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Langmuir 1997, 13, 1850-1852
Adsorption Kinetics of Nonradiolabeled Lysozyme via Surface Pressure-Area Isotherms Brent S. Murray* Food Colloids Group, Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, U.K. Received October 10, 1996. In Final Form: December 17, 1996
Introduction At first sight the determination of the amount of adsorbed protein at a fluid interface as a function of time might seem to be one of the less difficult problems facing physical chemistry and surface science. To the initiated it soon becomes clear that unfortunately this is not the case. Proteins are complex molecules which exhibit complex folding/unfolding behavior at interfaces which is strongly affected by all the other molecular details of the interface, such as the distribution of ions, nonionic molecules, and segments of the protein itself.1 The approach to the true equilibrium conformation may be very slow and may be associated with the slow accumulation of secondary layers of protein.2 In addition, the nature and amount of the adsorbed protein may depend on the initial concentration of the solution from which the protein adsorbs.3 To this extent protein adsorption, over the usual time scales of observation, may appear to be not determined by thermodynamics.4 In most real systems where protein adsorption is important, for example in biological systems, mixtures of proteins usually occur and the picture is even more complex, for example with a cascade of different proteins adsorbing and then being replaced by others.5 In such situations, reliable, noninvasive methods of obtaining the individual protein surface concentrations are few. In this laboratory many studies6 have been made of the competitive adsorption of a number of pure food proteins, such as the caseins and whey proteins of milk. Thus, for example, the disordered protein β-casein has a much greater tendency to displace a globular milk protein such as β-lactoglobulin.6 On the other hand, a protein such as gelatin seems able to form secondary layers even when the initial adsorbed gelatin layer is displaced by sodium caseinate.2 Recently Damodaran and co-workers4,7-12 have provided some new measurements of competitive adsorption of a number of proteins, including β-casein,4,7,11 Rs1-casein,11 BSA,7,9 and lysozyme.4,8,9 One of the results of these studies is that they confirm that protein adsorption from mixtures of proteins with quite different surface activity is kinetically and not thermodynamically con* Address correspondence to author at Tel: 44 (0)113 2332962. Fax: 44 (0)113 2332982. E-mail:
[email protected]. (1) Haynes, C. A.; Norde, W. Colloids Surf., B 1994, 2, 517. (2) Dickinson, E.; Murray, B. S.; Stainsby, G. In Advances in Food Emulsions and Foams; Dickinson, E., Stainsby, G., Eds.; Elsevier Applied Science: London, 1988; p 123. Galazka, V. B.; Dickinson, E. J. Texture Stud. 1995, 26, 401. (3) Mitchell, J. R. In Developments in Food Proteins; Hudson, B. J. F., Ed.; Elsevier Applied Science: London, 1986; Vol. 4, p 291. (4) Xu, S. Q.; Damodaran, S. Langmuir 1994, 10, 472. (5) Brash, J. L.; Horbett, T. A. ACS Symp. Ser. 1995, 602, 1. (6) Dickinson, E. ACS Symp. Ser. 1991, 448, 114. (7) Cao, Y. H.; Damodaran, S. J. Agric. Food Chem. 1995, 43, 2567. (8) Xu, S. Q.; Damodaran, S. J. Colloid Interface Sci. 1993, 159, 124. (9) Anand, K.; Damodaran, S. J. Colloid Interface Sci. 1995, 176, 63. (10) Damodaran, S.; Xu, S. Q. J. Colloid Interface Sci. 1996, 178, 426. (11) Anand, K.; Damodaran, S. J. Agric. Food Chem. 1996, 44, 1022. (12) Xu, S.; Damodaran, S. In Food Proteins Structure and Functionality; Schwenke, K. D., Mothes, R., Eds.; VCH: Weinheim, Germany, 1993; p 270.
S0743-7463(96)00983-3 CCC: $14.00
trolled. Thus the protein which arrives at the interface first may dominate the composition of the resultant film, regardless of the overall final composition of the bulk phases. Attention is drawn in this note, however, to some rather anomalous results obtained by these workers4 for the adsorption of lysozyme to the air-water (A-W) interface, where negative adsorption was apparently observed at low protein concentrations. The authors have provided arguments4,12 involving the electrostatic barriers to adsorption, due to the high charge on lysozyme at neutral pH. However, the adsorption results are quite at odds with those obtained by de Feijter and Benjamins13 under very similar conditions. Here we argue that the route of this discrepancy may lie in the use of radiolabeled proteins. There are not many noninvasive methods for studying adsorption at fluid interfaces. Ellipsometry is perhaps the most widely used at A-W interfaces. Many techniques have been developed for adsorption at solid-fluid interfaces, based on total internal reflectance of UV-vis or IR radiation. In principle such techniques may be used at A-W or oil-water interfaces14 when the surface species are highly absorbing or fluorescing, but for proteins this necessitates covalent linking of chromophores/fluorophores to the polypeptide chain, which may alter the structure and surface properties of the native protein. In this work we have used a Langmuir trough to study the adsorption kinetics of nonradiolabeled lysozyme at the A-W interface under exactly the same conditions as those of Damodaran et al.4, to investigate whether or not radiolabeling could be the source of anomalous results. This is an important issue to clarify since the radiotracer method is potentially an extremely powerful way of monitoring adsorption from a mixture of proteins. Materials Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and hydrochloric acid were all AnalR grade reagents from BDH Merck. Lysozyme (chicken egg white, product code L6876, lot number 111H7010) was from Sigma Chemicals. All water used was from a Millipore alpha-Q purification system with a surface tension of 72.0 mN m-1 at 25 °C. Phosphate buffer was made up to an ionic strength of 0.1 mol dm-3 and adjusted to pH 7 with HCl. Lysozyme was dissolved in this buffer to give a protein concentration of 1.5 × 10-4 wt % and used immediately. Mica plates were used for measurement of the surface pressure.
Methods The technique of measuring protein surface concentrations, Γ, via the trough is based on one principal assumption: that at a certain high area per molecule (low Γ), A*, the surface pressure, π, has a value, π*, that is a unique function of A*, i.e., independent of the adsorption time and bulk concentration of protein, Cb. A new type of Langmuir trough was used for these measurements, described in detail elsewhere.15,16 The trough was filled (13) de Feijter, J. A.; Benjamins, J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987; p 72. (14) Perera, J. M.; McCulloch, J. K.; Murray, B. S.; Grieser, F.; Stevens, G. W. Langmuir 1992, 8, 366. (15) Murray, B. S; Nelson, P. V. Langmuir 1996, 12, 5973. (16) Murray, B. S. Colloids Surf., A, in press. (17) Xu, S. Q.; Damodaran, S. J. Colloid Interface Sci. 1992, 157, 485. (18) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403, 415, 427. (19) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 227. (20) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76, 240.
© 1997 American Chemical Society
Notes
Langmuir, Vol. 13, No. 6, 1997 1851
(Dtπ )
Γ ) 2Cb
Figure 1. Surface concentration of lysozyme, Γ, versus t1/2. Different symbols indicate separate experiments. Dashed lines indicate results due to de Feijter and Benjamins (A) and Xu and Damodaran (B).
with protein solution and the A-W interface was reduced to low area. The surface was then sucked off via a Pasteur pipet and a vacuum line until the surface pressure was zero, i.e., the surface tension was that of the pure buffer. (Separate measurements showed that the surface tension of the buffer did not change with time.) At zero time, t, the surface was then expanded (in 10 s) to its full area. At certain time intervals the interface was relatively rapidly (in 10-30 s) compressed until π ) π*, and then the interface quickly re-expanded to the full, original film area. As Γ increases with time, the trough area at which π ) π* increases with time. From the area of the interface when π ) π* and provided the corresponding value of A* is known, Γ may be calculated. In experiments conducted here, films were compressed to π* ) 3 mN m-1. Experiments were conducted at 25 ( 0.3 °C. Results and Discussion The results of several repeat experiments are shown in Figure 1. Γ has been plotted against t1/2. The results due to de Feijter and Benjamins,13 who used ellipsometry to determine Γ, and the results due to Xu and Damodaran,4 who used radiolabeled protein, have also been indicated, for comparison. It is seen that the agreement between the results obtained in this study and those of de Feijter and Benjamins is reasonable, whereas the results of Xu and Damodaran are quite different. Surface concentrations have been calculated using a value of A* ) 1870 Å2 (Γ* ) 1.25 mg m-2), corresponding to π* ) 3 mN m-1, from the study due to de Feijter and Benjamins. de Feijter and Benjamins used slightly different solution conditions: Cb ) 1.0 × 10-4 wt %, pH 6.7, ionic strength 0.02 mol dm-3. The justification for using A* ) 1870 Å2 is that over the concentration range of Cb ) 10-4-10-1 wt % A was shown by these workers to be independent of Cb at π ) 3 mN m-1. Part of the slight discrepancy with de Feijter and Benjamins’ result may be explained by the difference in Cb. At low Cb and at short adsorption times the rate of adsorption should obey the equations13
dΓ D ) Cb dt πt
( )
1/2
(1)
and (21) Adams, D. J.; Evans, M. T. A.; Mitchell, J. R.; Phillips, M. C.; Rees, P. M. J. Polym. Sci., Part C 1971, 34, 167. (22) Clark, D. C.; Husband, F.; Wilde, P. J.; Cornec, M.; Miller, R.; Kragel, J.; Wustneck, R. J. Chem. Soc., Faraday Trans. 1995, 91, 199. (23) Izmailova, V. N.; Yampol’skaya, G. P.; Lapina, G. P.; Sorokin, M. M. Colloid J. USSR 1982, 44, 195.
1/2
(2)
where D is the diffusion coefficient and π ) 3.141.... Taking the line of best fit through the experimental data gives D ) (1.5 ( 0.2) × 10-11 m2 s-1, which is only slightly lower than the value of D ) 2.0 × 10-11 m2 s-1 obtained by de Feijter and Benjamins. Both of these values, however, are lower than the accepted value for the bulk diffusion coefficient of lysozyme of 10-10 m2 s-1.13 This may indicate an electrostatic barrier to diffusion to the interface,24,25 the high charge (on the lysozyme molecules at pH 7) at the interface preventing the adsorption of further molecules, except when Γ is so low, and therefore the surface charge density so low, that this effect is not important (i.e., in the early stages of adsorption). Xu and Damodaran12 invoked arguments based on electrostatics to explain their results, obtained under exactly the same conditions as in this work, but using the radiolabeled protein. It should also be noted that the results of Xu and Damodaran were obtained using a calibration based on monolayers of radiolabeled β-casein spread from chloroform/methanol/hydrochloric acid solution.17 This was used because the results calculated with this calibration were seen to be closer to the results of the same study of de Feijter and Benjamins,13 though the final adsorbed amounts estimated were still 58% lower than the de Feijter and Benjamins result. The calibration method produced very different calibration curves depending on whether spread monolayers of β-casein, lysozyme, or stearic acid were used. The authors suggested this was in part due to the different configurations adopted by the three types of molecule and the consequent different spatial distribution of the radionucleotide at the interface. This led to different dissipation of the radiation between the film and the detector. This is a serious difficulty, since if protein conformation changes with time on adsorption, the calibration at one conformation, i.e., of the spread film, will not be applicable to any other conformation. The classic study of protein adsorption by Graham and Phillips18-20 also employed radiolabeled proteins for the determination of surface concentrations. Earlier work21 indicated that even the lowest level of labeling used produced significant differences in surface activity compared with the native protein. Maxima in the surface rheology as a function of Cb were also observed (discussed previously2), and similar behavior noted recently was attributed as “impurities” in the system.22 An objection which may be raised against the trough method described above is that proteins unfold with time, and therefore although π may remain constant, A may not necessarily be the same. For lysozyme at pH 7 the molecule is quite compact and is not thought to unfold particularly quickly or easily.23 This is evidenced by the distinct lag before the onset of any observed π on adsorption from low bulk concentrations.13 Zero surface pressure is not necessarily indicative of zero surface concentration unless substantiated by reliable independent means. This is because the protein film may be in a “gaseous monolayer state”, with large areas of bare interface between isolated adsorbed protein molecules. A pronounced lag in the surface shear viscosity has also been observed when π is still low,26 under similar conditions as studied here, again indicating a time lag before protein unfolding and crosslinking occurs at the interface. At high protein surface (24) Sharpe, D.; Eastoe, J. Langmuir 1996, 12, 2303. (25) Bonfillon, A.; Sicoli, F.; Langevin, D. J. Colloid Interface Sci. 1994, 168, 497. (26) Murray, B. S.; Dickinson, E. Food Sci. Technol. Proc. Int. 1996, 2, 131.
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concentrations/long adsorption times, however, the trough method will be invalid because surface concentrations are known to continue to change after π has reached an equilibrium value, due to continued unfolding and rearrangements in the interface. In principle, a lysozyme monolayer could be spread onto the buffer and the π-A isotherm used to determine A* corresponding to π*. However, it was found that quantitative spreading was impossible under these conditions (pH 7, 0.1 mol dm-3 ionic strength); the protein appeared to be too soluble in the aqueous phase. This is in agreement with the work of Adams et al.,21 who found that quantitative spreading was impossible except at very high salt concentrations in the bulk and closer the isoelectric point, to reduce the electrostatic charge on the molecule. Since radiolabeled lysozyme can be successfully spread to provide a calibration curve,17 this in itself may indicate that the solubility and, therefore, the structure is substantially different from the native protein. In any case, spreading from a monolayer may be very different from adsorption from solutionsleading to a much more unfolded state,27 and there may be concerns when proteins are spread from solvents rather than from aqueous solution (as here) due to protein denaturation.27 Other objections which may be raised against the trough method are that the intermittent compression and expansion may introduce convection (which will affect the rate of adsorption) and that brief compression of adsorbed molecules to surface pressures even as low as π ) 3 mN m-1 may irreversibly change the conformation of the proteins in the adsorbed layer. However, the results (27) MacRitchie, F. Adv. Colloid Interface Sci. 1986, 25, 341.
Notes
obtained using different time intervals are in general agreement, and the linear dependence of Γ on t1/2 does not suggest that convection was significant. Although the compression to π* was rapid (taking 10-30 s) compared to the overall time scale of adsorption, this compression speed was still not sufficient to cause any visible turbulence or stirring in the aqueous phase. This compression time would also help any π gradients due to high surface viscosities to dissipate, though surface viscosities are thought to be low (see earlier) in the early stages of adsorption under these conditions. There is evidence that initial flow disturbances, particularly at low Cb, may influence adsorption kinetics, though this evidence is often from measurements where the formation of the initial, clean interface involves a considerably more drastic disturbance, e.g., the more rapid formation of a droplet or bubble,28 or deliberate stirring to introduce the protein into solution.29 Also, if convective transport was induced and had a significant effect on the adsorption kinetics, then one would not except agreement with the results of de Feijter and Benjamins, where the interfacial area was kept constant throughout. Overall then, the agreement with the results of de Feijter and Benjamins and the disagreement with the results of Xu and Damodaran seem to validate the trough measurements but cast significant doubt over the use of radiolabeled lysozyme to determine the adsorption kinetics of this protein. LA9609837 (28) Hansen, F. K.; Myrsvold, R. J. Colloid Interface Sci. 1995, 176, 408. (29) Paulsson, M.; Dejmek, P. J. Colloid Interface Sci. 1992, 150, 394.
