Micellization and Adsorption Characteristics of CHAPS - American

The adsorption of CHAPS on hydrophobic latex particles was studied at 22 and 36 °C by determining the adsorbed amount and the enthalpy of adsorption...
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Langmuir 2000, 16, 4853-4858

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Micellization and Adsorption Characteristics of CHAPS Carla E. Giacomelli,*,†,‡ Arnouldus W. P. Vermeer,§ and Willem Norde| Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands, and Bayer AG, Central Research AG, ZF-FP Biophysik (Building E41), D-51368 Leverkusen, Germany Received October 18, 1999. In Final Form: February 14, 2000 The adsorption of CHAPS on hydrophobic latex particles was studied at 22 and 36 °C by determining the adsorbed amount and the enthalpy of adsorption. The adsorption process was compared to the micellization of the surfactant. Therefore, the critical micelle concentration (cmc) and the heat of micellization were also determined at both temperatures. From these two quantities the Gibbs energy and the entropy of the process were calculated. The cmc and the heat of micellization are temperature dependent; the cmc increases as the temperature rises, and the heat of micellization goes from positive at 22 °C to negative at 36 °C. There is an entropy-enthalpy compensation in the micellization process, characteristic of hydrophobic interactions. The maximum adsorbed amount is independent of the temperature, while the initial slope of the isotherms is slightly steeper at 22 °C. Although the adsorption process is exothermic at both temperatures, the enthalpy of adsorption is more negative at 36 °C. Since the adsorption process is more favorable at 22 °C, there is a substantial entropy contribution to the overall process, suggesting that hydrophobic interactions are also dominating the adsorption of CHAPS on latex particles. The orientation of the hydroxyl groups in the steroid nucleus of the surfactant is mainly responsible for the aggregation and adsorption behavior of CHAPS. Indeed, the mechanisms of the micellization and the adsorption processes are strongly related: both are driven by hydrophobic interactions between the apolar faces of the CHAPS molecules (micellization) or between the hydrophobic parts of the molecules and the hydrophobic latex particle surface (adsorption).

Introduction CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate) was designed for membrane protein purification in the early 1980s.1 In addition, it is often used for protein solubilization2 and disaggregation3 and in hydrophobic interaction chromatography as an eluting agent to provide selectivity4,5 or to prevent nonspecific adsorption on column materials.6 In most of these studies, CHAPS has been presented as a mild surfactant or as a constituent of a chromatography medium without discussing in much detail its own physicochemical properties or its adsorption behavior. The micellization of CHAPS is, perhaps, the most studied and well-established characteristic of the surfactant. In fact, the critical micelle concentration (cmc) has been studied by different techniques under a wide range of conditions7-11 and some features about the micellization mechanism have emerged. † To whom correspondence should be addressed. Tel: +31-317483288. Fax: +31-317-483777. E-mail: [email protected]. ‡ On leave of absence from the Departamento de Fisicoquı´mica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Co´rdoba, Argentina. § Bayer AG. | Wageningen University.

(1) Hjelmeland, L. M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6368. (2) Chattopadhyay, A.; Harikumar, K. G. FEBS Lett. 1996, 391, 199. (3) Dingley, A. J.; Mackay, J. P.; Chapman, B. E.; Morris, M. B.; Kuchel, P. W.; Hambly, B. D.; King, G. F. J. Biomol. NMR 1995, 6, 321. (4) Buckley, J. J.; Wetlaufer, D. B. J. Chromatogr. 1989, 464, 61. (5) Buckley, J. J.; Wetlaufer, D. B. J. Chromatogr. 1990, 518, 111. (6) Hronowski, L. J. J.; Anastassiades, T. P. Anal. Biochem. 1990, 191, 50. (7) Hjekmeland, L. M.; Nebert, D. W.; Osborne, J. C., Jr. Anal. Biochem. 1983, 130, 72. (8) Chattopadhyay, A.; London, E. Anal. Biochem. 1984, 139, 408. (9) Stark, R. E.; Leff, P. D.; Milheim, S. G.; Kropf, A. J. Phys. Chem. 1984, 88, 6063. (10) Schu¨rholz, T.; Kehne, J.; Gieselmann, A.; Neumann, E. Biochemistry 1992, 31, 5067. (11) Schu¨rholz, T. Biophys. Chem. 1996, 58, 87.

Since it is known that CHAPS can prevent nonspecific protein adsorption and that it has only a mild effect on the protein structure (i.e., it is a nondenaturing surfactant), it may be used as a surface modifier in systems where proteins are adsorbed to change the surface properties for a specific purpose. This kind of surface modification is applied to systems or techniques of commercial and technological importance, such as biosensors, diagnostic tests (e.g., immunoassays), or chromatographic procedures. Therefore, the adsorption behavior of such a modifier and the driving forces involved in the process are important keys in understanding the whole system. The aim of this work is to investigate the adsorption behavior of CHAPS on latex particles that can be used as a sorbent surface for immunoassays. The adsorption of CHAPS was studied at two temperatures by determining not only the adsorbed amount but also the enthalpy of adsorption, which gives a good indication of the forces involved in the process. The micellization of CHAPS is described in this paper as well. Experimental Section Materials. The latex particles were of the core-shell type. The latex sample was kindly donated by Bayer AG, Leverkusen, Germany. The hydrodynamic diameter of the particles, as measured by dynamic light scattering, was 192 ( 3 nm, and the specific surface area was 35 m2/g. The latex particles were characterized by a negative electrokinetic potential that is essentially invariant with pH and ranges from -25 mV in 0.1 M NaCl to -53 mV in 1 × 10-3 M NaCl. CHAPS (Figure 1) was obtained from Calbiochem (purity g98% by TLC). For the NAD-linked 3R-hydroxy steroid dehydrogenase reaction, we used NAD from Boehringer and the enzyme from Sigma. All other chemicals were of analytical grade. The buffer solution

10.1021/la9913708 CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000

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Figure 1. Schematic structure of the CHAPS ((3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)) molecule.

was prepared by mixing appropriate volumes of 10 mM Na2HPO4 and 10 mM NaH2PO4. Determination of the cmc. The cmc of CHAPS was determined by surface tension measurements at 22 and 36 °C. CHAPS solutions (ranging from 0.5 to 20 mM) were prepared in 10 mM buffer solutions of pH 8.1. The surface tensions of the surfactant solutions were determined by the static Wilhelmy plate method. Adsorption Isotherms. Adsorption isotherms were determined at two temperatures (22 and 36 °C) at pH 8.1 (phosphate buffer 10 mM). Different volumes of 32.5 mM CHAPS solution were added to 5 mL of the latex suspension containing the appropriate volume of buffer to reach a final volume of 10 mL. The samples were incubated at the desired temperature during 4.5 h and centrifuged at 50000g for 45 min. The concentration of CHAPS in the supernate was determined as described below, and the adsorbed amount was calculated from the mass balance. CHAPS Determination. The concentration of CHAPS in solution can be determined by the same procedure as used for bile acids, which is based on the quantitative oxidation of the C(3)-hydroxyl group (3R) using the NAD-linked 3R-hydroxy steroid dehydrogenase (3R-HSD) reaction:12 3R-HSD

3R-hydroxy bile acid + NAD 98 3-oxo bile acid + NADH2 The resulting NADH2 may be determined both by UVvisible spectroscopy and by fluorescence. The enzymatic determination was made by adding two reagents to the samples containing CHAPS. The first reagent was prepared by mixing 3 mg of NAD with 10 mL of buffer (0.1 M Na4P2O7/1.0 M hydrazine, 1:1). This buffer was used to give the adequate pH (9.5), and the hydrazine was added to the reaction mixture as a trapping reagent for the reaction product.12 The second reagent was a 1 mg/mL solution of the enzyme in 0.03 M Tris-EDTA buffer. The enzymatic reaction was completed after 15 min at room temperature. The concentration of CHAPS was determined by UV-visible spectroscopy in the 2002000 µM range or by fluorimetry in the 10-500 µM range. Isothermal Titration Microcalorimetry. Isothermal titration calorimetry (ITC) was carried out in an LKB Instruments model 2277 thermal activity monitor (TAM). The TAM permits titration of the sample with a single titrant solution. All samples were continuously mixed with a propeller mixer during measurements. The titration cell was filled with 1 mL of the latex suspension and 0.9 mL of buffer, pH 8.1. The titrant solution was 32.5 mM CHAPS, and the titration was performed by adding 10 µL (12) Iwata, T.; Yamasaki, K. J. Biochem. 1964, 56, 424.

Figure 2. Surface tension measurements as a function of CHAPS concentration carried out at 22 °C (squares) and 36 °C (circles).

aliquots of this solution to the titration cell. The reference cell contained 2 mL of deionized water. The calorimetric output signal was integrated to yield the heat effect by using the TAM software. The experiments in solution, i.e., in the absence of latex particles, were performed in a Microcal ITC apparatus. The titrant solution was 32.5 mM CHAPS. The calorimeter, although more sensitive than the TAM, allows only titrations of solution because the cells cannot be removed, which obstructs cleaning from adhered latex particles. The two calorimeters give results that differ only within a few percent. Results Figure 2 shows the surface tension (γ) of CHAPS solutions plotted against the logarithm of CHAPS concentration at 22 and 36 °C. The cmc values were obtained from the sharp break in these curves. The cmc increases as the temperature increases, from 4.6 mM at 22 °C to 7.1 mM at 36 °C. Comparison of these values with those reported in the literature is not straightforward because the cmc of a surfactant is affected by a number of conditions such as pH, ionic strength, temperature, type of medium, etc. Moreover, somewhat different values for the cmc may originate from using different experimental methods. Surveying the literature reveals that the cmc of CHAPS ranges between 3 and 10 mM, depending on the conditions and the applied techniques.7-11 At conditions close to the ones in this work, a cmc value of 8.2 mM is reported at 30 °C4 and values in the range 3-4 mM are reported at room temperature;10 these data are in fair agreement with the results obtained here. Adsorption isotherms, i.e., plots of the adsorbed amount of CHAPS (ΓCHAPS) vs the equilibrium concentration of

Micellization and Adsorption Characteristics of CHAPS

Figure 3. Adsorption isotherms of CHAPS adsorbed on latex particles at pH 8.1 recorded at 22 °C (squares) and 36 °C (circles).

CHAPS in solution (cCHAPS), are presented in Figure 3, for both 22 and 36 °C. Because the adsorbed amount was measured by the depletion method, the error associated with its determination becomes larger as the difference between the concentrations of CHAPS in solution before and after adsorption decreases. Indeed, the points in the first part of the isotherms are of the same size as the error bars, while in the plateau region the experimental error is larger. The inset in Figure 3 shows in more detail the initial part of the isotherms. The adsorption isotherms of CHAPS have the same plateau value (0.85 µmol/m2) at both temperatures but the initial slope is steeper at 22 °C. Figure 4 gives ITC results, namely, the heats of titration of the latex particles with CHAPS (qt is the heat per titration step) as a function of the amount of CHAPS added, measured at 22 and 36 °C. For both temperatures, qt is large and negative (exothermic) during the first part of the titration, and it becomes constant and small in the last part of the titration. At 22 °C, qt remains negative, but at 36 °C, it attains slightly positive (endothermic) values in the last trajectory of the titration. Because the titration was performed with a concentrated CHAPS solution (32.5 mM, which is beyond the cmc), the measured heat is the sum of the heat of dilution (qdil), the heat of demicellization (qdem), and the heat of adsorption (qads). qdil and qdem were obtained by titrating 32.5 mM CHAPS solution into the buffer under the same conditions as in the adsorption experiment. When the micellar CHAPS solution is added to the buffer, the measured heat is due to demicellization and dilution. During this titration the surfactant concentration in the cell increases up to the cmc. If the titration is continued after reaching the cmc in the cell, no further demicellization takes place and only dilution of the micelles occurs. The general trend is that

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Figure 4. Total heats per titration step as a function of the initial added amount of CHAPS at 22 °C (squares) and 36 °C (circles).

a more or less constant heat value is observed for each titration step in the premicellar region (related to the breaking of the micelles and to the dilution effect) and a different, but also constant, heat effect is observed in the postmicellar region (determined only by the dilution of the micelles). The difference between these constant values corresponds to the heat of micellization.13,14 Hence, from such a titration curve, the cmc and the heats of dilution and micellization of the surfactant can be determined. At constant pressure, which is the case in our experiments, these heats equal the respective enthalpy effects ∆Hdil and ∆Hmic. Figure 5 shows the heat of titrating a concentrated CHAPS solution into the buffer at pH 8.1 as a function of CHAPS concentration, at 22 and 36 °C. Table 1 summarizes the values derived for ∆Hdil and ∆Hmic at both temperatures. It is noted that the micellization of CHAPS is endothermic at 22 °C and exothermic at 36 °C. The plot of the cumulative heat, q(cum), against the concentration of the surfactant (Figure 6) shows a discontinuity in the slope in going from qdil + qdem to qdil, at cCHAPS ) cmc. The values thus estimated are in the ranges 7-8 mM at 36 °C and 3-5 mM at 22 °C, which are in line with the results obtained from surface tension measurements. In our experiments, the concentration of CHAPS after adsorption is never higher than 1 mM (see Figure 3), much smaller than the CHAPS concentration in Figure 5. Then, for a given temperature the correction that has to be performed to qt (Figure 4) in order to obtain qads is constant (13) van Os, N. M.; Daane, G. J.; Haandrikman, G. J. Colloid Interface Sci. 1991, 141, 199. (14) Mehrian, T.; de Keizer, A.; Korteweg, A. J.; Lyklema, J. Colloids Surf., A 1993, 71, 255.

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Figure 5. Enthalpies of dilution and demicellization as a function of CHAPS concentration at 22 °C (squares) and 36 °C (circles). Table 1. Thermodynamic Data and Cmc Values for CHAPS in 10 MM Phosphate Buffer, pH 8.1 T (°C)

cmc (mM)

∆Hdil (kJ/mol)

∆Hmic (kJ/mol)

∆G°mic (kJ/mol)

T∆Smic (kJ/mol)

22 36

4.6 7.1

0.1 0.5

0.4 -1.6

-23.0 -22.9

23.4 21.3

for the whole adsorption range. The value and the sign of this constant depend on the temperature; it is negative at 22 °C and positive at 36 °C, as seen in the last part of the titration curve depicted in Figure 4. When the heat of adsorption is obtained, the molar enthalpies of adsorption (∆Hads) as a function of surface coverage can be calculated if the adsorbed amount of CHAPS is known for every titration step. It is not possible to directly measure the adsorbed amount in the calorimeter cell, but from the adsorption isotherms, ΓCHAPS can be calculated for every step in the calorimetric experiment. Figure 7 shows the cumulative heats of adsorption, qads(cum), as a function of the adsorbed amount of CHAPS at 22 and 36 °C. ∆Hads values, expressed in J/mol, follow from the slopes of these plots. The adsorption process is exothermic at both temperatures. There is a gradual change in ∆Hads to less exothermic values as the adsorbed amount increases; i.e., the adsorption is (enthalpicaly) less favorable as the surface becomes more crowded. Discussion CHAPS is a sulfobetaine derivative of cholic acid. Its hydrophilic group, although zwitterionic, behaves essentially as a nonionic compound. It does not have a net charge at any pH between 2 and 12, it exhibits no conductivity or electrophoretic mobility, and it does not bind to ion-exchange resins.1 Its hydrophobic group is the

Figure 6. Cmc calculations from calorimetric data at (A) 22 °C and (B) 36 °C.

same as that of cholic acid, i.e., a trihydroxy bile acid.15 Bile acids are rigid molecules, shaped like flattened ellipsoids possessing dissimilar sides whose properties depend mainly on the orientations of the hydroxyl groups in the steroid nuclei. Cholic acid and, hence, CHAPS have the 3-hydroxyl groups at the same side of the molecule (in the R configuration), forming a triangle in the R face of the molecule. The particular distribution of the hydroxyl groups of the steroid nucleus gives to the molecule one water-soluble and one lipophilic water-insoluble side. These hydrophobic and hydrophilic faces are responsible for the aggregation behavior and surface configuration at an air-water interface of 3-R-hydroxyl bile acids. In aggregate formation, the hydrophobic faces are considered to contact each other, while the hydrophilic ones remain exposed to the aqueous environment.15,16 In the air-water interface, the R side is in contact with water, while the other one faces the air.15 The R configuration of the three hydroxyl groups determines these processes, so they are almost independent of the polar side chain. For instance, when a hydroxyl substituent is changed from an R to a β conformation, the cmc goes from 13 (cholic acid) to 60 mM (ursocholic acid), while amidation of the side chain of cholic acid with glycine or taurine only causes a decrease from 13 to 12 or 10 mM, respectively.16 In line with these arguments, the cmc of CHAPS is within the range of the conjugate cholic acid compounds. (15) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, London, 1971; Vol. 1, p 249. (16) Roda, A.; Hofmann, A. F.; Mysels, K. J. J. Biol. Chem. 1983, 258, 6362.

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The calorimetric titration of a micellar surfactant solution into a buffer directly provides the enthalpy of micelle formation (∆Hmic). The standard Gibbs energy (∆H°mic) of the process can only be be calculated if a model for micelle formation is assumed. For that purpose, the mass action approach was adopted.14,17,18 This model has been used for other surfactants, including some of the bile salts.15 The values for the standard entropy of micellization, ∆S°mic, is derived from ∆Gomic) ∆H°mic - T∆S°mic. ∆Hmic as obtained by calorimetry was assumed to be equal to the standard enthalpy (∆H°mic) because the experiments were carried out with dilute solutions. The values of ∆G°mic and ∆S°mic, thus obtained are included in Table 1 for both temperatures. The enthalpy of CHAPS micellization goes from negative at 36 °C to positive at 22 °C. The ensuing negative value for (∆Cp)mic (≡∂∆Hmic/∂T) is a strong indication of the contribution from hydrophobic bonding to the micelle formation. At lower temperatures, the interaction between the water molecules surrounding the apolar compound is stronger and more heat is required to break that structure. However, as the temperature increases, that structure is less rigid and the process becomes less endothermic or, for that matter, more exothermic. This effect is also reflected in the temperature dependence of ∆S°mic: there is a larger gain in entropy at lower temperature because the water molecules released from contact with the apolar face of the surfactant molecules are more structured. Thus, the temperature dependence of ∆H°mic is compensated by

the temperature dependence of the T∆S°mic term, resulting in a ∆G°mic that is almost invariant with temperature. Such an enthalpy-entropy compensation was also observed for the micellization of sodium taurocholate (NaTC), the taurine conjugate of cholic acid. Moreover, the (∆Cp)mic values are almost the same for both surfactants, i.e., -0.15 kJ/(mol K) for CHAPS and -0.14 kJ/(mol K) for NaTC,15 reinforcing the idea that the micellization process is mainly determined by the steroid nucleus and almost independent of the polar group. The adsorption isotherms of CHAPS on the latex particles show the same plateau values at both temperatures but a slightly steeper initial slope at 22 °C (Figure 3). The plateau value is primarily determined by the orientation and intermolecular (lateral) interaction of the molecules at the surface. Long-range lateral interactions between the CHAPS molecules are not expected because CHAPS is essentially noncharged. It may, therefore, be concluded that CHAPS molecules adsorb in the same orientation and conformation at both temperatures. The initial part of the isotherm reflects the affinity of the CHAPS molecules for the latex surface. The higher affinity at the lower temperature implies an exothermic adsorption process. The adsorption of CHAPS onto the hydrophobic latex particles may occur similarly to the adsorption of 3-R-hydroxyl bile acids at the air-water interface. Cholic acid molecules adsorb flat at the interface with the hydroxyl and the carboxyl groups in the water,15 occupying an area per molecule in the range 1-2 nm2 depending on the surface pressure of the monolayer. The plateau value of the adsorption isotherm of CHAPS adsorbing onto latex particles is 0.85 µmol/m2, i.e., 1.9 nm2/molecule, suggesting that the conformation of CHAPS molecules on the latex particles does not deviate largely from that of a cholic acid monolayer at the air-water interface. It is then expected that, on the hydrophobic latex particles, CHAPS adsorbs with its β face toward the surface and the hydroxyl and sulfobetaine groups toward the solution, suggesting that hydrophobic interactions are dominating the adsorption process. Furthermore, displacement of fibrinogen by CHAPS is more efficient at a hydrophobic surface than at a hydrophilic surface,19 CHAPS prevents nonspecific adsorption of glucocorticoid receptors onto hydrophobic interaction chromatography material,20 and CHAPS competes with some proteins for binding sites on the stationary phase in hydrophobic interaction chromatography.4 Interaction between the hydrophobic face of CHAPS molecules and the hydrophobic surface may be decisive for these phenomena. This particular orientation of CHAPS molecules on the hydrophobic latex particles renders the interface more hydrophilic, and as proteins generally have a higher affinity for hydrophobic surfaces than hydrophilic ones, preadsorption of CHAPS on hydrophobic surfaces may reduce or prevent nonspecific protein adsorption. The adsorption of CHAPS on the latex particles is exothermic at both temperatures with a gradual change to less negative values for the molar adsorption enthalpy as the adsorbed amount increases (Figure 7). This trend suggests either that, at higher surface coverage, the adsorption takes place at energetically less favorable sites or that there is an increased repulsion between adsorbed molecules. As CHAPS is essentially uncharged at any pH between 2 and 12, repulsion between adsorbed molecules cannot be of electrostatic nature.

(17) Couper, A. In Surfactants; Tadros, Th. F., Ed.; Academic Press: London, 1984; p 19. (18) Zana, R. In Cationic Surfactants. Physical Chemistry; Rubingh, D. N., Holland, P. M., Eds.; Marcel Decker, Inc.: New York, 1991; p 41.

(19) Welin-Klintstro¨m, S.; Askendal, A.; Elwing, H. J. Colloid Interface Sci. 1993, 158, 188. (20) Warren, B. S.; Kusk, P.; Wolford, R. G.; Hager, G. L. J. Biol. Chem. 1996, 271, 11434.

Figure 7. Cumulative heats of adsorption as a function of the adsorbed amount of CHAPS at 22 °C (squares) and 36 °C (circles).

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∆Hads is more negative at 36 °C than at 22 °C, but as inferred from the initial part of the isotherms, the affinity of CHAPS for the surface is higher and, hence, the Gibbs energy of the adsorption process (∆Gads) is more negative at 22 °C. Therefore, even when the adsorption is enthalpically favorable, there is a nonnegligible entropy contribution to the overall process, which is larger at 22 °C. This, together with the temperature dependence of ∆Hads, is another strong indication of a major role for hydrophobic interactions in the adsorption process. As discussed above for the micellization process, the shift of the enthalpy to more negative values and of the entropy to more positive ones as the temperature increases is due to the release of structured water adjacent to an apolar (hydrophobic)

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substance (surface). Indeed, the mechanisms of the micellization and the adsorption processes are strongly related: both are driven by hydrophobic interaction between the apolar faces of the CHAPS molecules (micellization) or between the hydrophobic parts of the molecules and the hydrophobic latex particle surface (adsorption). Acknowledgment. This work was made possible by the financial support of Bayer AG, Leverkusen, Germany. We thank Ir. J. Goossens and Dr. K. Sievert from Bayer AG for helpful discussions. LA9913708