Adsorption of Modified HIV-1 Capsid p24 Protein onto

9002

Langmuir 2000, 16, 9002-9008

Adsorption of Modified HIV-1 Capsid p24 Protein onto Thermosensitive and Cationic Core-Shell Poly(styrene)-Poly(N-isopropylacrylamide) Particles† David Duracher, Abdelhamid Elaı¨ssari,* Franc¸ ois Mallet, and Christian Pichot Unite´ Mixte CNRS-bioMe´ rieux, ENS de Lyon, 46 Alle´ e d’Italie, 69364 Lyon Cedex 07, France Received March 15, 2000. In Final Form: May 23, 2000 The adsorption of HIV-1 capsid p24 protein bearing six histidine residues (named RH24) onto wellcharacterized thermosensitive and cationic poly(styrene)-poly(N-isopropylacrylamide) core-shell particles was investigated as a function of temperature, pH, incubation time, and salinity. The maximum amount of adsorbed RH24 was observed when the temperature was above the lower critical solution temperature (LCST) of the hydrogel, whereas a negligible adsorbed amount occurred below the LCST. Adsorption isotherms were then determined above the LCST and exhibited well-defined plateaus, which were pH and ionic strength dependent. Isotherm data were tentatively discussed using the Freundlich power law, from which the standard free enthalpy of protein adsorption was estimated. The adsorption behavior of protein was mainly governed by hydrophobic interactions above the LCST; however, differences between the two latexes gave evidence that electrostatic forces also played a significant role.

I. Introduction The choice of latex particles as biomolecule carriers has given rise to a great amount of research on the adsorption of protein molecules onto such dispersed material.1,2 The adsorption study of proteins onto latexes is indeed quite relevant for at least two aspects. First, the adsorption studies can provide new and relevant information on the affinity between both species involved in the immobilization process. Second, the latex particles bearing proteic materials, such as antigen, antibody, enzyme, etc., can be used for immuno-separation and immuno-detection of viruses, antibodies, and antigens in biomedical diagnosis. Before any real application is considered in the biomedical field, it is fundamental to know how the selected biomolecules interact with the polymeric supports. Biomolecule immobilization at the solid/liquid interface is usually affected by factors such as pH, ionic strength, temperature, surface tension of the medium, charge, and nature of the polymer matrix. Therefore, any variation in one or more of these parameters may cause a drastic change in the adsorption-desorption processes of the considered biomolecules. The adsorption of proteins onto colloidal polymer particles has been extensively investigated using bovine serum albumin (BSA), immunoglobulins (IgG), fibrinogen, and enzymes.3-7 The maximum amount of protein adsorbed onto such particles was found to be at the isoelectric point of the * To whom correspondence should be addressed. Tel: (33) 7272-83-64. Fax: (33) 72-72-85-33. E-mail: [email protected]. † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millennium”. (1) Andrade, J. D.; Hlady, V.; Wei, A. P. Pure Appl. Chem. 1992, 64, 1777. (2) Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1979, 71, 350. (3) Kondo, A.; Higashitani, K. J. Colloid Interface Sci. 1992, 150, 344. (4) Betton, F.; Theretz, A.; Elaı¨ssari, A.; Pichot, C. Colloids Surf. 1993, 1, 97. (5) Elgersma, A. V.; Zsom, R. L.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1990, 138, 145. (6) Galisteo-Gonzalez, F.; Martin-Rodriguez, A.; Hidalgo-Alvarez, R. Colloid Polym. Sci. 1994, 272, 352. (7) Puig, J.; Fernandez- Barbero, A.; Bastos-Gonzalez, D.; Serra Domenech, J.; Hidalgo-Alvarez, R. Surf. Prop. Biomater. 1992, 13, 9.

protein-covered colloids, rather than that of the isoelectric point of the protein as generally observed. The observed behavior has been interpreted by taking into account the ion participation in the protein adsorption process.5 In addition, the exchange process between adsorbed and free protein has been studied by Ball et al.8 who demonstrated the dynamics involved in the exchange event. Globally, the adsorption of proteins onto colloidal polymer particles is governed by hydrophobic interactions3,6 with low or negligible contribution of attractive electrostatic interactions.9 In fact, protein adsorption can be reduced by increasing the hydrophilicity character of the solid support as reported by several authors.10-12 Recently, several academic studies have been devoted to the adsorption of proteins onto thermally sensitive latexes. Pioneering work has been reported by Kawaguchi et al.13 related to human γ-globulin (HGG) adsorption onto negatively charged poly(N-isopropylacrylamide (NIPAM)) microgel particles. They pointed out that the amount of protein increased when increasing the hydrophobic character of the particles which was induced by raising the adsorption temperature. The desorption of protein by lowering the temperature (below the lower critical solution temperature (LCST) of poly(NIPAM)) was found to depend on the incubation time for the adsorption above the LCST. As part of a systematic work dealing with the covalent immobilization of recombinant proteins onto synthetic polymers, this paper aims at reporting on a preliminary study of the adsorption of the modified HIV-1 capsid p24 protein (named RH24, Mw ∼ 27 500) onto two cationic poly(styrene)-poly(NIPAM) core-shell particles as a function of time, temperature, protein concentration, salinity, and pH. The adsorption behavior of this protein was tentatively (8) Ball, V.; Huetz, P.; Elaı¨ssari, A.; Cazenave, J.-P.; Voegel, J.-P.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7330. (9) Maramatsu. N.; Kondo. T. J. Colloid Interface Sci. 1992, 153, 23. (10) Okubo, M.; Yamamoto, Y.; Uno, M.; Kamei, S.; Matsumoto, T. Colloid Polym. Sci. 1987, 265, 1061. (11) Suzawa, T.; Shirahama, H. Adv. Colloid Interface Sci. 1991, 35, 139. (12) Revilla, J.; Elaı¨ssari, A.; Carriere, P.; Pichot, C. J. Colloid Interface Sci. 1996, 180, 405. (13) Kawaguchi, H.; Fujimoto, K.; Mizuhara, Y. Colloid Polym. Sci. 1992, 270, 53.

10.1021/la0004045 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/09/2000

Adsorption of Modified HIV-1 Capsid p24 Protein

Langmuir, Vol. 16, No. 23, 2000 9003

interpreted using the power law (Freundlich adsorption representation), allowing one to discuss the magnitude of the driving forces involved in the adsorption process of such a complex system. II. Materials and Methods 1. Materials. The recombinant-modified protein (RH24) was synthesized according to the method of Cheynet et al.14 The prepared protein (Mw ∼ 27 500 and calculated isoelectric point IEP ∼ 5.9) is a recombinant HIV-1 capsid p24 protein modified by introducing six histidine residues at the N terminus. The protein solution was purified using column affinity.14 Disodium hydrogen phosphate and sodium phosphate from Prolabo were used as a buffer. Deionized water (Milli-Q) was purified by a Millipore Q-T M system. 2. Preparation of Poly(styrene)-Poly(NIPAM) CoreShell Microspheres. Thermosensitive core-shell latexes were prepared by a shot growth process combining emulsifier-free emulsion and precipitation polymerization as described elsewhere.15 The first step was a batch polymerization of styrene and N-isopropylacrylamide (NIPAM) using 2′-azobis(2-amidinopropane) dihydrochloride (V50) as initiator and carried out at 70 °C. Then, the shot process was performed at 70% and 90% of total conversion of the batch step for DD11 and DD4 latex, respectively. This shot addition consisted of a mixture of NIPAM, aminoethylmethacrylate hydrochloride (AEM) as a cationic functional monomer, V50, and methylene bisacrylamide (MBA) as a cross-linker. 3. Characterization of Latex Microspheres. The two latexes were cleaned by repeated centrifugation and redispersion in deionized water in order to remove water-soluble polymers produced during the polymerization process. Next, the cleaned latexes were characterized in terms of particle size as a function of temperature and electrophoretic mobility as a function of pH and temperature. Particle size was determined using quasi-elastic light scattering (N4 apparatus from Coultronics) and the polydispersity index was estimated from the transmission electron microscopy (TEM) measurements. The electrophoretic mobility as a function of pH was performed in 1 mM NaCl solution at 20 °C using a Zetasizer III from Malvern Instrument. 4. Adsorption of Protein onto Latex Particles. All adsorption experiments were performed using phosphate buffer and low protein binding tubes. The amount of protein adsorbed onto latex particles was determined from the difference between the initial and the final protein concentration in the supernatant. The supernatants were collected after a centrifugation step (10 000 rpm for 10 min at a given temperature). The protein concentration was measured using Bradford titration based on the establishment of a calibration curve and using the well plate analysis method. The pH of the adsorption medium was controlled by the phosphate buffer mixture. The effect of ionic strength was investigated by adding a small volume of concentrated NaCl to a highly diluted phosphate buffer of 10 mM at a given pH. The effect of temperature on the adsorption of protein was performed using the batch process by mixing both protein solution and latex particles in appropriate conditions (pH and salinity) and stirring and incubating the mixture at a selected temperature. Adsorption kinetics of protein onto the latex particles were performed by preparing different samples. After a given incubation time, only one sample was analyzed by determining the adsorbed amount of protein as described above.

III. Results and Discussion 1. Characterization of Latex Particles. Thermosensitive and cationic core-shell latex particles were monodisperse (polydispersity index PDI ∼ 1) with a mean diameter at 20 °C of 551 and 358 nm for DD4 and DD11, respectively. The hydrodynamic diameter of the particles was found to be temperature-dependent according to the (14) Cheynet, V.; Verrier, B.; Mallet, F. Protein Expression Purif. 1993, 4, 367. (15) Duracher, D.; Sauzedde, F.; Elaı¨ssari, A.; Perrin, A.; Pichot, C. Colloid Polym. Sci. 1998, 276, 219.

Figure 1. Diameter of both latexes (DD4 and DD11) as a function of temperature in 1 mM NaCl solution and at pH 6.

Figure 2. Electrophoretic mobility of DD11 and DD4 as a function of pH at 20 °C and in 1 mM NaCl solution.

well-known properties of poly(NIPAM), as reported in Figure 1. The measured particle size for both latexes decreased continuously when the temperature increased from 20 to 50 °C, reflecting a sharp variation close to the LCST (∼32 °C) of poly(NIPAM).16 The magnitude in the volume phase transition can be attributed to the charge distribution in the shell and the thickness layer of the interfacial poly(NIPAM) hydrogel network, as recently reported.17 The electrophoretic mobility of both latexes was measured as a function of pH, and the results obtained are reported in Figure 2. The electrophoretic mobility of the latexes was found to be 0.5 × 10-8 and 2 × 10-8 m2/(V s) below pH 9 and at 20 °C for DD4 and DD11, respectively. The observed behavior in electrophoretic mobility can be attributed to both differences in the charge density and the shell structure such as thickness and volume charge distribution in the hydrogel. The positive electrophoretic mobility found in the case of the two latexes originates both from the functional monomer introduced during the polymerization and from the amidine charges coming from the initiator decomposition. The electrophoretic mobility exhibits a plateau in a large pH range (from pH 3 to 9). Then, a dramatic change is observed around pH 10-11 which corresponds to the (16) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (17) Kim, J. H.; Ballauff. M Colloid Polym. Sci. 1999, 277, 1210.

9004

Langmuir, Vol. 16, No. 23, 2000

Duracher et al.

Table 1. Colloidal Characteristics of Latexes: Particle Size, Hydrogel Thickness Layer, and Charge Density Dh (nm) sample

20 °C

50 °C

DTEM (nm)

δa (nm)

charge density (µmol/g)

DD4 DD11

551 358

380 320

303 303

85 19

14.6 23.7

a Hydrodynamic thickness determined from the difference between Dh (20 °C) and Dh (50 °C).

isoelectric point of the latex particles bearing primary amine groups.18 The negative electrophoretic mobility observed above pH 10 is probably caused by the presence of carboxylic groups derived from hydrolysis of amidine groups.19 On the whole, both latexes exhibit positive and constant electrophoretic mobility below pH 9 and the same curvature. The higher electrophoretic mobility of DD11 compared to that of DD4 can be attributed to the higher charge density (as illustrated in Table 1) and also to the smaller thickness of the poly(NIPAM) shell. In brief, these two thermosensitive latexes were chosen in order to point out the effect of shell structure composition and properties on the adsorption of the modified HIV-1 capsid p24 protein. In fact, both latexes have the same polystyrene core, but DD4 exhibits a thick poly(NIPAM) based shell with a low charge density and DD11 displays a smaller hydrophilic thickness bearing higher charge density. The colloidal characteristics of the latexes are reported in Table 1. Charge density values (σ) were estimated based on a chemical reaction titration method.20 A more detailed study of the electrokinetic and colloidal properties of such core-shell latexes has been recently reported.15,21,22 2. Effect of Temperature on the RH24 Adsorption onto Latex Particles. The effect of temperature on the adsorption of protein onto both latexes was investigated at constant pH ) 6.1 and in 10 mM phosphate buffer. As expected, the adsorption of protein was dramatically influenced by the operating temperature as illustrated in Figure 3a. In fact, there is a large increase in the adsorbed amount of protein when the temperature is increased from 20 to 50 °C. In addition, it is interesting to notice that at low temperatures (i.e., below 25 °C), protein adsorption is almost below 3 mg‚g-1, reflecting that the adsorption is negligible when the particles shell are highly hydrated. In contrast, when the temperature was increased from 25 to 40 °C, the adsorbed amount of protein increased linearly in this LCST region. The same behavior is observed when the amount of adsorbed protein is expressed in mg‚m-2 (as reported in Figure 3b) and the maximum values of the adsorption (above the LCST) are lower compared to a closely packed monolayer (when the equivalent hard spherical particles are considered at a given temperature). The adsorbed amount expressed in mg‚m-2 was calculated by taking into account the surface area developed by latex particles using hydrodynamic particle size at a given adsorption temperature. This adsorption behavior versus temperature reflects the effect of temperature on the hydrophilic-hydrophobic balance of the interface shell of these thermally sensitive core-shell latexes. (18) Sauzedde, F.; Ganachaud, F.; Elaı¨ssari, A.; Pichot, C. J. Appl. Polym. Sci. 1997, 65, 2331. (19) Guthrie, W. H. PhD Thesis, Lehigh University, 1985. (20) Delair, T.; Marguet, V.; Pichot, C.; Mandrand, B. Colloid Polym. Sci. 1994, 272, 962. (21) Duracher, D.; Sauzedde, F.; Elaı¨ssari, A.; Pichot, C.; Nabzar, L. Colloid. Polym. Sci. 1998, 276, 920. (22) Castanheira, E. M. S.; Martinho, J. M. G.; Duracher, D.; Charreyre, M. T.; Elaı¨ssari, A.; Pichot, C. Langmuir 1999, 15, 6712.

Figure 3. (a) Temperature-dependent adsorption of modified HIV-1 capsid p24 protein onto core-shell microspheres at pH 6.1 and 10 mM phosphate buffer. (b) Protein-adsorbed amount (in mg/m2) versus temperature at pH 6.1 and 10 mM phosphate buffer.

Indeed, it is worth mentioning the relevant work of Zhang et al.23 dealing with the contact angle of a water drop onto cross-linked poly(N-isopropylacrylamide) gels as a function of temperature. The authors pointed out that below the LCST, the low contact angle value (40°) is indicative of hydrophilic material (the gel indeed contains approximately 90% water). In contrast, when the gel is heated above the LCST (∼32 °C), the contact angle jumped to 90°, revealing the hydrophobic character of the crosslinked poly(N-isopropylacrylamide) gel above the LCST. Such a variation in the hydrophilic-hydrophobic balance versus temperature may partially explain the temperature dependence of protein adsorption as given in Figure 3. Considering the effect of temperature on the adsorption phenomenon and on the core-shell properties, it seems that hydrophobic interactions are the main forces governing protein adsorption onto such thermosensitive latexes, but it does not exclude contribution of electrostatic interactions. The adsorption of RH24 protein onto such coreshell poly(styrene)-poly(NIPAM) is thus attributed to the change in the shell structure of the poly(NIPAM) shell, which depends on both the temperature and the accessible interfacial adsorption sites. In fact, the increase in temperature also affects the charge density and distribution (23) Zhang, J.; Pelton, R.; Deng, Y. Langmuir 1995, 11, 2301.

Adsorption of Modified HIV-1 Capsid p24 Protein

Langmuir, Vol. 16, No. 23, 2000 9005

Figure 4. Kinetics of RH24 protein adsorption onto coreshell microspheres at 40 °C and 10 mM phosphate buffer, pH 6.1.

in the thermosensitive shell, as evidenced by the electrophoretic mobility variation versus temperature.21,24 To point out the role of electrostatic interactions in this adsorption process, both pH and ionic strength are studied as below presented. 3. Adsorption Kinetics of RH24 onto Latex Particles. The adsorption kinetics of RH24 protein onto both latexes were performed at pH 6.1, close to the IEP of the protein (pH ≈ 5.9), 10 mM sodium phosphate, and at 40 °C. The adsorption kinetics were only investigated above the LCST since the adsorption is more significant than below the LCST as discussed above. The results obtained are given in Figure 4 in which the adsorbed amount of RH24 is reported versus incubation time. Two adsorption domains can be well evidenced. (i) The first part (below 120 min) reflects a linear dependence between the proteinadsorbed amount and incubation time irrespective of latex particles. In addition, this illustrates that the adsorption rate was found to be latex nature independent at the investigated conditions (short adsorption time). (ii) The second domain is observed above 120 min and exhibits a well-defined plateau in the protein adsorption process, corresponding to the adsorption equilibrium state. The plateau value was found to be 56 mg/g (∼3.2 mg‚m-2) and 64 mg/g (∼4 mg‚m-2) for DD11 and DD4, respectively. The difference in the plateau values may be attributed either to the interfacial structure of the particles (distribution of accessible sites) or to the rearrangement of adsorbed protein molecules. 4. Effects of pH on the Maximum Adsorbed Amount of RH24 onto Latex Particles. Adsorption isotherms of RH24 onto both latexes at a constant ionic strength and at various pH values were first carried out in order to demonstrate the effect of protein concentration and pH on the adsorbed amount. Then, the data were treated using the Freundlich adsorption representation with a view to estimating the standard free enthalpies of protein adsorption. The results obtained on the adsorption isotherms of RH24 protein onto DD4 and DD11 latex particles above the LCST are shown in parts a and b of Figure 5, respectively. The adsorbed amounts are strongly dependent on the pH of the medium for both latexes. For adsorption investigated at 20 °C (below LCST) irrespective (24) Nabzar, L.; Duracher, D.; Elaissari, A.; Chauveteau, G.; Pichot, C. Langmuir 1998, 14, 5062.

Figure 5. Adsorption isotherms of RH24 onto DD4 (a) and onto DD11 (b) core-shell microspheres at 40 °C and as a function of pH.

of pH, no marked adsorption was evidenced because of the hydrophilic character of the latex surface as discussed in the temperature effect section. For both latexes, typical adsorption isotherms were obtained and exhibited two marked domains: (i) a rapid increase in the adsorbed amount for low protein concentrations, and (ii) a maximum adsorbed amount corresponding to a plateau value. The plateau values deduced from the adsorption isotherms were found to be pH dependent when the adsorption was investigated at 40 °C (i.e., above the LCST). As depicted in Figure 6, the maximum RH24 adsorbed amount versus the pH exhibits two domains: (i) At pH values lower than pH ∼ 7.5 (which is close to the isoelectric point for the modified protein) the maximum adsorbed amount of RH24 shows a pseudoplateau. (ii) Above pH 7.5, the RH24 adsorbed amount decreases on increasing the pH. If electrostatic interactions between proteins and latex particles were the driving forces in the adsorption process, the maximum amount of protein adsorbed should appear above pH 6 where the proteins and latex particles are oppositely charged as expected, which is not the case. Similar behavior has been reported and discussed by Elgersma et al.5 by investigating the adsorption of bovine serum albumin (BSA) onto positively and negatively charged polystyrene latexes. Briefly, the amount of BSA adsorbed versus pH shows a maximum at the isoelectric point of the protein-coated particles rather than that of the BSA protein itself. The observed behavior has been

9006

Langmuir, Vol. 16, No. 23, 2000

Duracher et al.

Figure 6. pH dependence of RH24 adsorption onto (DD4 and DD11) core-shell microspheres below and above the LCST of poly(NIPAM). Adsorption using phosphate buffer at different pH values, at ionic strength 10 mM, and at adsorption temperatures 20 and 40 °C.

attributed to the ion participation in the protein adsorption process. The decrease in the amount of RH24 adsorbed above pH ∼ 7.5 may also be explained by a decrease in the cationic sites when the pH was increased (at 10 mM salinity concentration), then leading to a reduction in the attractive electrostatic interactions. The initial part of the obtained isotherms (protein adsorbed amount Ns versus bulk protein Cb concentration) reflects a poor linearity when Ns-1 versus Cb-1 representation was plotted, which explains the nonvalidity of the Langmuir isotherm model. Then, an attempt was made to use the Freundlich adsorption representation generally available for low bulk protein concentrations.25,26 The adsorption isotherms are presented as a log-log plot (Figure 7) and discussed using the following empirical power law equation NS ) KiCbRi, where Ki is the affinity constant, Cb is the bulk protein concentration, and Ri (i ) 1, 2) is the exponent of the power law.27 The free energy of adsorption (∆Ga) can be calculated from the determined affinity constant (K1) and the interfacial volume concentration (Ns*/δ) using the following equation (for R1 ≈ 1): Ns*/δ ) KCb*/δ ) Cb* exp(-∆Ga/RT), where Cb* and Ns* are the values corresponding to the change in slope and δ is the thickness of the adsorbed protein layer (δ1 ) 45 Å and δ2 ) 90 Å hypothesis of side-on and end-on adsorption protein conformation, respectively).28 The determined parameters (K1 and R1) and the calculated standard free enthalpy ∆Ga are reported in Table 2. Several points of interest emerge from the above considerations: (i) The K1 constants for Cb < Cb* were found to be nearly the same for both latexes. The observed behavior reflects the noneffective influence of the interface nature on the affinity constant. In addition, the intercept of the initial adsorption values coincides perfectly with the plateau of the adsorption isotherms, showing how easy it is for the protein molecules to approach the vicinity of the latex surface. (ii) The determined R1 exponent values in the low protein concentration domain are close to 1 for DD11 latex, (25) Schmitt, A.; Varoqui, R.; Uniyal, S.; Brash, C.; Pusineri, J. J. Colloid Interface Sci. 1983, 92, 25. (26) De Baillou, N.; Voegel, J. C.; Schmitt, A. J. Colloid Interface Sci. 1986, 16, 271. (27) Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988, 125, 246. (28) Duracher, D. Ph.D. Thesis, Claude Bernard University, 1999.

Figure 7. Freundlich plot adsorption isotherms of RH24 onto DD4 (a) and DD11 (b) at 40 °C and as a function of pH. Table 2. Adsorption Isotherm Parameters of RH24 onto Latexes at 40 °C Assuming a Freundlich Type Modela ∆Ga pH

K1

R1

δ ) 45 Å

4.8 6.1 9.0

8.60 9.70 8.80

0.62 0.60 0.60

DD4 7.3b 7.4b 7.4b

4.8 6.1 9.0

9.00 8.80 7.80

1.04 1.04 0.88

DD11 7.6 7.6 7.3

δ ) 90 Å

R2

6.9b 6.9b 6.9b

0.068 0.060 0.060

6.7 6.9 7.7

0.06 0.003 0.02

a ∆G (kcal/mol) is the standard free enthalpy, K is the affinity a 1 constant, and R1 and R2 are the exponents of the power law. b Calculated from R ) 1. 1

showing that the protein adsorption process is mainly controlled by protein-surface interactions as reported by De Baillou et al.26 The low R1 values (R1 < 1) in the case of DD4 indicate a discard from the Langmuir adsorption model and suggest strong interactions between adsorbed proteins and heterogeneous adsorbent surface at the protein dimension scale. In fact, DD4 exhibits a thicker cross-linked poly(NIPAM) shell compared to DD11 (see Table 1). In the high protein concentration domain, the exponent R2 was found to be close to 0 (for both latexes) reflecting roughly the latex surface saturation or saturation of available adsorption sites.25,26

Adsorption of Modified HIV-1 Capsid p24 Protein

Langmuir, Vol. 16, No. 23, 2000 9007

adsorption mechanism of such a protein (RH24) onto cationic latex supports, the effect of ionic strength was examined above the LCST and at a constant pH (∼6.1). The effect of salt on the adsorption behavior was investigated by diluting the adsorption buffer and by adding a given amount of concentrated NaCl solution. As shown in parts a and b of Figure 8 for DD4 and DD11 latex particles, respectively, the results were obtained by using the plateau value of the adsorption isotherms at 40 °C (above the LCST) since below the LCST the adsorbed amounts are negligible or nil. For both latexes, the amount of protein adsorbed decreases on increasing the ionic strength reflecting the drastic effect of salinity in the adsorption process of proteins onto such thermosensitive latexes. The increase in ionic strength causes a reduction in the attractive electrostatic interactions (screening effect) between low negatively charged protein and positively charged latex particles and, consequently, a decrease in the amount of protein adsorbed onto latex particles. Briefly, the influence of ionic strength on the adsorption of protein onto such thermally sensitive latex particles suggests the contribution of electrostatic interactions in the adsorption process above the LCST when the particle surface exhibits a hydrophobic character. IV. Conclusion

Figure 8. Effects of electrolyte concentration [NaCl] on RH24 adsorption onto DD4 (a) and DD11 (b) core-shell microspheres at pH 6.1 both at 20 and 40˚C.

(iii) The values of standard free enthalpies ∆Ga (≈7 kcal/mol), estimated using a power law (RH24 adsorption for Cb < Cb*) as explained above, are given in Table 2 and found to be in a range similar to that of fibrinogen (10.2 kcal/mol) and albumin (8.4 kcal/mol) adsorption onto negatively charged polystyrene latexes, as reported by De Baillou et al.26 The estimated values of ∆Ga (for DD11) are suggestive of an adsorption mainly governed by hydrophobic interactions. The energies calculated in the case of DD4 latex (by assuming that the exponent R1 ≈ 1) are given for sake of illustration only and cannot be discussed here. (iv) At the surface (Ns*) and bulk (Cb*) concentrations, the protein adsorption process is modified. In fact, the values of Ns* (for given latex and adsorption conditions) correspond to a saturated RH24 layer in the conformation of highest interaction with the considered surface (sideon conformation). Above the transition adsorption state (Ns* and Cb*), the protein-adsorbed amounts are roughly constant (R2 exponents are close to zero) presumably revealing the saturation of the adsorption sites limited by: the possible change in protein conformation (from side-on to end-on adsorption) and the alteration in the surface affinity lead to a modified power law between Ns and bulk Cb. 5. Effect of Ionic Strength on the Maximum Adsorbed Amount of RH24 onto Latex Particles. With a view to obtaining further information on the

The static adsorption of modified HIV-1 capsid p24 protein (named RH24) onto thermosensitive and positively charged core-shell latex particles was examined in view of both electrostatic and hydrophobic interactions by investigating the effect of temperature, pH, adsorption time, and salinity on the protein-adsorbed amount. The following outcomes may be emphasized. The adsorption of RH24 onto such core-shell latexes principally reflects the effect of temperature on the hydrophilic-hydrophobic property of such colloids. The amount of RH24 adsorbed below the LCST (i.e., 20 °C) was negligible due to the hydrophilic character of the shell which retained a large excess of water and also to the cationic charges diluted in the hydrophilic layer. The observed behavior was found to be pH and salinity independent. However, the amount of protein increases on raising the temperature, as expected. The maximum amounts of protein adsorbed onto core-shell latexes observed above LCST were found to be lower compared to a close-packed monolayer as generally observed for protein adsorption onto polystyrene latexes. In addition, the adsorbed amount of RH24 protein onto such positively charged thermosensitive latexes was found to be pH and salinity dependent when the adsorption was investigated above the LCST at which the particles were in a shrunken state and exhibited high surface charge density. The adsorption isotherms above the LCST were discussed on the basis of the empirical power law approach using the Freundlich representation. The standard free enthalpy of adsorption ∆Ga estimated from low protein concentration was found to be close to 7 kcal/mol irrespective of the pH and adsorption conformation used, revealing weak hydrophobic interactions compared to highly hydrophobic supports such as polystyrene latexes. Finally, protein adsorption onto thermally sensitive latex particles was principally controlled by two main factors: (i) changes in the hydration state of each species, protein and adsorbent, and (ii) the electrical double layers of the protein molecules and the adsorbent leading to the alteration of charge distribution. In fact, these two parameters are directly related to the adsorption temperature which monitors the hydrophilic-hydrophobic

9008

Langmuir, Vol. 16, No. 23, 2000

balance and charge density distribution in the interfacial layer. Such behavior will be beneficial for performing both protein purification and covalent binding of protein biomolecules (such as antibodies, enzymes, and antigens) by thermally controlling the adsorption-desorption process.

Duracher et al.

Acknowledgment. The authors acknowledge the Fondation Me´rieux for financial support of David Duracher’s thesis. The authors are also indebted to Dr. V. Cheynet for HIV-1 capsid p24 preparation and purification. LA0004045