Removal of T4 Lysozyme from Silicon Oxide Surfaces by Sodium

Dec 1, 1996 - proteins were investigated, wild type T4 lysozyme and a mutant protein ... at a somewhat slower rate than wild type protein, but the ads...
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Langmuir 1997, 13, 8-13

Articles Removal of T4 Lysozyme from Silicon Oxide Surfaces by Sodium Dodecyl Sulfate: A Comparison between Wild Type Protein and a Mutant with Lower Thermal Stability Marie Wahlgren* and Thomas Arnebrant Department of Food Technology, University of Lund, P.O. Box 124, 221 00 Lund, Sweden Received February 26, 1996. In Final Form: September 16, 1996X The adsorption and surfactant induced removal of T4 lysozyme was studied by in situ ellipsometry. Two proteins were investigated, wild type T4 lysozyme and a mutant protein where isoleucine 3 had been replaced by tryptophan (tryptophan mutant). The amount adsorbed is higher for the wild type protein than for the tryptophan mutant at the protein concentration employed (0.2 mg/mL). Furthermore, the adsorption kinetics differ between the two proteins, and the tryptophan mutant initially seems to adsorb at a somewhat slower rate than wild type protein, but the adsorbed amount levels off faster. Sodium dodecyl sulfate removes adsorbed proteins of both types from the silicon oxide surface, provided the surfactant concentration is high enough. The surfactant concentration needed to start removal was found to be well below cmc (3-11% of cmc in solution). The effect of ionic strength on this critical concentration follows the trends expected for surfactant aggregation and is not what would be expected for electrostatic interaction between protein and oppositely charged surfactant. This means that the removal starts at a lower surfactant concentration when the ionic strength is increased. Thus, the removal is thought to start at the concentration where the surfactant associates cooperatively to the adsorbed protein. The critical concentration for protein removal is higher, and the rate of protein removal is lower, at discrete surfactant concentration for wild type protein than for tryptophan mutant. This indicates that the onset of cooperative binding occurs at lower concentration for the tryptophan mutant, which is believed to be due to its larger changes in the conformation upon adsorption. These conformational changes might facilitate surfactant binding, for example, by exposing hydrophobic groups.

During the past few years we have extensively studied the removal of protein by surfactants,1-10 and a review of this work has recently been presented.11 So far, these studies have mainly concerned systems where the surfactants’ concentrations were above cmc. The degree of surfactant-induced removal of proteins is controlled by a balance between surface, surfactant, and protein properties, and the mechanism of removal is dependent on whether the surfactant can interact with the protein or the surface or both.6 When the surfactant adsorbs to the surface, the removal of protein will in some cases be due to a replacement mechanism and will take place if the surfactant-surface interaction is stronger than the proX Abstract published in Advance ACS Abstracts, December 1, 1996.

(1) Wahlgren, M.; Arnebrant, T. Colloids Surf. B 1996, 6, 63. (2) Wahlgren, M. C.; Paulsson, M. A.; Arnebrant, T. Colloids Surf. A 1993, 70, 139. (3) Wahlgren, M.; Arnebrant, T.; Askendal, A.; Welin-Klintstro¨m, S. Colloids Surf. A 1993, 70, 151. (4) Wahlgren, M. Adsorption of Proteins and Interaction with Surfactants at Solid/Liquid Interfaces; University of Lund: Sweden, 1992. (5) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1992, 148, 201. (6) Wahlgren, M. C.; Arnebrant, T. J. Colloid Interface Sci. 1991, 142, 503. (7) Wannerberger, K.; Wahlgren, M.; Arnebrant, T. Colloids Surf. B, 1996, in press. (8) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 193. (9) McGuire, J.; Krisdhasima, V.; Wahlgren, M.; Arnebrant, T. Proteins at Interfaces; Brash, J., Horbett, T., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995; p 52. (10) McGuire, J.; Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1995, 170, 182. (11) Arnebrant, T.; Wahlgren, M. Proteins at Interfaces; Brash, J., Horbett, T., Eds.; ACS Symposium Series 602; American Chemical Society: Washington DC, 1995; p 239.

S0743-7463(96)00171-0 CCC: $14.00

tein-surface interaction. Recently, we have investigated the removal of adsorbed lysozyme at methylated silica surfaces, as a function of surfactant concentration (above and below cmc), for pentaethylene II glycol mono ndodecylether. In this system the removal is likely to be an effect of replacing the adsorbed protein with surfactant, and a strong correlation was found between the increase in removal of protein and the adsorption of surfactant to clean surface.1 In cases where surfactant only binds to the protein, the mechanism must be different, and it has been suggested that it is due to a solubilization of the protein.6 Examples of such systems are those that remove lysozyme from silica and mica by sodium dodecyl sulfate (SDS).6,12 Surfactant and protein interactions in solution have been reviewed recently by Ananthapadmanabhan.13 SDS is known to extensively bind to most proteins. Its binding isotherm usually contains one region at low surfactant concentration where the surfactant binds to the protein at a specific site, such as a charged amino acid group or a hydrophobic pocket and one region where the surfactant binds in a cooperative mode at a critical concentration, likely to involve self-association of the surfactants. The latter is similar for most proteins and is accompanied by structural changes in the protein and some type of aggregate formation of the surfactant, while the first one is strongly dependent on protein type.13,14 Lysozyme, which is positively charged at pH 7, binds SDS, which in the first phase results in a precipitation of the complex. (12) Blomberg, E. Surface Force Studies of Adsorbed Proteins; Royal Institute of Technology: Stockholm, Sweden, 1993. (13) Ananthapadmanabhan, K. P. In Interactions of Surfactants with Polymers and Proteins, Ananthapadmanabhan, K. P., Goddard, E. D., Eds.; CRC Press: Boca Raton, FL, 1993; p 319. (14) Jones, M. M. Biochem. J. 1975, 151, 109.

© 1997 American Chemical Society

Removal of T4 Lysozyme from Silicon Oxide Surfaces

This complex is dissolved upon further addition of surfactant.15 It is of interest to know in which of these regions the surfactant-induced removal of proteins starts. Increase in electrostatic screening by addition of electrolyte affects the two regions differently and may be used to distinguish between them.15 Surfactant does not usually remove all of the adsorbed protein, and the amount removed seems to be strongly dependent on the character of the proteins. Thus, provided that the surfaces investigated are similar enough and that the mechanism of removal is the same, the degree of removal (elutability) can be used to describe the binding strength of proteins to a surface.2,8-10,16-22 This approach has, for example, been used to characterize the blood compatibility of different biomaterials.16-22 These studies have mainly been above cmc, where the amount of protein removed by surfactant usually has reached a plateau. Usually there are several properties, such as size, charge, hydrophobicity, etc., that differ between two proteins. One better way to control which property influences the surface behavior is to use mutant proteins. In this work we have used lysozyme from phage T4. The two proteins investigated are wild type T4 lysozyme and a mutant where isoleucine 3 has been substituted by tryptophan (tryptophan mutant). The substitution does not seem to change the structure of the protein, but it is known to decrease its heat stability in solution to 2.8 kcal/ mol lower than that of the wild type protein.23 The heat stability was in this case defined as the difference in Gibbs free energy for unfolding between wild type and mutant protein at the melting temperature of the wild type protein and at pH 6.5. The mutant also shows larger conformational changes upon adsorption compared to wild type protein, as indicated by decrease in secondary structure24 and change in molecular dimensions.25 Materials and Methods Wild type T4 lysozyme and tryptophan mutant were kindly provided by Joseph McGuire, Oregon State University, Corvallis. They were produced from transformed cultures of Escherichia coli strain RR1 according to established methods.26-28 The sodium dodecyl sulfate (Fluka Chemi AG, no. 71727) was recrystallized three times from a water solution prior to use. The water used was distilled, passed through an ion exchanger and active charcoal (Millipore Corporation, Bedford, MA), and then distilled twice in a glass still. All chemicals were of analytical grade. The silica surfaces, from Okmetic OY, Finland (resistivity 1-20 Ω cm (100) orientation, type P, boron doped), were kindly provided by Stefan Welin-Klintstro¨m, Linko¨ping, Sweden, and (15) Jones, M. N.; Manley, P. In Surfactant Solution, Mittal, K. L., Lindman, B., Eds.; Plenum Press: New York, 1984; Vol. 2, p 1403. (16) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989, 133, 148. (17) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1988, 124, 535. (18) Bohnert, J. L.; Horbett, T. A. J. Colloid Interface Sci. 1986, 111, 363. (19) Ertel, S. I.; Ratner, B. D.; Horbett, T. A. J. Colloid Interface Sci. 1991, 147, 433. (20) Rapoza, R. J.; Horbett, T. A. J. Biomater. Sci., Polymer Ed. 1989, 1, 99. (21) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480. (22) Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1990, 24, 1263. (23) Matsumura, M.; Becktel, W. J.; Matthews, B. W. Nature 1988, 334, 406. (24) Billsten, P.; Wahlgren, M.; Arnebrant, T.; McGuire, J.; Elwing, H. J. Colloid Interface Sci. 1995, 175, 77. (25) Fro¨berg, J. C.; Arnebrant, A.; McGuire, J.; Claesson, P. M. 1996, in press. (26) Alber, T. Methods Enzymol. 1987, 154, 511. (27) Poteete, A. R.; Dao-pin, S.; Nicholson, H.; Matthews, B. W. Biochemistry 1990. (28) Muchmore, D. C.; McIntoch, L. P.; Russel, C. B.; Anderson, D. E.; Dahlquist, F. W. Methods Enzymol. 1989, 177, 44.

Langmuir, Vol. 13, No. 1, 1997 9 were prepared as described in ref 29 to give a 300 Å thick oxide layer. The surfaces were cleaned, at 80 °C, for 5 min with NH4OH:H2O2:H2O (1:1:5) (v:v:v) and HCl:H2O2:H2O (1:1:5) (v:v:v), respectively, and then were washed with water and ethanol.29 The surfaces were stored in ethanol, and before use they were treated for 5 min in low-pressure air plasma (0.2-0.3 Torr) using an rf glow discharge unit (Harrick PDC 3 XG, Harrick Scientific Corp., Ossining, NY). The adsorbed amount was measured using in situ nullellipsometry, a technique that is based on the change of polarized light upon reflection in an interface.30 This gives the ellipsometric angles ∆ and ψ from which the refractive index and the thickness of the adsorbed layer were calculated according to a three-phase model (surface, adsorbate, and ambient)31 using the measured pseudo-refractive index for the silicon with the oxide layer. The amount adsorbed was calculated according to Cuypers et al.32 using 0.75 mL/g and 4.1 g/mL as the values for the partial specific volume and the ratio between molar mass and molar refractivity, respectively (eq 1).

Γ)

3d1f(n1)

(nf - nm) Ad n2m - 1 - vd 2 Md nm + 2 f)

(1)

(nf + nm) (n2m

+ 2)(n2f + 2)

where Γ ) adsorbed amount, nm ) refractive index of the medium, nf ) refractive index of the film, df ) film thickness, A ) molar refractivity, M ) molar mass, and v ) partial specific volume. The instrument used was a Rudolph thin film ellipsometry type 43603-200E. It had been automated by adding stepping motors to the polarizer and analyzer which are controlled by a personal computer. The ellipsometric angles (∆ and ψ) were determined from the settings of polarizer and analyzer that gave a minimum in light intensity. The minima were obtained by measuring the light intensity for different positions of either polarizer or analyzer, keeping the other component fixed, and the obtained data were then fitted to a second-degree polynomial. The experiment was performed in a cuvette using 4.5 mL of saline solution to which 0.5 mL of the sample solution was added. The saline solutions used were 0.01 and 0.1 M NaCl. The temperature was 25 ( 0.1 °C. The pH was around 5.6, but as no buffer was used, it varied slightly between 5.5 and 5.7. The system was stirred by a magnetic stirrer at a rate of 325 rpm. After the pseudo-refractive index of the bare surface had been measured, the experiment was started by addition of the sample. The adsorption was carried out for 60 min, after which the cuvette was rinsed at a flow rate of 20 mL/min with saline solution for 5 min. Desorption was then monitored for an additional 10 min after which surfactant solution was added. The surfactant concentration in the cuvette was, unless stated otherwise, initially 1/ 100 of cmc in water (80 µM) and was then increased every 15 min with an additional 1/100 cmc. Before a final rinse of the cuvette, the surfactant concentration was increased to cmc to investigate whether or not all of the protein could be removed.

Results and Discussion The adsorption of the wild type T4 lysozyme and tryptophan mutant and the effects of sequential addition of SDS to adsorbed proteins are shown in Figures 1 and 2. The adsorption of the two proteins differs with respect to adsorbed amounts, adsorption rate, and salt dependence. The tryptophan mutant was found to adsorb in (29) Wahlgren, M.; Arnebrant, T. J. Colloid Interface Sci. 1990, 136, 259. (30) Azzam, R. M. A.; Bashara, N. M., Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1977. (31) McCrackin, F. L. Technical Note 478; U.S. Department of Commerce, National Bureau of Standards: Washington, DC, 1969. (32) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. C. J. Biol. Chem. 1983, 258, 2426. (33) Wahlgren, M.; Arnebrant, T.; Lundstro¨m, I. J. Colloid Interface Sci. 1995, 175, 506.

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Wahlgren and Arnebrant Table 1

adsorbed amount after 60 min adsorbed amount after rinsing with buffer start of removal between % of cmc in watera aValues

wild type protein, 0.1 M NaCl

tryptophan, 0.1 M NaCl

wild type protein, 0.01 M NaCl

tryptophan, 0.01 M NaCl

2.2 ( 0.16 (n ) 10) 1.86 ( 0.24 2-3 (10.7-16)

1.1 ( 0.21 (n ) 8) 0.74 ( 0.19 1-2 (5.33-10.7)

2.3 ( 0.24 (n ) 3) 1.95 ( 0.29 5-6 (6.7-8)

1.3 ( 0.11 (n ) 3) 0.91 ( 0.18 2-3 (2.7-5.3)

in parentheses in % of cmc corresponding to the NaCl concentration used.

Figure 1. Adsorption of 0.2 mg/mL wild type protein of T4 lysozyme (open circle) and tryptophan mutant (isoleucin 3 f tryptophan) (open diamond) to silicon oxide surfaces from a 0.1 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (rinsing is marked with closed arrow), and after 75 min, SDS (1/100 of cmc in water, open arrow) is added. After this, sequential additions of SDS are made every 15 min (195 min total) when surfactant concentration is increased to cmc in water (black arrow). A final rinse is performed 15 min after the last addition of surfactant. The temperature was 25 °C.

Figure 2. Adsorption of 0.2 mg/mL wild type protein of T4 lysozyme (open circle) and tryptophan mutant (isoleucin 3 f tryptophan) (open diamond) to silicon oxide surfaces from a 0.01 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (rinsing is marked with closed arrow), and after 75 min SDS (1/100 of cmc in water, open arrow) is added. After this, sequential additions of SDS are made every 15 min (255 min total) for wild type protein and for tryptophan mutant (225 min total) until surfactant concentration is increased to cmc in water (black arrow). A final rinse is performed 15 min after the last addition of surfactant. The temperature was 25 °C.

lower amounts than wild type protein (Table 1). This is in line with the findings by McGuire et al.10 who observed similar differences in behavior for the two proteins at pH 7 and at a higher protein concentration. The differences in adsorption kinetics for wild type protein and tryptophan mutant are also in agreement with these earlier investigations.10 The tryptophan mutant seems initially to adsorb somewhat slower than wild type protein, but the adsorbed amount levels off faster. Although neither of the proteins seems to reach their plateau levels in adsorbed amount, after 1 h of adsorption the increase in adsorbed

amount is higher for wild type protein than for tryptophan mutant. In all cases some of the protein is removed upon rinsing with saline solution. The amount of protein removed was similar for both proteins, see Table 1. However, the amount of wild type protein adsorbed was larger than that tryptophan, and thus the fraction removed becomes slightly lower, around 20% for the wild type and 30% for the mutant. It could be noted that the desorption of tryptophan mutant is initially slightly faster than that of wild type protein, but it also levels off faster. The large difference in adsorption behavior is interesting, considering that the proteins only differ in one amino acid. On the basis of x-ray studies of other similar T4 lysozyme mutants, it is likely that the structures of the mutant and wild type proteins are comparable in solution. Furthermore, the two proteins have very similar circular dichroism spectra in the far UV,24 indicating an identical secondary structure. Consequently, the maximal amount of protein adsorbed in a monolayer could theoretically be the same for both proteins and the expected value for hexagonal close-packed layers of proteins are 3.96 and 2.05 for end-on and side-on adsorption. respectively.10 Although the adsorption plateaus are not reached in these experiments, the adsorbed amount of wild type protein is in the range of side-on monolayer, while at least at these low concentrations the tryptophan mutant seems to adsorb below this value. The lower value for tryptophan mutant can be due to differences in surface area of the adsorbed protein as a consequence of changes in conformation or reflecting differences in organization of the molecules in the adsorbed layer due to orientation or packing of the adsorbed proteins. The tryptophan mutant is known to change conformation to a larger extent upon adsorption than wild type protein,24 and this could influence both orientation and packing efficiency.9 For wild type protein the adsorbed amount in 0.01 M NaCl is close to the value in 0.1 M NaCl, while there is a small increase in the adsorbed amount of tryptophan mutant from 1.1 ( 0.21 at 0.1 M to 1.3 ( 0.11 mg/m2 (n ) 3) at 0.01 M NaCl, see also Figures 1 and 2. It is not possible to explain the difference between the two proteins from the available data. However, one possible reason is that the amount of wild type protein is closer to a densely packed monolayer than the tryptophan mutant, and it has been observed for hen lysozyme that the effect of ionic strength is larger further away from a close packing.33 The ionic strength also influences the onset of surfactant-induced removal, which occurs earlier at a high ionic strength (Figures 1 and 2 and Table 1). The effect of increasing ionic strength has been earlier observed by Rapoza and Horbett.21 The effect can be ascribed to the influence of salt on surfactants associative behavior, which is reflected in changes in the concentration of selfassociation and in the surfactants binding to protein. Jones and Manly found that the two stages of surfactant binding to lysozyme are affected differently by increasing ionic strength.15 The first stage, which reflects the electrostatic interaction between SDS and oppositely charged amino acids, is shifted to a higher concentration. The second stage, where SDS binds cooperatively to the protein, occurs at a lower concentration. The latter is in line with the

Removal of T4 Lysozyme from Silicon Oxide Surfaces

similarity between this cooperative binding and the selfassociation of surfactants in solution. This is also in agreement with the effect of increased ionic strength on binding of surfactant to synthetic polyelectrolytes.34 The observed effect of salt makes it likely that the start of surfactant-induced protein removal is affected by the onset of cooperative binding of SDS to the protein. Still, the start of removal occurs well below cmc (Table 1). For SDS, the cmc will decrease from 6.0 mM at 0.01 M NaCl (interpolated from 0 and 0.02 M NaCl) to 1.5 mM in 0.1 M NaCl.35 The shift in surfactant concentration for desorption is not as large as the shift in cmc at corresponding change in ionic strength. However, the effect of increased salt concentration is also smaller for binding of SDS to hen lysozyme than for the shift in cmc.15 When the surfactant concentration is lower than that needed to start removal of the protein, the addition of SDS leads to an increase in the adsorbed amount (Figures 1 and 2). This probably corresponds to the specific binding of SDS to lysozyme. The highest amount of SDS bound to the proteins prior to the start of removal is difficult to estimate since desorption is induced by only a small increase in surfactant concentration. Thus the amount of SDS estimated to be bound per protein varied strongly and was between 12 and 26, giving a mean value of 18 ( 5 (for 10 different measurements) SDS molecules per protein. The amino acid sequence of T4 lysozyme contains 13 each of lysine and arginine and 1 histidine,36 and the molecule has a net charge of +9 at pH 7. Thus the amount of SDS bound is closer to the number of positively charged amino acids than to the excess number of positive charges. At concentrations above cmc, SDS removes all of the preadsorbed protein, and in this respect, no difference can be observed between the two types of T4 lysozyme. However, the onset of protein removal differs and, regardless of ionic strength, removal was observed to start at a lower surfactant concentration for the tryptophan mutant than for wild type protein (Table 1). To verify that the difference in start of removal between wild type and mutant T4 lysozyme is not due to the lower amount of protein adsorbed for the tryptophan mutant, an adsorption experiment was carried out at a higher concentration (Figure 3). The adsorbed amount from this concentration is considerably higher than that from 0.2 mg/mL. The increased amount does not affect the concentration at which removal of protein starts. In fact, if the results are normalized by the maximal adsorbed amount, the two curves are nearly superimposed on each other (Figure 4). The removal kinetics is also unaffected by the amounts adsorbed, and the observed differences between the two proteins must be due to their interaction with the surface or the surfactant. The obtained results indicate that tryptophan mutant is easier to remove than wild type protein, and this is somewhat contradictory to earlier observation made for this system. For example, the fraction of protein that is removed by 2 × cmc of DTAB (a positively charged surfactant) was lower for the tryptophan mutant,10 and this was taken as an indication of stronger interactions between the mutant and silicon oxide surfaces than those involving wild type protein. Furthermore, the tryptophan mutant changes its conformation upon adsorption to a (34) Lindman, B.; Thalberg, B. In Interactions of Surfactants with Polymers and Proteins; Ananthapadmanabhan, K. P., Goddard, E. D., Eds.; CRC Press: Boca Raton, FL, 1993; p 203. (35) Williams, R. J.; Phillips, J. N.; Mysels, K. J. Trans. Faraday Soc. 1955, 51, 728. (36) Tsugita, A.; Inouye, M. J. Mol. Biol. 1968, 37, 201.

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Figure 3. Adsorption of T4 lysozyme tryptophan mutant (isoleucin 3 f tryptophan) 0.2 mg/mL (open diamond) and 1 mg/mL (filled diamond), to silicon oxide surfaces from a 0.1 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (rinsing is marked with closed arrow), and after 75 min, SDS (1/100 of cmc in water, open arrow) is added. After this, sequential additions of SDS are made every 15 min (175 min total). The temperature was 25 °C.

Figure 4. Normalized adsorption of T4 lysozyme tryptophan mutant (isoleucin 3 f tryptophan), 0.2 mg/mL (open diamond) and 1 mg/mL (filled diamond), to silicon oxide surfaces from a 0.1 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (closed arrow), and after 75 min, SDS (1/100 of cmc in water, open arrow) is added. After this, sequential additions of SDS are made every 15 min (175 min total). The temperature was 25 °C. Normalization is made by division with the maximum amount of proteins adsorbed.

larger degree than does the wild type protein.24,37 This could lead to more contact between protein and surface and thus support the assumption of stronger binding. The reason for the inconsistency could be found either in a difference in mechanism in removal between SDS and DTAB or in the difference in the measured quantities. In the DTAB experiments the quantity measured is the maximum amount of lysozyme removed, while in the work presented here it is the minimum surfactant concentration needed to start the removal. Surfactants remove protein from solid surfaces according to two mechanisms: by replacing the protein at the surface or by the surfactants binding to the protein and desolving the complex into solution.6 SDS will, unlike DTAB, not adsorb to the clean silica surface; thus its only possibility of removing protein is by solubilization. DTAB can interact both with the surface and the protein, and thus the removal could be by both mechanisms. It is known that cationic surfactants bind to a lower degree than SDS to lysozyme, and this might be the reason that (37) Claesson, P. M.; Blomberg, E.; Fro¨berg, J. C.; Nylander, T.; Arnebrant, T. Adv. Colloid Interface Sci. 1995, 57.

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removal is more incomplete when DTAB is used.38 The replacement mechanism might be more important for DTAB than solubilization as it is bound in lower amounts to the protein and has a positive charge, which makes electrostatic repulsion between complex and surface unlikely. In the replacement mechanism the removal will be controlled by the interaction between surface and the adsorbing molecules; in the case of DTAB that would be protein, surfactant, and protein-surfactant complex. In the solubilization mechanism the removal will be controlled by the interactions between protein and surfactant and between the complex and the surface. Proteins will, in the case of SDS, start to leave the surface when they have formed a complex containing sufficient amounts of surfactant molecules. The number of surfactants may be the same for all adsorbed lysozyme molecules or the number may vary due to differences in adsorption mode. In the latter case the start of removal will, to some extent, reflect the strength of protein-surface interaction. In the former, it will only reflect the binding of surfactant to the protein and the strength of interaction between the formed complex and the surface. The difference observed between wild type T4 lysozyme and its tryptophan mutant may be due to a portion of the wild type protein molecule interacting more strongly with the surface than do any of the tryptophan mutants or that the mutant protein binds with the surfactant easier. It is plausible that the binding of surfactant might differ between the two adsorbed proteins. This could be due to the tryptophan group, which is slightly more hydrophobic than the isoleucine, but it is more probably caused by a difference in the structure of the two adsorbed proteins. The difference in conformational changes was observed both by circular dichroism, where the proteins decreased their R-helix content upon adsorption to silica nano particles by 29% for tryptophan mutant and only 12% for wild type protein24 and by surface force measurement, where the layer thickness of adsorbed tryptophan mutant on mica is much smaller than that of wild type protein.37 It is possible that this change in structure facilitates surfactant binding, for example, by exposure of hydrophobic groups. The results in Figures 1 and 2 are obtained by a sequential addition of surfactant, which makes it difficult to evaluate the adsorption-desorption kinetics for specific surfactant concentrations, so the investigation was complemented by experiments where single additions of surfactants and longer equilibration times were used (Figures 5 and 6). The concentrations used, 1/100 and 3/100 of cmc, were chosen to study the effect of surfactant above and below the concentration needed for onset of removal in the previous experiments. In the case of the low surfactant concentration, Figure 5, the increase in adsorbed amount due to surfactant is larger for wild type protein than for tryptophan mutant, but this is likely to be due to the differences in amounts of protein adsorbed. If the data are scaled by the amount adsorbed before surfactant addition, then the increase is quite similar. The similarity between the two proteins in this region of adsorption is in agreement with the earlier suggestion that this is specific binding of SDS to charged amino acid groups. This indicates that electrostatic attraction between surfactant and protein is not influenced by differences in adsorption mode for the two proteins. Since the desorption after the second rinse is quite slow, and during the time of the experiment the adsorbed amount did not decrease to the same level as prior to surfactant addition, it is (38) Subramanian, M.; Sheshadri, B. S.; Venkatappa, M. P. J. Biosci. 1986, 10, 359.

Wahlgren and Arnebrant

Figure 5. Adsorption of 0.2 mg/mL wild type protein of T4 lysozyme (open triangle) and tryptophan mutant (isoleucin 3 f tryptophan) (open diamond) to silicon oxide surfaces from a 0.1 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (rinsing is marked with closed arrow), and after 75 min, SDS (1/100 of cmc in water, open arrow) is added. A final rinse is performed 30 min after the addition of surfactant. The temperature was 25 °C.

Figure 6. Adsorption of 0.2 mg/mL wild type protein of T4 lysozyme (open triangle) and tryptophan mutant (isoleucin 3 f tryptophan) (open diamond) to silicon oxide surfaces from a 0.1 M saline solution of pH 5.6. Rinsing with buffer is performed after 60 min (rinsing is marked with closed arrow), and after 75 min, SDS (3/100 of cmc in water, open arrow) is added. A final rinse is performed 30 min after the addition of surfactant. The temperature was 25 °C.

indicated that at these low surfactant concentrations the adsorbed SDS is strongly bound to both proteins. The concentration of 3/100 of cmc is above the concentration where protein removal starts. The results are presented in Figure 6, and as can be seen, the rate of removal is much faster for tryptophan mutant than for wild type protein. The removal does in both cases level off after a while, which might indicate that there is an array of proteins that needs higher surfactant concentration to be removed from the surface. Multiple states of adsorbed proteins are considered to be a common characteristic of adsorbed protein layers, which has been suggested to be a reason for incomplete removal of adsorbed proteins.39 The amount of protein left at the surface is higher for wild type protein than for mutant. Thus, the trend seen for the onset of removal, that SDS removes wild type protein easier, continues also above this threshold value. Conclusion T4 lysozyme is removed from silica surfaces at surfactant concentrations well below cmc (between 3 and 11% (39) Horbett, T. A.; Brash, J. L. In Proteins at Interfaces-Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 343, American Chemical Society: Washington, DC, 1987; p 1.

Removal of T4 Lysozyme from Silicon Oxide Surfaces

of cmc in solution). The removal does not start until the surfactant concentration is above a critical value, and this concentration is believed to be where cooperative binding of surfactant to adsorbed protein occurs. In line with associative behavior of ionic surfactants, the critical concentration for removal decreases with increasing ionic strength. Prior to removal, SDS adsorbs strongly to adsorbed protein, probably due to binding by positively charged amino acid groups. The surfactant concentration where protein removal starts is higher for wild type protein than for tryptophan mutant. Furthermore, at the same surfactant concentration the rate of protein removal and the removed fraction are lower for wild type protein than for tryptophan mutant. This was taken as an indication that the onset of cooperative binding of the tryptophan

Langmuir, Vol. 13, No. 1, 1997 13

mutant occurs at lower concentration than that in the binding of the wild type protein. Acknowledgment. The authors acknowledge the Swedish Research Council for Engineering Science for financial support. We also thank Stefan Welin-Klintstro¨m, University of Linko¨ping, Sweden, for providing the silica surfaces. Joseph McGuire and co-workers at Bioresource Engineering, Oregon State University, are acknowledged for supplying the protein as is Brian Matthews, Institute of Molecular Biology, Oregon State University, for providing the bacteria strains for mutant and wild type lysozyme. LA960171A