Langmuir 1995,11, 3542-3548
3542
Conformational Changes in Adsorbed Proteins A. Ball and R. A. L. Jones* University of Cambridge, Cavendish Laboratory, Madingley Road, Cambridge CB3 OHE, U.K. Received February 6, 1995. In Final Form: June 9, 1995@ Attenuated total reflection Fourier transform infrared (FTIR-ATR) spectroscopy was used to follow the adsorption of the globular protein lysozyme from aqueous (D20)solution onto a silicon surface. The effect of changing temperature andor pD on the structure of protein in the adsorbed layer is observed and compared with the effect of these changes on concentrated solutions. We show that the same spectral changes as those occurring in the heat-set gelation of concentrated solutions of globular proteins can also be observed in adsorbed proteins, but at lower temperatures. We suggest that this is due to the presence of the surface having the effect of promoting molecular structural rearrangements.
The adsorption of proteins at interfaces is a well-known and widespread phen~menon.l-~ The particular case of adsorption of proteins at the solid-liquid interface is important in, for instance, industrial applications such as the fouling of food processing equipment, and in medicine, in the determination of biocompatibility (Le., the suitability of materials for medical applications4). The issue we concentrate on in this work is the question of the degree to which the conformation of the polymer is modified on adsorption. The possibility of such structural rearrangement has been known for some time,3p5and its importance is 2-fold. Firstly, such rearrangements will have a significant effect on the thermodynamics of adsorption. The structural rearrangement will usually produce more disordered states, and the corresponding change in entropy provides an extra driving force for adsorption. This extra driving force can sometimes even overcome apparently unfavorable protein-surface interactions, as in the case of myoglobin and a-lactalbumin,6 which were shown to adsorb on hydrophilic surfaces even under unfavorable electrostatic interactions, as compared with lysozyme and ribonuclease. These latter proteins behave like hard particles and interact with an interface under the influence of both hydrophobic and electrostatic effectsa6This contrast is attributed to the relatively high structural stability of lysozyme and ribonuclease and the low structural stability of a-lactalbumin and myoglobin. Secondly, the properties of the adsorbed layer may be profoundly influenced by the occurrence of such structural changes. For example, the existence of intermolecular interactions is likely to lead to an adsorbed layer with viscoelastic properties; this in turn will be of great importance if such an adsorbed layer is stabilizing an emulsion or a foam. Before considering the question of how a surface might promote structural rearrangement, it is worth briefly reviewing the types of structural rearrangement that might occur in globular proteins in bulk solutions. Firstly, in solutions at very low concentrations, there is the unfolding transition, to which much attention has been
given.7 This is essentially a transition of an isolated molecule from a compact, highly ordered (and biochemically active) state to an unfolded, disordered state. This transition is in many ways essentially analogous to a melting transition in that it is a first-order transition with a well-defined latent heat; among many potential complications is the possibility of an intermediate state, the "molten globule" state, which is compact but more disordered than the native state.8 If denaturation is carried out reversibly, e.g., by dissolving the protein in guanidinium hydrochloride, the unfolding transition is reversible, and the protein moleculewill refold to its native state once the denaturing conditions are removed. The second class of transitions are simple liquid-liquid unmixing transitions, in which the protein solution separates into two phases, one rich in protein, and one poor. These transitions have been shown by careful experiments on lysozyme solutions to be in the same universality class as liquid-liquid unmixing transitions in small molecule mixture^;^ in fact the unique complexity of proteins plays little role in these transitions, which are probably best understood as being analogous to liquidliquid phase transitions in charged colloid suspensions. The third class oftransitions occur in proteins at higher solution concentrations and involve aggregation or precipitation of many protein molecules. These transitions are much less well characterized and understood than the unfolding transitions. They are, however, familiar as ubiquitous factor in cooking operations (e.g., the setting of egg white), as well as being potentially important in biotechnology. A typical example is the heat-set gelation that occurs in some globular proteins, such as lysozyme. On heating a reasonably concentrated solution of such a protein to about 70 "C,the solution changes to aviscoelastic gel which shows strong light scattering. This change is irreversible, in contrast to the simple unfolding transition. In lysozyme, spectroscopic and other evidence suggests that heat-set gelation involves the formation of intermolecular P-sheets,lo-12 though other factors, such as hydrophobic interactions, are likely to be important, too. How might we classify the possible structural transitions that occur at interfaces? In other areas of condensed
* Abstract published inAdvance ACSAbstracts, August 15,1995. (1)Andrade, J. D. Surface and interfacial aspects of biomedical polymers. Vol.2. Protein adsorption; Plenum Press: New York, 1985. (2)Cohen Stuart, M. A.; Fleer, G. J.; Lyklema, J.; Norde, W.; Scheutjens, J. M. H. M. Adv. Colloid Interface Sci. 1991, 34, 477. (3) Norde, W.Adv. Colloid Interface Sci. 1986,25, 267. (4)Hubbell, J . A. Trends Polym. Sci. 1994,2, 20. ( 5 ) Jakobsen, R. J.;Strand, S. W. In Internal Reflection Spectroscopy. Theory and Applications; Mirabella, F. M., Jr., Ed.; Marcel Dekker: New York, 1993,Chapter 5. (6)Arai, T.;Norde, W. Colloids Surf. 1990, 51, 1.
(7) Creighton, T. E. Protein Folding; W. H. Freeman and Company: New York, 1992. (8)Kuwajima, K. Proteins: Struct., Funct., Genet. 1989,6 , 87. (9)Schurtenberger,P.; Chamberlin, R. A.; Thurston, G. M.; Thomson, J . A,; Benedeck, G. B. Phys. Rev. Lett. 1989,63, 2064. (10)Clark, A. H.; Saunderson, D. H. P.; Suggett, A. Int. J. Pept. Protein Res. 1981, 17, 353. (11)Clark, A. H.; TufTnell, C. D. Int. J.Pept. Protein Res. 1980,16, 339. (12)Clark,A.H.; Judge, F. J.;Richards, J . B.; Stubbs, J. M.; Suggett, A. Int. J. Pept. Protein Res. 1981, 17, 380.
Introduction
0743-7463/95/2411-3542$09.o0/0 0 1995 American Chemical Society
Conformational Changes in Adsorbed Proteins IR stainless steel trough
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Figure 1. Schematic view of attenuated total reflection spectroscopy trough, viewed from above.
matter, it is a familiar notion that surface transitions occur which are closely analogous to bulk transitions, though possibly shifted in temperature or concentration. Examples of such events include the transition from single chains to micelles in block copolymers (i.e,, the critical micelle composition),which is shifted to lower concentrations by the presence of a surface which favors one component of the block over the other,13 and the phenomenon of prewetting.14 Possibly most relevant to our work is the idea of surface gelation. Some polymer solutions form a gel at some givep concentration; if a wetting layer is formed at a surface or interface, then, although the bulk concentration may be too low for a gel to form, the local concentration at the surface may exceed the threshold for gelation, allowing a thin layer of gelled material to form at the interface. Such surface gelation was first observed by Kim et al.15 in polystyrene sulfonate solutions. The potential importance of surface gelation in proteins has already been pointed out by Dickinson.16 The adsorption of proteins, or other macromolecules, to surfaces can be studied by Fourier transform infrared spectroscopy, in the attenuated total reflection mode (FTIR-ATR).5 In ATR spectroscopy,17the sample under investigation is placed in a cell, in contact with a crystal, such that when an infrared beam is shone through the crystal, total internal reflection takes place at each contact of the beam with the crystallsample interface (Figure 1). Each time this occurs, a small amount of energy is lost to the sample, in the form of a rapidly decaying evanescent standing wave. The absorbance spectrum results from loss of the IR frequencies absorbed by material within the range of this evanescent wave. Under appropriate conditions, macromolecules will adsorb to the crystal itself, and it is thus possible to observe the adsorption process as it O C C U ~ S . ~ J ~The J ~ main IR frequencies ofinterest in the case ofproteins are the amide I, 11, and I11 regions, centered on 1650, 1550,and 13501200 cm-l, respectively. An increase in the area under the absorbance peak can be used to determine adsorbed amounts, and any changes in peak shape can be correlated to structural changes in the molecules of the adsorbed layer. Fourier transform infrared (FTIR)spectroscopy is one of the methods frequently used for determination of the structural content (a-helices, /?-sheets, and turns) of protein molecule^.^,^^ The amide I peak, in particular, is used to determine the conformations present in proteins. (13)Shull, K.R.Macromolecules 1993,26,2346. (14)Sullivan, D. E.; Telo da Gama, M. M. In Fluid Interface Phenomena; Croxton, C. A., Ed.; Wiley: New York, 1986;p 45. (15)Kim, M. W.;Peiffer, D. G.; Pincus, P. J. Phys. Lett. 1984,45, L-953. (16)Dickinson, E.An Introduction toFood Colloids;Oxford University Press: Oxford, 1992. (17)Mirabella,F. M., Jr. In Internal Reflection Spectroscopy. Theory andApp1ication.s; Mirabella,F. M., Jr., Ed.;Marcel Dekker: New York, 1993;Chapter 2. (18)Fu, F.-N.;Fuller, M. P.; Singh, B. R.Appl. Spectrosc. 1993,47, 9R
(19)van der Beek, G. P.; Cohen Stuart, M. A.; Fleer, G. I. Macromolecules 1991,24,3553. (20)Byler, D. M.; Susi, H. Biopolymers 1988,25,469.
Langmuir, Vol. 11,No.9, 1995 3543 Absorbance at this frequency is due primarily to the amide CO bond stretching vibration, coupled to the in-plane NH bending and the CN stretchingmodes.21 The frequencies of the bond vibrations are affected by hydrogen bonding and the different conformations found in the protein, so the amide I peak is a composite absorbance band. The individual absorbance frequencies are not normally resolvable, except by resolution enhancement techniques, for instance, Fourier self-deconvolution and/or secondderivative spectroscopy,20-22or factor analysis meth0ds.23924 In some circumstances, there may be gross changes in the shape of the peak, which can be correlated with structural reorganization in the sample molecules. Changes in the amide I peak on heating of concentrated solutions were used by Clark et al.1° to investigate the heat-set gelation of a number of globular proteins under a range ofsolution conditions. The results were compared with small-angleX-ray scattering and electron microscopy studies.11J2 They found that, in general, the amide I peak of the FTIR spectra of the proteins shows the appearance ofshoulders at 1621cm-l, and 1684cm-l at temperatures above 70 "C, coincidingwith the onset ofheat-set gelation. This combination was attributed to the formation of antiparallel p-sheet structure.1° Other instances of this change in the amide I peak on heat-set gelation of globular proteins have also been ~ b s e r v e d and , ~ ~it~ has ~ ~ been shown not to occur in globular proteins which do not form gels on heating.25 One aspect of FTIR-ATR adsorption studies which is potentially of great interest is the effect of changes in the sample environment on the structure of the adsorbed material. This can easily be followed, for instance by changing the bulk solution, or heating the cell. In the case of adsorbed materials, the absorbance is derived from three contributing element^:^ the irreversibly adsorbed material, the reversibly adsorbed material, and the bulk sample. Only in cases where a protein solution concentration is below 1 mg/mL will all the absorbance signal be derived from irreversibly adsorbed p r ~ t e i n . ~ The adsorption of the globular protein lysozyme at the solid-liquid interface has been widely studied, using various techniques, under a range of conditions of substrate, protein concentration, pH, and ionic ~ t r e n g t h . ~ J ~ ,It~ is ' - ~generally ~ found that electrostatic interactions play a part in the adsorption behavior, and that adsorption will occur as a monolayer at lower concentrations, but that multilayers may also be f~rmed.~ The ~ ?adsorption ~~ of lysozyme (on graphite) appears to be in a regular 2-D array.28 In this study, we use ATR to observe the adsorption of the globular protein lysozyme from solution in D2O onto a silicon ATR crystal, using the capabilities of ATR first to determine the amount adsorbed with time, and then changing the bulk solution to remove irreversiblyadsorbed protein and protein in solution. We then alter conditions (21)Surewicz, W. K.;Mantsch, H. H. Biochim. Biophys. Acta 1988, 952,115. (22)Kumosinski, T.F.; Farrell, H. M.,Jr. Trends Food Sci. Technol. l993,4,169. (23)Haaland, D. M. In Practical Fourier Transform infrared spectroscopy. Industrial and laboratory chemical analysis; Ferraro, J. R., Krishnan, K., Eds.;Academic Press: New York, 1990;Chapter 8. (24)Lee, D.C.; Haris, P. I.; Chapman, D.;Mitchell,R. C.Biochemistry 1990,29,9185. (25)Byler, D. M.;Purcell, J. M.Proc. SPIE-Int. Soc. Opt. Eng.1989, 1145,415. (26)Herald, T. J.;Smith, D. M. J.Agric. Food Chem. 1992,40,1737. (27)Arai, T.;Norde, W. Colloids Su$. 1990,51,16. (28)Haggerty, L.; Lenhoff, A. M. Biophys. J. 1993,64,886. (29)Ivarsson, B. A.; Hegg, P.-0.; Lundstrom, K. I.; Jonsson, U. Colloids Surf 1985,13,169. (30)Wahlgren, M. C.; Arnebrant, T.; Paulsson, M. A. J. Colloid Interface Sci. 1993,158,46.
3544
Langmuir,
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Vol. 11, No. 9, 1995
in t h e cell, by changing pD a n d o r heating, and study the effect on the spectrum of irreversibly adsorbed layer.
Experimental Section Hen egg white lysozyme (L6876) was obtained from Sigma Chemical Co. and used without further purification. Solutions were made up in 0.1 M NaCl in D2O (99.9% deuterium, Sigma) and the pD adjusted, where acidic solutions were used, with DC1, pD values were determined as pH 0.4.31 It was decided to work in D2O rather than H2O as the amide I peak in DzO is not obscured by the solvent absorbance, as is the case in H2O. Although digital subtraction of solvent spectra is now common, making the use of HzO possible in many applications, it was found here that unacceptable levels of noise were introduced, due to the extremely low sample absorbances obtained. The equivalent absorbance frequency of D2O to that at 1650 cm-' of HzO is shifted to lower frequencies, and solvent subtractions are consequently easier to perform. In DzO solution, some of the hydrogen atoms in the protein will exchange with deuterium atoms in the solution, affecting the absorbance frequencies of the protein itself; in particular, amide hydrogen atoms, both on the backbone nitrogen atoms and on side-chain nitrogens, can exchange on quite fast time scales. The amide I1 frequency is shifted by about 100 cm-l, from 1550to 1450cm-1, but the amide I region is hardly affected, frequencies changing by about 5, certainly less than 10, wavenumbers.20 We can distinguish between amide hydrogen atoms located close to the surface of the native structure, which will exchange very fast at room temperature, and amide hydrogen atoms located deep within the native structure, which at room temperature can only exchange very slowly, due to the inability of the solvent to reach the core of the protein, but which on unfolding will exchange f a ~ t . 3All ~ solutions were made up the day before use so that all H-D exchange in the first class of groups occurs before the experiments. Exchange of H atoms of the second class, which is expected after unfolding ofthe protein on heating, manifests itself in the infrared spectrum primarily as the disappearance of the residual amide I1 peak (due to N-H bending) at 1550 cm-1. The disappearance of this peak gives an indication of the unfolding temperature.1° Infrared Spectroscopy. Infrared spectra were collected using a Mattson Galaxy 4020 FTIR spectrometer with a room temperature deuterated triglycerine sulfate (DTGS) detector. The spectrometer was thoroughly purged with dry air to remove water vapor, which has IR absorbance frequencies in the area of interest. (a) ATR Spectroscopy. ATR spectra were collected using a 10 internal reflection, 45" k e d angle of incidence silicon crystal with a native oxide layer. The cell was based on the Specac squarecol ATR cell, modified in our laboratory to allow filling and drainage from outside the spectrometer sample compartment. This meant that a good purge could be set up and maintained throughout the experiment, and that the cell did not have to be moved for changes of solution. Heating was carried out using a Specac Eurotherm temperature controller. This gave stable temperatures to within h0.5 "C. The temperature control was calibrated using a thermocouple. Data were collected as interferograms at 2 cm-l resolution and Fourier transformed into the frequency domain via the Mattson FIRST software package. Typically,250 interferograms were collected and coadded during heating scans, less while following adsorption. All spectra were ratioed against the background of an empty cell and then converted to absorbance units. Appropriate solvent spectra were taken for subtraction from the sample spectra. In heated experiments, the cell was heated at the rate of 1"C/min,with 3 min allowed for equilibration at each temperature at which a spectrum was taken. Solvent spectra were taken at correspondingtemperatures and subtracted from the sample spectra. The adsorbed amount per unit area was determined by exchanging the bulk solution for a series of solutions of known concentration and using the results of absorbance versus concentration to give adsorbed amounts per
+
(31) Covington,A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Anal. Chem. 1968,40, 700. (32)Delepieme, M.; Dobson, C. M.; Karplus, C. M.; Poulsen, F. M.; States, D. J.;Wedin, R. E. J. Mol. Biol. 1987,197, 111.
unit area, according to19
(1) where
A = area under infrared absorbance peak k = constant c = concentration of adsorbing solution
d , = depth of penetration of evanescent wave
r = adsorbed amount (mg/m2) (7~) Transmission Spectroscopy. Spectra of the bulk solutions were produced from interferograms collected in transmission mode at 1 cm-1 resolution and treated as for the ATR interferograms. Typically, 120 interferograms were collected and coadded. Specac semipermanent liquid cells were used wth 4 mm CaFz windows and tin spacers giving path lengths of either 6 or 12 pm. Again, heating was carried out via the Eurotherm temperature controller. Up to temperatures of about 80 "C, the temperature was stable towithin 0.5 "C;for higher temperatures, the temperature was stable to within 2 "C. The cell temperature versus the set temperature was calibrated by measurement with a thermocouple. Heating was carried out in the same manner as for the adsorbed protein, but solvent spectra were not subtracted from the concentrated solution spectra. Adsorption Experiments. Adsorption was carried out from a solution of lysozyme at 5 mg/mL in 0.1 M NaCl in D20, with a pD of 6.9. A background spectrum ofthe empty cell was taken, followed by the spectrum of 0.1 M NaCl in D20, after which the protein solution was introduced into the cell and spectra were taken continuously. Afbr the required time for adsorption, the protein solution in the cell was exchanged twice for 0.1 M NaCl and left to equilibrate. The cell could then be heated, or else the solution could be exchanged again, for 0.1 M NaCl at pD 2, left to equilibrate, and heated. The exchange of solution ensured that the adsorption process was stopped, the reversibly adsorbed material would be removed and so, as far as possible, only the irreversibly adsorbed layer would be observed.
Results and Discussion Lysozyme is known to have a relatively high level of structural stability and to act as a hard particle in solution.6 Under the conditions of adsorption, Le., in 0.1 M NaCl (pD around 6.91, the molecules will have a charge of +7.6 The surface of the silicon crystal, which will in fact be silicon oxide, will be negatively charged under these conditions. It is therefore to be expected that adsorption will take place under the influence of electrostatic attraction, even though experiments were conducted in 0.1 M NaCl solution, to reduce intermolecular interactions. The adsorption process was followed as a function of time, by the increase in the area under the amide I peak, and is shown in Figure 2. It is clear that, after the initial rapid adsorption phase, a near plateau develops, although there is a general trend of increase in the adsorbed amount. In the long term this may be due partly to a change in the spectrum of the adsorbed material, as an increase in peak a r e a w a s also observed, in some cases, on standing in salt solution overnight. Calibration of adsorbed amounts was attempted for individual experiments, by exchanging the protein solution, in the near plateau region of adsorption, for a series of solutions oflysozyme oflower concentration. Spectra were taken as quickly as possible to avoid a n y further adsorption during the course of the calibration. Adsorbed amounts per unit area were calculated according to eq 1. Adsorbed amounts are typically around 4 mg/m2in the plateau region of adsorption. This level of adsorption corresponds to more than monolayer adsorption, as also after 1800 s, on observed by o t h e r ~ . ~For ~ tadsorption ~~
Conformational Changes in Adsorbed Proteins
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Langmuir, Vol. 11,No. 9, 1995 3545
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Figure 2. Adsorbed amount as a functionof time for lysozyme from a 5 mg/mL solution in 0.1 M NaCl in D20, onto silicon. wavenumber (cm-')
silica, from phosphate-buffered saline, Wahlgren et al. obtained adsorption levels of 3.7 mg/m2?0 Here, we obtain levels around 3.0 mg/m2. Depending on the orientation of the molecules, and assuming no change of shape, monolayer adsorption can be calculated as being from 1.8 to 2.7 mg/m2,assuming molecular dimensions of3.0 x 3.0 x 4.5 nm3 and a molecular weight of 14 600.0 Assuming, for the sake of argument, a layer thickness of 5 nm, 3.0 mg/m2 would be roughly equal to a concentration in the layer of 600 mg/mL, or about 60%by weight. Thermal Denaturationof Lysozyme Solutions. An extensive study of the thermal denaturation and heat-set gelation of concentrated solutions of globular proteins'O included the effects of heat on a 13% (w/w) solution of lysozyme in 0.1 M NaCl in D2O at pD 2. This showed that a pronounced shoulder appeared in the amide I peak at 1621 cm-', at raised temperatures, accompanied by a less pronounced shoulder at 1684 cm-l. This was attributed to the formation of intermolecular antiparallel /?-sheet and was shown to be accompanied by gelation of the protein. Another study of thermal denaturation of lysozyme, this time by microcalorimetry of very dilute solutions,33showed that a denaturation (unfolding) step took place on heating, and that the temperature of the denaturation dropped as the solution pH was lowered. At pH 2, denaturation occurred at 55 "C. We have studied the transmission spectra of lysozyme at aroung 13%(w/w) in 0.1 M NaCl in D20 both with pD unadjusted and with the pD adjusted to about 2 with the addition of DCl. We assume that a concentrated solution with the pD unadjusted, i.e., with the protein acting as buffer (final pD around 5.91, is the nearest possible approximation to the conditions in the concentrated surface layer surrounded by a 0.1 M NaCl bulk solution, supposing that the adsorbed layer is also self-buffering. Unfortunately, it is not really feasible to measure the pD in the surface layer. We have studied the transmission spectra of lysozyme at around 13%(w/w)in 0.1 M NaCl in DzO both with the pD unadjusted and at pD 2. The same changes in the amide I peak as observed by Clark et al.l0 can be seen at 68 "C in the unadjusted solution (Figure 3a) and somewhat lower, at 60 "C, in the solution at pD 2. In additibn, we observe the residual amid I1 region (that is, the residual peak at 1550cm-l due to nonexchanged hydrogen atoms). At around 53 "C,in both cases, this peak starts to decrease in size and rapidly disappears (Figure 4a,b). This can be attributed to an unfolding process in the protein at this temperature, allowing further H-D exchange to take (33) Privalov, P. L.; Khechinashvili,N. N. J . Mol. B i d . 1974,86,666.
b
al
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; 0
n
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1650
1600
1550
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Figure 3. (a) Changes in the amide I peak with temperature for lysozyme in solution at 13%(w/w)in 0.1 M NaCI/D20 with no adjustment of pD. (b) Changes in the amide I peak with temperature for lysozyme in soiution at 13% (w/wj in 0.1 M NaCI/D20 at pD 2.
place. This is clearly not associated with the alteration in shape of the amide I region, firstly because it occurs at a markedly lower temperature, and secondly because the peak shiR in the amide I maximum necessary to cause the appearance of this shoulder is larger than the very small effect that H-D exchange has on the amide I peak. It would appear, then, that there are two processes occurring at both pDs, in these concentrated solutions. The first, occurring at 53 "C, involves some degree of unfolding, which leads to rapid H-D exchange in the interior of the molecule. This is followed by intermolecular interactions, probably including formation of/?-sheet,and subsequent gelation, occurring at 68 "C when the pD is not adjusted and at 60 "C a t pD 2. In both cases, there is a clear shifi in the peak position toward lower wavenumbers on cooling, and there is an increase in the overall peak area. This is illustrated in Figure 5, for the unadjusted solution, where the peak maximum shifts from 1654 to 1667 cm-l. This is presumably a result of further changes in the overall structure of the protein molecules. Thermal Denaturation of Adsorbed Lysozyme. This was carried out on lysozyme adsorbed to silicon for 3 h, Le., within the near plateau region of the adsorbance
Ball and Jones
3546 Langmuir, Vol. 11, No. 9, 1995
- -4OC - -5OC - - 59c
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Figure 4. (a) The residual amide I1 peak as a function of temperature for lysozyme in solution at 13% (w/w) in 0.1 M NaCYDzO with no adjustment of pD. (b)The residual amide I1 peak as a function of temperature for lysozyme in solution at 13%(w/w) in 0.1 M NaCYD20 at pD 2. 0.4
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Figure 5. Spectral changes on cooling a solution of 13%(w/w) lysozyme in 0.1 M NaCl in D2O from 87 to 25 "C.
curve. Adsorption was followed by calibration, exchange ofthe bulk solution for solvent, and equilibration with the solvent in the cell. The cell was then heated as described above. The effects of heat on the adsorbed layer after equilibration with solvent overnight, equilibration for 1
h, and no equilibration were compared. The changes in the shape of the amide I peak with temperature could be followed, but due to the relatively low signal and high level of noise, the residual (hydrogenated)amide I1 region at 1550 cm-I could not be observed, nor could the deuterated amide I1 region at 1450 cm-l be observed, as silicon is opaque to IR radiation below 1470 cm-l. Figure 6 is for a series of spectra taken at increasing temperature in a cell equilibrated overnight before heating. The spectra clearly show the appearance ofa shoulder at 1621 cm-l, accompanied by a smaller shoulder at 1684 cm-'. These are consistent with those occurring on gelation in the bulk, but appear at much lower temperatures, 43-49 "C, intensifjingup to 87-89"C, with a slight decrease in intensity as the temperature is raised further. The overall peak area increases with temperature, from about 40 "C, up to about 80 "C. The shoulder becomes much more pronounced on cooling back to room temperature, the peak area increases further, and there is a shift in the position of the peak maximum from about 1648 to 1641 cm-l (Figure 8). The same results were obtained if the cell was equilibrated with solvent for 1h or not at all before heating. Next we consider the effect of heating the cell with the bulk solution exchanged for a solution of 0.1 M NaCl in D20 at pD 2. In this case, the protein solution is first exchanged for the normal solution of 0.1 M NaCl in D20 and, &r equilibration, is exchanged again for the solution at pD 2. This is aliowed to equilibrate for 1h and then heated as described above. The results are shown in Figure 7. Figure 7 shows changes similar to those in Figure 6, but occurring at still lower temperatures. In this case, the shoulders first appear at 35-37 "C, increase in size up to about 65 "C, remain constant up to 99 "C, and again increase on cooling to room temperature. In both the cases (pD 6.9 and 2) above, a significant increase in the amide I peak area is observed on heating, the temperature of the initial rise corresponding to the temperature at which the spectral changes are first observed. A small shift in the peak position toward lower wavenumbers is observed on cooling. The peak maximum appears to shift from 1648 to 1641 cm-l. The data collected for lysozyme adsorbed over 3 h was also compared with that for short adsorption times of 1-3
Conformational Changes in Adsorbed Proteins
1700
1650
1600
Langmuir, Vol.11, No. 9, 1995 3547
1550
1700
1650
wovenumber (cm-')
Figure 7. Changes in the amide I peak with temperature for lysozyme adsorbed at high levels onto a silicon ATR crystal with bulk solution exchanged for 0.1 M NaCI/DzO at pD 6.9, followed by 0.1 M NaCI/DzO at pD 2.
c ~
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wovenumber (cm-')
Figure 8. Spectral changes on cooling adsorbedlysozyme from 95 to 25 "C.
min. Although calibration of these experiments was not possible, due to the risk of further adsorption from the calibrating solutions, we estimate that the adsorbed amount (using the calibrated runs) cannot be more than 2 mg/m2. If this is so, and monolayer coverage is between 1.8 and 2.7 mg/m2, we are looking at about monolayer coverage, or less, as compared to greater than monolayer coverage with 3 mg/m2 in the longer adsorption times. Adsorption was followed by the exchange of the bulk solution for 0.1 M NaCl in D2O and 1h equilibration time, before heating, as above. The effects of heating are as in the longer term adsorbed material, except that the spectral changes are first observed at higher temperatures, i.e., about 53 "C (Figure 9). This strongly suggests that the conformational changes responsible for the spectral change are due to intermolecular interactions and are facilitated by higher surface concentrations. Again, an increase in the peak area on heating and a shift in the peak maximum toward lower wavenumbers, plus a further increase in the peak area on cooling, are observed. Reversibility of Thermally Induced Changes. In order to probe the extent to which the spectral changes we see were reversible, we heated the samples to temperatures below 99 "C and then cooled them to room temperature again. If the adsorbed layer is heated to a temperature just below the temperature (up to 42 "C) at which spectral changes first occur, on cooling, the spectrum
0.001
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"
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Figure 10. Spectra of adsorbed lysozyme heated t o 42 "C and cooled to room temperature.
reverts to one similar to the original (Figure 10). However, if the h a l heating temperature is only a few degrees above that at which the spectral changes occur (e.g., 50 "C), the spectrum does not revert to the original on cooling (Figure l l ) , indicating that the conformational changes which occur are immediately irreversible nd that the irreversibility detected in the higher temperature experiments is not a function of the temperature. The changes in the spectrum at temperatures a few degrees above 45 "C are not so pronounced as at higher temperatures, however, which suggests that the conformational change occurring here is one which increases with increasing temperature. This could be due either to more change per molecule or to a greater number of molecules changing conformation. Unfortunately, it is not possible to identify which is the case from FTIR experiments alone. Comparison of the Spectral Changes Occurring on Heating of Lysozyme in Solution and Adsorbed on Silicon Oxide. We compare the overall spectral changes observed in the ATR and transmission experiments and correlate these with structural changes in the protein. We do not attempt direct correlation of the frequencies or intensities observed, as differences in the optical systems mean that these are not always exactly
Ball and Jones
3548 Langmuir, Vol. 11, No.9, 1995
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we could detect no conclusive evidence of dichroism on taking polarized spectra of the adsorbed layer, either unheated or heated. No evidence was obtained for an increase in the peak area with temperature in the transmission experiments, suggesting that, if the phenomenon is due to molecular ordering, it is either surface induced or not ordered over a sufficiently long range in the bulk to affect the transmission spectrum.
0
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0.03
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0 02
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Figure 11. Spectra of adsorbed lysozyme heated to 50 "Cand cooled t o room temperature. the same.34 Neither do we attempt to make structural assignments of the sub-bands; rather it is the correlation of the overall shape changes between protein in the bulk and adsorbed at the surface that we have studied. Consequentlywe cannot be certain that the conformational changes occurring in the bulk and in solution are identical; we can only say that they appear similar in character. In adsorbed (ATR) spectra, an increase in the size of the absorbance peak is observed on heating. This is consistent with other ATR work on heated proteins.35In the case of the adsorbed lysozyme here, the increase is marked and initially occurs at about the same temperature as the first signs of /3-sheet formation (Figures 6 and 7). It appears, therefore, to be connected with structural changes in the protein; it has in fact been suggested that peak intensities could be the future means of determining structural changes.34 The changes in intensity may be due to changes in orientation of a significant number of amide groups,possibly combinedwith a change in mobility of the groups leading to rotational averaging. However, (34) Koenig, J. L. Spectroscopy of Polymers; American Chemical Society: Washington, DC, 1992. (35) Kirsch, J. L.; Koenig, J. L. Appl. Spectrosc. 1989, 43,445.
The amide I region of the infrared spectrum of lysozyme adsorbed on silicon changes on heating, in a manner which corresponds to that observed on gelation of heated concentrated solutions, but at much lower temperatures. The temperature at which this change takes place is further reduced by lowering the pD of the medium surrounding the adsorbed protein from 6.9 to 2. It appears that heat-set gelation takes place at a lower temperature for an adsorbed protein than for bulk solutions;moreover, gelation takes place at a lower temperature for an adsorbed layer with a higher adsorbed amount than for a layer with relatively little protein adsorbed at the surface. Concentrations of material adsorbed at the surface will be relatively high, even in material adsorbed from low bulk concentration solutions; and this high local concentration may promote gelation in a way analogous to that observed for synthetic polymers.15J6 We conclude that although our own experiments show no clear evidence of structural rearrangement of lysozyme molecules on adsorption at ambient temperatures, and other experiments clearly suggest that this does not occur,G the effect of surface adsorption is to allow molecular rearrangement as a result of applied stresses such as increased temperature to take place more easily than would normally be the case (i.e., at lower temperatures).
Acknowledgment. We would like to acknowledge helpful discussions with Dr. D. C. Clark of the BBSRC Food Research Institute, Norwich. The data used in Figures 3b and 4b were collected by Dr. I. Hopkinson. This project is funded by the Agricultural and Food Research Council (now Biotechnology and Biological Sciences Research Council). LA950085V
