, and the relative permittivity of the medium. The adsorption constant k2 can be calculated from the equation
1 ) k2 c
(
zeN1
x8n0kBT[sinh(zeζ1/2kBT) - sinh(zeζ2/2kBT)]
-1
)
(3) Here c is chosen as the concentration at the ζ-potential midpoint between ζ1 and ζ2. The standard free energy of adsorption, ∆G°ads, can be obtained from the equation
k2 ) exp(-∆G°ads/kBT)
(4)
Table 1 shows that the number of available adsorption sites per unit area obtained from eq 2 for each pH value is very similar at the three pHs under study and also of similar magnitude for the two proteins. The N1 values obtained for HSA/propranolol can be compared with that (29) Kayes, J. B. J. Colloid Interface Sci. 1976, 56, 426. (30) Stadilis, G.; Avranas, A.; Jannakoudakis, D. J. Colloid Interface Sci. 1990, 135, 313.
Interaction of Propranolol Hydrochloride with HH and HSA
Figure 7. ζ-potential (9) and Gibbs energies of adsorption (O) of human haemoglobin (0.125% w/v) in aqueous glycine-saline solution (ionic strength 0.0312) as a function of logarithm of propranolol hydrochloride concentration at pH 2.5 and 25 °C.
Figure 8. ζ-potential (b) and Gibbs energies of adsorption (O) of human serum albumin (0.125% w/v) in aqueous glycinesaline solution (ionic strength 0.0312) as a function of logarithm of propranolol hydrochloride concentration at pH 6.8 and 25 °C.
from capillary electrophoresis/frontal analysis measurements31 for the same system (N1 ) 5.48 × 1015 m-2 at pH 7.4). Representative plots of the standard Gibbs energies of adsorption evaluated from eq 4 are shown in Figures 7 and 8. Calculations were restricted to a propranolol concentration range of 0.05-0.07 mol kg-1 for HH at pH 2.5 (nonzero gradient of ζ-potential versus c); calculations for other systems were carried out over the whole concentration range. Comparison of values of ∆G°ads and ζ-potential in Figures 7 and 8 shows that the Gibbs energies are large and negative at low values of drug concentration where binding to the high-energy sites takes place and become less negative as more drug molecules bind. Similar behavior was found for the system sodium n-dodecylsulfate/histone (HI).32 As expected, a good agreement between the changes in the number of adsorption sites and the Gibbs energy with pH was found, showing, for both proteins, maximum values of each parameter at the same pH (Table 1). The values calculated of Gibbs energies of adsorption for albumin at the three pHs were (31) Ding, Y. S.; Zhu, X. F.; Lin, B. C. Chromatographia 1999, 49, 343. (32) Moosavi-Movahedi, A. A.; Housaaindokht, M. R. Physiol. Chem. Phys. Med. NMR 1990, 22, 19.
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very similar at each propranolol concentration over the whole concentration range. In contrast, larger differences can be observed in the haemoglobin system. These results may indicate a higher homogeneity in the albumin structure in the presence of propranolol in the three media. B. Complex Stability. The ζ-potential and UV difference spectroscopy measurements have highlighted a difference of behavior of the human serum albumin and human haemoglobin systems, the former showing a much greater stability to conformational change in the presence of propranolol over the pH range studied. In an attempt to evaluate the interactions between the HSA/propranolol complexes, we have used dynamic light scattering to determine diffusion coefficients and have interpreted the data using the Corti and Degiorgio33 treatment based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. This theory permits us to quantify the interactions between complexes by means of the interaction potential, which consists of a hard-sphere repulsive part, an electrostatic long-range repulsion, and a Londonvan der Waals attraction. It contains only two unknown parameters, the electric potential at the shear surface of the aggregate and the Hamaker constant, which may be calculated by an iterative computational procedure. This treatment has been widely used to obtain interactions between micelles in electrolyte solution but to our knowledge has not previously been applied to systems of the type considered here. In applying this theoretical treatment, we consider the protein with its adsorbed drug surface layer to be the only type of particle in the solution (i.e., no micelles of drug are present), and we assume a uniform distribution of drug molecules over all of the protein molecules. The concentrations of propranolol were kept below its critical concentration, so its behavior could be considered that of a 1:1 electrolyte. A previous study of the system HSA/propranolol by dynamic light scattering at pH 7.4 has shown that propanolol binds to the protein molecule, causing an increase in the globular size from 3 to 4 nm.34 According to crystallographic studies of human serum albumin, there are two helical rods close together in the C-terminal region of the protein, whose sequence contains 18 residues after Cys567, making the last disulfide bridge with Cys558. This C-terminal helical rod is amphiphilic with hydrophilic residues on one side of the rod and hydrophobic ones on the other.35 It is possible that stabilization of the native conformation of the protein occurs by cross-linking of propranolol ions between nonpolar and charged residues located on different loops of the protein. Apparent diffusion coefficients, D, of HSA in water and aqueous solutions propranolol (below cmc) are presented in Figure 9 as a function of protein concentration. Experimental data have been fitted with the linear function
D ) D0[1 + kDc]
(5)
where D0 is the limiting diffusion coefficient. The concentration dependence of D is due to interactions between the positively charged complexes. Hydrodynamic radii were calculated from D0 using the Stokes-Einstein equation (33) Corti, M.; Degiorgio, V. J. Phys. Chem. 1981, 85, 711. (34) Luik, A. I.; Naboka, Y. N.; Mogileviche, S. E.; Hushca, T. O.; Mischenko, N. I. Spectrochim. Acta Part A 1998, 54, 1503. (35) Moriyama, Y.; Takeda, K. Langmuir 1999, 15, 2003.
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Ruso et al. Table 2. Limiting Diffusion Coefficient, D0, and Hydrodynamic Radius, rh, of the HSA-Propranolol Hydrochloride Complex in Aqueous Solution at 25 °C [propranolol] (mol kg-1)
1010D0 (m2 s-1)
rh (nm)
0.00 0.02 0.05 0.08
0.80 0.74 0.65 0.57
3.04 3.31 3.76 4.29
VA ) -
[
A 2 (x + 2x)-1 + (x2 + 2x + 1)-1 + 12 2 ln(x2 + 2x) (x2 + 2x + 1)
Figure 9. Diffusion coefficient, D, as a function of the human serum albumin concentration in (9) water and in aqueous solutions of propanolol of concentration (b) 0.02, ([) 0.05, and (4) 0.08 mol kg-1 at 25 °C.
kBT rh ) 6πηD0
(6)
where kB is the Boltzmann constant and η the solvent viscosity. Table 2 shows an increase of the globular size with added propranolol concentration, in agreement with the increased number of adsorbed drug molecules on the protein surface. Diffusion data were analyzed according to the treatment proposed by Corti and Degiorgio33 to quantify the interactive forces between complexes. In this treatment, the concentration dependence of D for interacting particles can be expressed in terms of the volume fraction φ of the particles:
D ) D0(1 + k′Dφ)
(7)
where k′D ) kD/νj and νj is the specific volume of the solute particles, as determined from density measurements. kD may be related to the pair-interaction potential, V(x), between spherical particles of radius a using the expression proposed by Felderhof:36
kD ) 1.56 +
∫0∞[24(1 + x)2 - F(x)][1 exp(-V(x)/kBT)] dx (8)
where x ) (R - 2a)/2a, R is the distance between the centers of two particles, and F(x) is given as
F(x) ) 12(1 + x) -
27 15 (1 + x)-2 - (1 + x)-4 + 8 64 75 (1 + x)-5 (9) 64
(36) Felderhof, B. U. J. Phys. 1978, 11, 929.
(10)
where A is the attractive Hamaker constant. Two approximate expressions have been proposed for the repulsive interaction, VR(x), for the limiting cases of κa < 1 and κa > 1. We have used the expression
VR(x) )
aΨ02 ln[1 + exp(-2κax)] 2
(11)
which is appropriate for values of κa > 1. The aggregate charge, q, is related to the surface potential, Ψ0, by the expression37
Ψ0 ) (2kBT/e) sinh-1[2πeκ-1qe/4πa2kBT]
(12)
The computational procedure involved the iterations of values of A and Ψ0 to give the best fit of computed and experimental values of kD over the range of surfactant concentration. The value of q derived from eq 12 was 2.2 units of electron charge (uec), and the Hamaker constant was 0.41 × 10-22 J. Agreement between computed and experimental kD values was reasonable in view of the assumptions inherent in these calculations (Table 3). In Figure 10 we show the V(x)/kBT curves for a set of propranolol concentrations showing the predominance of electrostatic repulsion at each concentration, which leads to a very stable dispersion. Increased adsorption with increasing propranolol concentration leads to screening of the electrostatic potential and the increasing importance of London-van der Waals attraction. The interaction potential can be related to the stability ratio W by the relationship:38
W)2
∫0∞
exp(V/kBT) (x + 2)2
dx
(13)
It can be seen from Figure 10 that the position of the maximum (xm) is not dependent on propanolol concentration, and hence W is determined almost entirely by the value of this maximum (Vm/kBT). Hence, eq 13 can be written
W) The interaction potential V(x) as it is usually written in the DLVO theory is the sum of an attractive Londonvan der Waals interaction VA(x) and a repulsive interaction due to the electric charge of the spheres. The expression for VA(x) derived by Hamaker for the case of two spheres is
]
∫0∞exp(V/kBT) dx
2 (xm + 2)2
(14)
On expanding V in a Taylor series around Vm and neglecting terms higher than 2, eq 14 becomes (37) Szajdzinska-Pietek, E.; Maldonado, R.; Kevan, L.; Jones, R. R. M. J. Colloid Interface Sci. 1986, 110, 514. (38) Ottewill, R. H.; Rastogi, M. C.; Watanabe, A. Trans. Faraday Soc. 1960, 56, 854.
Interaction of Propranolol Hydrochloride with HH and HSA Table 3. Experimental and Theoretical Slopes, kD, and Reduced Potential, eΨ0/kBT, As a Function of Propranolol Concentration kD [propranolol] (mol kg-1) 0.00 0.02 0.05 0.08
exptl 22.47 14.18 11.89 10.51
theor 22.92 15.71 10.89 10.35
eΨ0/kBT
Vm 2π1/2 exp kBT (xm + 2)
Table 4. Maximum Values of Interaction Potential (Vm/kBT) and Stability Ratios (W) of the HSA-Propranolol Hydrochloride Complex As a Function of Propranolol Concentration at 25 °C [propranolol] (mol kg-1)
Vm/kBT
W
0.00 0.02 0.05 0.08
1.81 0.91 0.37 0.22
4.91 1.98 1.16 1.00
7.81 4.46 2.28 1.70
Figure 10. Plots of the pair interaction potential V(x) as a function of the reduced and normalized distance x at the propranolol concentrations (mol kg-1) indicated.
W)
Langmuir, Vol. 16, No. 26, 2000 10455
(15)
Values of Vm/kBT and W obtained from eq 15 using a value of xm ) 0.1033 (Table 4) show a decreasing stability of the albumin/drug dispersion as propranolol is adsorbed onto the protein. C. Conclusions. The ζ-potential and UV absorption of human serum albumin (HSA) increased smoothly with increase of concentration of added propranolol hydro-
chloride at all pHs as a consequence of adsorption of the drug ions. In contrast, an abrupt change in these properties was observed for the human haemoglobin (HH)/propranolol system at a concentration of approximately 0.05 mol kg-1 at pH 2.5. This change has been attributed to a tetramer to dimer transition arising from the increase of concentration of drug which functions as an electrolyte. Increases of the ζ-potential with drug concentration for this system at higher pH were more gradual, the electrolyte concentration (which includes the H+ ions) now being insufficient to cause conformational change. The number of adsorption sites on both proteins and the Gibbs energies of adsorption were determined by analysis of the ζ-potential-drug concentration plots. The Gibbs energies of adsorption for albumin at the three pHs were very similar at each propranolol concentration over the whole concentration range; larger differences observed in the haemoglobin system may indicate a higher homogeneity in the albumin structure in the presence of propranolol in the three media. The interactions between HSA-propranolol complexes in the presence of propranolol hydrochloride at concentrations below its critical concentration were interpreted from dynamic light-scattering data using DLVO theory. The results indicated a decrease of the stability of the albumin/drug dispersion with increased adsorption of propranolol. Acknowledgment. The authors thank the Xunta de Galicia for financial support and EPSRC for funds for the purchase of light-scattering equipment. LA000965W
