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Langmuir 2001, 17, 3734-3740
Reversibility of Structural Rearrangements in Bovine Serum Albumin during Homomolecular Exchange from AgI Particles Tina Vermonden, Carla E. Giacomelli,*,† and Willem Norde Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands Received January 31, 2001. In Final Form: March 24, 2001 The reversibility of the homomolecular exchange of bovine serum albumin (BSA) from AgI particles was studied by differential scanning calorimetry, the binding of 8-anilino-1-naphthalene-sulfonic acid, and circular dichroism spectroscopy. The structure of BSA in solution before adsorption, in the adsorbed state, and in solution after exchange was analyzed. To investigate the influence of electrostatics and surface crowding effects, the experiments were performed at pH 4 and 7, two conditions of charge density at the AgI surface, and three degrees of surface coverage. BSA adsorbed on hydrophobic AgI particles adopts a perturbed state. After release from the surface, the protein aggregates through surface-induced hydrophobic patches as indicated by a decrease in hydrophobicity together with an increase in β-sheet content. This surface-induced aggregation is more pronounced at low pH and when the ratio of adsorbed/dissolved BSA increases but is independent of the AgI surface charge. A lower BSA concentration allows more time for the adsorbed molecules to relax at the sorbent surface. At pH 4, the native BSA is less stable than at pH 7. Both these conditions promote structural changes upon adsorption, which results in released BSA molecules having a different conformation than that of the protein before adsorption.
* Corresponding author: Dr. Carla E. Giacomelli, Laboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands. Tel: +31317-483288. 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.
The adsorption process comprises many steps, starting with the transport of the protein from the solution to the surface, attachment and relaxation at the sorbent surface, and release of the protein from the surface back into the solution.4 The relaxation step, the optimization of the protein molecule-surface interactions that may result in a change in the protein conformation, is held responsible for the possible irreversibility of the exchange process. In general, the relaxation of the adsorbed proteins involves some degree of spreading, which tends to be more extensive on hydrophobic than on hydrophilic surfaces.5 Accordingly, the homomolecular exchange of serum albumin proceeds reversibly from hydrophilic silica particles and irreversibly from hydrophobic polystyrene particles.6 The extent of the relaxation is also modified by the pH and the ionic strength of the solution and by the degree of surface coverage.7 Thus, the reversibility of the exchange process may be controlled by varying these parameters. The aim of this work is to investigate the reversibility of the homomolecular exchange of bovine serum albumin (BSA) from colloidal silver iodide (AgI) particles. This particular system was selected as a model system because the attachment stage and the kinetics of homomolecular exchange have been previously described.8 Furthermore, the BSA-AgI system is pre-eminently suitable for studying the effect of electrostatic interactions in the overall adsorption process. The electrical charge of the protein is determined by the pH, and the surface charge density of AgI is controlled by the concentration of I- (or Ag+) in solution. Hence, the charges on the AgI and the protein can be changed independently. The structure of the BSA
(1) Ball, V.; Schaaf, P.; Voegel, J.-C. In Biopolymers at interfaces; Malmsten, M., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 75, p 453. (2) Slack, S. M.; Horbett, T. A. In Proteins at interfaces II. Fundamentals and applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995; Vol. 602, p 112. (3) Norde, W. In Biopolymers at interfaces; Malmsten, M., Ed.; Surfactant Science Series; Marcel Dekker: New York, 1998; Vol. 75, p 27.
(4) Norde, W. In Physical chemistry of biological interfaces; Baszkin, A., Norde, W., Eds.; Marcel Dekker: New York, 2000; p 115. (5) Norde, W.; Haynes, C. A. In Interfacial phenomena and bioproducts; Brash, J. L., Wojciechowski, P. W., Eds.; Marcel Dekker: New York, 1996; p 123. (6) Norde, W.; Giacomelli, C. E. J. Biotechnol. 2000, 79, 259. (7) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (8) Fraaije, J. G. E. M.; Norde, W.; Lyklema, J. Biophys. Chem. 1991, 41, 263.
1. Introduction The adsorption of proteins onto solid surfaces is a dynamic process during which the adsorbed proteins are continuously being exchanged against proteins in solution. The occurrence of homomolecular and heteromolecular exchange processes has been proven by a large number of experiments1 and appears clearly in the Vroman effect.2 Depending on conditions, proteins more or less change their conformation upon adsorption onto solid surfaces.3 The protein that is released from the surface may either retain the conformation it had in the adsorbed state or (partly) refold into the original structure. From this point of view, if the protein moving back into the solution regains the native structure the exchange process is reversible, but if the conformation of the exchanged protein is different from the original one it is irreversible. The mere occurrence of exchange processes and, moreover, the possibility of proteins in solution having conformations different from the native molecules implies many practical consequences. Examples of such processes include the interaction of whole blood or plasma with surfaces of artificial materials as for example in hemodialysis, proteins released from drug targeting systems, and the purification of proteins by chromatographic methods.
10.1021/la010162o CCC: $20.00 © 2001 American Chemical Society Published on Web 05/17/2001
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before adsorption, while adsorbed, and when released into the solution was analyzed at two pH and pI values and at three different degrees of surface coverage. The protein structure in solution (before adsorption and after being exchanged) was studied by differential scanning calorimetry (DSC), by the binding of 8-anilino-1naphthalene-sulfonic acid (ANS), and by circular dichroism (CD) spectroscopy. These three techniques are complementary. DSC provides information on the thermal stability and ANS binding reflects the surface hydrophobicity of the protein, both on a macroscopic scale. CD allows insight into the secondary structure of the protein, giving information on a molecular level. Thus, by comparison of the structural properties of the protein before adsorption with those after exchange, information is obtained on the reversibility of the homomolecular exchange process. The structure of the protein on the AgI sorbent surface was evaluated by DSC. 2. Materials and Methods 2.1. Materials. BSA was purchased from Sigma (A-7030) and used as received. Acetate buffer was prepared by adding a few drops of CH3COOH to a 10 mM CH3COONa solution to give pH 4. Phosphate buffer pH 7 was obtained by mixing appropriate volumes of 10 mM Na2HPO4 and 10 mM NaH2PO4 solutions. ANS was purchased from Aldrich. The other chemicals were of analytical grade. 2.2. AgI Preparation. AgI sol was prepared following the procedure described by Koopal.9 A portion (500 mL) of 0.15 M AgNO3 was added dropwise to 1 L of 0.0825 M KI in the dark under vigorous stirring. The sol was cleaned by dialysis against water for 2 or 3 days until the conductivity of the dialysate was less than 10 µS. After that, the sol was aged at 80 °C for 3 days, filtered through glass wool to separate coagulated particles from the sol, and stored in the dark at pI ∼ 4. The pI was determined using an AgI-covered platinum electrode and a Ag/AgCl reference electrode previously calibrated with KI and AgNO3 solutions. The specific surface area of this sol as determined by methylene blue adsorption was 19.4 m2/g at pI 4.3 and 15.3 m2/g at pI 7.0 (some precipitation occurs at this pI9). 2.3. Adsorption Isotherms. Protein adsorption experiments were performed at pH 4 and 7 and at pI 4.3 and 7.0. These pI and pH values were selected not only to study the electrostatic effect on the adsorption process but also to minimize the Ag+ binding by the protein.10 The adsorption isotherms were carried out by adding different amounts of a BSA stock solution to a given amount of AgI sol (containing ca. 1 m2 surface area) in 10 mL polypropylene centrifuge tubes. The final volume (10 mL) was reached by adding the appropriate buffer solution. The tubes were rotated end-over-end for 16 h at room temperature. The solutions were separated from the solid by centrifugation, and the BSA concentration in solution was determined spectrophotometrically at 278 nm. The amount of protein adsorbed was calculated from mass balance. 2.4. Exchange Experiments. In the exchange experiments, the amount of protein molecules was always in excess of the available sorbent surface. In such a situation, both adsorbed and dissolved BSA molecules that continuously exchange from the one state into the other are present. From the exchange kinetics,8 it follows that after 12 h of incubation essentially all the molecules have been adsorbed, released, and so on. The samples of exchanged BSA were obtained by leaving a protein solution in contact with AgI particles for 16 h under conditions as described for the adsorption isotherms. After incubation, the solid was separated from the solution. The supernatant solutions containing BSA molecules that had been in contact with the sorbent surface and released into the solution were used for subsequent experiments. These samples may contain different populations of protein molecules depending on the number of times they had (9) Koopal, L. K. Ph.D. Thesis, Wageningen University, Wageningen, The Netherlands, 1978. (10) Fraaije, J. G. E. M. Ph.D. Thesis, Wageningen University, Wageningen, The Netherlands, 1987.
Langmuir, Vol. 17, No. 12, 2001 3735 been on and off the surface. As it is not possible to separate these populations, it is not possible to determine to what extent each of them changes conformation. However, by monitoring the thermal stability, secondary structure, and surface hydrophobicity of the protein in the supernatant solutions the average surfaceinduced structural rearrangement (if any) can be established. Three ratios of adsorbed/dissolved protein were employed in order to obtain exchanged BSA from different degrees of surface coverage at both pH and pI values. These ratios were calculated using the initial protein concentration (ci) and the BSA concentration remaining in the supernatant (cBSA) after adsorption. From the difference between ci and cBSA, the adsorbed amount was established, and cBSA provided the fraction of the protein in solution. They are hereafter denoted as low cBSA (ratio ) 0.95, 0.1 mg/mL of BSA in solution), middle cBSA (ratio ) 0.82, 0.4 mg/ml of BSA in solution), and high cBSA (ratio ) 0.65, 1 mg/mL of BSA in solution). 2.5. Differential Scanning Calorimetry. DSC thermograms of BSA adsorbed on AgI were recorded using a Setaram microDSC III calorimeter (Setaram, Caluire, France). Samples of 0.91.5 mg of adsorbed BSA were placed in a 1 mL measurement cell. The reference cell was filled with the same amount of AgI sol. The temperature range of the thermograms was between 20 and 90 °C, scanned at a rate of 0.5 °C/min. The samples of adsorbed BSA contained not more than 5% of the protein in the dissolved state. DSC thermograms of BSA in solution (before adsorption and exchange) were measured using a Microcal VP-DSC microcalorimeter (Microcal Incorporated). BSA solutions in the range 0.41.0 mg/mL were placed in a 0.5 mL measurement cell. The reference cell contained the same amount of appropriate buffer. The temperature range was 20-90 °C with a scan rate of 0.5 °C/min. It was not possible to measure at low cBSA because the BSA concentration was below the detection limit. The thermogram of the sample at pI 4.3 and pH 4 at middle cBSA could not be obtained because the supernatant still was contaminated with AgI particles. The denaturation temperature (Td) and the enthalpy of denaturation (∆dH) were determined using the software provided with the calorimeter. The DSC data reported are the mean values of at least two measurements deviating less than 10% for ∆dH and less than 1 °C for Td. The determination of ∆dH becomes less accurate as the protein concentration decreases; for middle cBSA the deviation is somewhat higher than 10%. 2.6. Circular Dichroism. The CD spectra were recorded on a JASCO spectropolarimeter, model J-715 (JASCO, Tokyo, Japan), using a 0.1 cm quartz cuvette. The wavelength range was between 190 and 260 nm with 0.2 nm resolution, a scan rate of 100 nm/min, and a time constant of 0.125 s. The protein concentration in the samples ranged between 0.05 and 0.2 mg/ mL. CD spectra for samples with a concentration below 0.1 mg/ mL were the average of 32 scans; for the other samples, 16 scans were recorded. All spectra were corrected for buffer background. The change in secondary structure of BSA molecules in the adsorption-desorption cycle was evaluated by comparing the ellipticity of the bands at 222 and 208 nm, representative of the R-helix content, for the protein before adsorption and after having been exchanged. 2.7. Protein Hydrophobicity. Protein surface hydrophobicity was determined using the hydrophobic fluorescent probe, ANS. The hydrophobicity index (HI) was quantified from the slope of the fluorescence intensity of the dye versus the protein concentration at a given pH. The HI can be compared only for samples prepared in the same buffer because of the interference of acetate and phosphate ions on the binding of ANS to BSA.11 Each sample was diluted to give a series of protein concentrations between 0.1 and 0.7 mg/mL or between 0.01 and 0.08 mg/mL. ANS was added to give a final concentration of 40 µM. The fluorescence intensity was recorded on a SPF-500 spectrofluorometer (SLM AMINCO) using an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
3. Results 3.1. Adsorbed BSA. Figure 1 shows the adsorption isotherms (adsorbed amount per unit sorbent surface area, (11) Daniel, E.; Weber, G. Biochemistry 1966, 5, 1893.
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Figure 1. Adsorption isotherms of BSA on AgI particles at (solid squares) pH 4, pI 4.3; (open squares) pH 7, pI 4.3; (solid circles) pH 4, pI 7.0; (open circles) pH 7, pI 7.0. The arrows indicate low cBSA (0.95 adsorbed/dissolved ratio), middle cBSA (0.82 adsorbed/dissolved ratio), and high cBSA (0.65 adsorbed/ dissolved ratio).
ΓBSA, versus protein concentration in solution, cBSA) of BSA adsorbed on hydrophobic AgI particles at pH 4 and 7 and pI 4.3 and 7.0. The arrows in the plot indicate the different protein concentrations at which the exchange experiments were performed. Because the isoelectric point (IEP) of BSA is ca. 4.7, the charge of the protein is negative at pH 7 and slightly positive at pH 4. The AgI surface charge density is independent of the pH, but it becomes more negative as the pI decreases. As judged by the initial slopes of the adsorption isotherms, BSA shows a very high affinity for the hydrophobic AgI particles at the four conditions studied. Electrostatic interactions do not determine the adsorption of BSA on AgI; the driving forces for the process may rather be ascribed to hydrophobic interactions and structural changes of the protein molecules.3,4 Three of the four isotherms show the same plateau value within experimental error. The only condition at which the isotherm has a significantly higher plateau value is pH 4.0 and pI 4.3. The plateau values are much lower than the maximum adsorbed amount that can be accommodated in a close-packed monolayer of native molecules (∼4 mg/ m2).6 This could be due to electrostatic repulsion between the adsorbed BSA molecules and/or to structural relaxation of the protein at the sorbent surface. DSC was the only technique to study the protein molecules in the adsorbed state; the presence of the AgI dispersion does not allow the use of spectroscopic methods because of strong light scattering. Figure 2 displays the thermograms of the BSA before adsorption and of the AgI particles covered with BSA at pH 4 (A) and pH 7 (B) for both pI values. At pH 4, the protein before adsorption showed two broad transitions at 44 and 65 °C, whereas at pH 7 there is only a single transition at 56 °C. The thermograms of the adsorbed protein are much noisier than those of the dissolved protein. However, it is clear
Vermonden et al.
Figure 2. DSC thermograms of BSA adsorbed on AgI at (A) pH 4 and (B) pH 7. The solid line refers to the protein before adsorption; the dashed line refers to BSA adsorbed at pI 7.0, and the dotted line refers to BSA adsorbed at pI 4.3.
from these plots that, under all conditions applied, there is no thermal transition of the protein in the adsorbed state. Although at high temperatures some “peaks” appear, they do not represent any real transition because it was not possible to reproduce them in separate experiments. This noisier signal may be related to aggregate formation among BSA-covered particles because flocculation was observed after the heat treatment. The absence of any thermal transition indicates that the conformation of the protein on the surface is different from that of the BSA before adsorption, that is, the protein is in a perturbed state when adsorbed on AgI. 3.2. Exchanged BSA. Figure 3 displays the thermograms of BSA before adsorption and after being exchanged at pH 4 (A, high cBSA; C, middle cBSA) and pH 7 (B, high cBSA; D, middle cBSA) for both pI values. Table 1 shows the temperatures and the enthalpy values of denaturation, Td and ∆dH, respectively. The thermostability of BSA before adsorption depends on the pH. At pH 4, there are two overlapping transitions at 43 and 65 °C, whereas at pH 7 a single and more cooperative transition is observed at 56 °C with a larger enthalpy of denaturation. The pH dependences of Td and ∆dH are in good agreement with those reported in the literature.12 The thermograms of exchanged BSA at pH 4 show the two transitions as the protein before adsorption but the Td values are shifted to higher temperatures by a few degrees. Although exchanged BSA at pH 4 is more thermostable than the protein before adsorption, there is no change in ∆dH (within experimental error) for the released molecules. (12) Yamasaki, M.; Yano, H.; Aoki, K. Int. J. Biol. Macromol. 1990, 12, 263.
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Langmuir, Vol. 17, No. 12, 2001 3737
Table 1. Thermostability, Secondary Structure, and Hydrophobicity of BSA before Adsorption and after Exchange from AgI Particles low cBSA pH 4 pH 7
a
before exchanged, pI 7.0 exchanged, pI 4.3 before exchanged, pI 7.0 exchanged, pI 4.3
middle cBSA
high cBSA
208/222a
HI, rub
Td (°C)
∆dH (J/g)
208/222a
Td (°C)
∆dH (J/g)
208/222a
HI, rub
1.05 0.93 0.99 1.06 0.96 1.00
1610 230 230 1710 910 910
43/65 47/65
8(1 8(1
55.8 55.5 55.6
10 ( 1 10 ( 2 8(2
1.05 1.05 1.07 1.06 1.07 1.06
43/64 46/67 45/66 55.9 55.3 55.9
8.6 8.4 8.1 11.0 11.5 10.5
1.05 1.06 1.03 1.06 1.04 1.05
1610 1610 1610 1710 1710 1710
208/222, ratio between the ellipticity at 208 and 222 nm. b ru, relative unities.
Figure 3. DSC thermograms of BSA exchanged from AgI at various combinations of pH values and protein concentrations: (A) pH 4, high cBSA; (B) pH 7, high cBSA; (C) pH 4, middle cBSA; (D) pH 7, middle cBSA. The solid line refers to the protein before adsorption; the dashed line refers to BSA exchanged at pI 7.0, and the dotted line refers to BSA exchanged at pI 4.3.
At pH 7, the thermograms of the exchanged BSA are similar to that of the protein before adsorption, that is, neither Td nor ∆dH change. However, the thermal transitions are broader and asymmetric, skewed to the hightemperature side. Indeed, the half-height width of the transition increases from 6 °C for the BSA before adsorption to 10 °C for the exchanged protein. For middle cBSA, the thermal transition not only becomes broader and asymmetric but also has a shoulder at higher temperatures. The lower cooperativity observed after homomolecular exchange at pH 7 and the occurrence of the shoulder in the thermogram suggest different populations of protein molecules: a nativelike fraction and a more thermostable one(s) whose ratio depends on cBSA. As a general trend, the Td of the exchanged protein increases while ∆dH remains essentially unaltered. It indicates that although the released BSA molecules are more (thermally) stable than before adsorption, their enthalpic intramolecular interactions are more or less the same. In Figure 4, the CD spectra of the BSA before adsorption
Figure 4. CD spectra of BSA exchanged from AgI at various combinations of pH and pI values: (A) pH 4, pI 4.3; (B) pH 7, pI 4.3; (C) pH 4, pI 7.0; (D) pH 7, pI 7.0. Squares refer to the protein before adsorption; diamonds refer to low cBSA, triangles refer to middle cBSA, and circles refer to high cBSA.
and after exchange at pH 4 (A, pI 4.3, and C, pI 7) and at pH 7 (B, pI 4.3, and D, pI 7.0) at the three cBSA values are compared. The spectrum of the protein before adsorption shows the two distinct negative minima at 222 and 208 nm and the positive band between 190 and 195 nm as expected for a protein with a high content of R-helix.13,14 For middle and high cBSA, the CD spectra of exchanged BSA molecules resemble those of the protein before adsorption, corroborating the conclusion that their intramolecular interactions are essentially the same as in the native protein. On the other hand, at low cBSA the spectra show a decrease in intensity of the characteristic R-helix bands indicating a loss of this structural element. Furthermore, the two negative bands largely overlap producing an almost single broad minimum together with (13) Woody, R. W. In Circular dichroism. Principles and applications; Nakanishi, K., Berova, N., Woody, R. W., Eds.; VCH Publishers: New York, 1994; p 473. (14) Manavalan, P.; Johnson, W. C., Jr. Nature 1983, 305, 831.
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tion) between exchanged protein molecules or to refolding into a slightly different conformation burying some of the apolar groups. Thus, the homomolecular exchange of BSA from AgI particles proceeds irreversibly. At least, a fraction of the released protein molecules do not regain the conformation they had before adsorption. The conformational change is more pronounced at pH 4 and as the ratio of adsorbed/ dissolved BSA is increased, but it is not affected by the particle surface charge. Following exchange, BSA molecules show (a) lower surface hydrophobicity, (b) a higher content of β-sheet structure, and (c) higher thermostability than the protein before adsorption. BSA released from negatively charged polystyrene particles undergoes similar alterations.6 On the other hand, BSA exchanged from hydrophilic silica surfaces showed the same structural characteristics as the protein before adsorption.6 Therefore, the sorbent surface hydrophobicity seems to be an essential parameter determining the extent of conformational change and, hence, the reversibility of the exchange process. 4. Discussion
Figure 5. Fluorescence intensity of ANS binding vs concentration of BSA exchanged from AgI at various combinations of pH values and protein concentrations: (A) pH 4, high cBSA; (B) pH 7, high cBSA; (C) pH 4, low cBSA; (D) pH 7, low cBSA. Squares refer to the protein before adsorption; circles refer to BSA exchanged at pI 7.0, and triangles refer to BSA exchanged at pI 4.3.
a change of the intensity ratios from 1.05 ( 0.02 for the protein before adsorption and exchanged at high and middle cBSA to about 0.96 for exchanged BSA at low cBSA (Table 1). This type of spectrum, still dominated by the R-helix portion but exhibiting a single broad minimum skewed to 220 nm, is characteristic of proteins in which R-helix segments are mixed with segments of β-sheet.14 Thus, at low cBSA the homomolecular exchange of BSA from AgI particles promotes β-structure formation. ANS has the property of being practically nonfluorescent in water but highly fluorescent when bound to hydrophobic sites (of proteins). An increase in the hydrophobicity of the exterior of the protein leads therefore to an increase in the fluorescence quantum yield of the probe. Figure 5 presents the intensity of the fluorescence emission of ANS as a function of BSA concentration at the four conditions at high and low cBSA. The surface hydrophobicity indices (HI), defined as the slope of these plots, are listed in Table 1. At high cBSA (Figure 5A,B), the HI of BSA does not change under any condition after being exchanged from AgI. Parts C and D of Figure 5 show the fluorescence intensity as a function of the protein concentration at low cBSA obtained at pH 4 and 7, respectively. The exchanged protein molecules show a significant decrease in HI. This reduction is invariant with pI, but it is more pronounced at pH 4. The loss of surface hydrophobicity indicates that in the exchanged BSA there are a smaller number of hydrophobic groups available to bind to ANS. This change may be related to intermolecular interactions (aggrega-
In response to changing conditions, BSA molecules show a large conformational adaptability. Transformations in BSA structure have been described for varying conditions of pH,12,15,16 ionic strength,12,17 temperature,16 pressure,18 and binding of ligands such as lipids and surfactants.17,19 Lowering the pH (
