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Langmuir 1996, 12, 3272-3275
Role of Protein Unfolding in Monolayer Formation on Air-Water Interface Andrey Tronin,*,† Timothy Dubrovsky,*,‡ Svetlana Dubrovskaya,§ Giuliano Radicchi,§ and Claudio Nicolini§ Institute of Crystallography of Academy of Sciences of Russia, Leninsky pr. 59, Moscow 117333, Russia, Institute of Biochemistry of Academy of Sciences of Russia, Leninsky pr. 33, Moscow 117071, Russia, and Institute of Biophysics, University of Genoa, via Giotto 2, Genoa Sestri Ponente 16153, Italy Received October 16, 1995. In Final Form: February 8, 1996X Molecular exchange kinetics between a monolayer of antibody molecules formed on the air-water interface and the protein solution was studied by means of fluorescent labeling. It was shown that there is no inclusion of dissolved molecules in the previously formed monolayer during even 6 h of exposure regardless of monolayer surface density. The surface activity of IgG and horseradish peroxidase molecules was studied by means of surface compression isotherms, and the specific biological activity of the monolayers formed from these proteins was measured by enzyme and immunoassay techniques. It was shown that the surface activity of the proteins increases while specific biological activity decreases with exposure of the molecules on the water surface. Since the same effects were caused by denaturing agents, we propose that the surface activity of the proteins and the absence of surface-volume exchange are due to partial unfolding of the molecules which takes place on the water surface. Two models of the partial unfolding are discussed: complete denaturation of some part of the molecules and partial unfolding of each molecule. The process of surface denaturation was shown to be slow and controllable. One can achieve a pronounced increase of protein surface activity with low degradation of the specific biological activity of the monolayer; thus, it can be used in the practice of protein Langmuir film deposition.
1. Introduction The process of protein monolayer formation on airwater interfaces is of great importance for LB film fabrication. Although globular proteins are water soluble, many of them adsorb to the water surface, forming rather dense monolayers. There are numerous reliable data on the matter such as compression isotherms,1,2 ellipsometric,3 and surface density4 measurements of the monolayers of different proteins. The monolayers can be transferred, usually by the Langmuir-Schaefer technique, onto solid supports. Despite well-known surface denaturation, proteins in the transferred films preserve their secondary and tertiary structure, as was shown by spectra of circular dichroism and specific activity measurements for immunoglobulins,5 reaction centers,6 and some enzymes,7 etc. The films on the air-water interface can be formed either by spreading some amount of protein solution on the water surface8 or upon specific adsorption to the surface of the protein molecules injected into the subphase.9,10 In both cases an equilibrium between the molecules in the * Corresponding authors. † Institute of Crystallography of Academy of Sciences of Russia. E-mail:
[email protected]. ‡ Institute of Biochemistry of Academy of Sciences of Russia. E-mail:
[email protected]. § University of Genoa. X Abstract published in Advance ACS Abstracts, May 1, 1996. (1) Ahluvalia, A.; DeRossi, D.; Ristori, C.; Schirone, A.; Serra, G. Biosens. Bioelectron. 1991, 7, 207. (2) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1992, 205, 113. (3) Tronin, A.; Dubrovsky, T.; Nicolini, C. Langmuir 1995, 11, 385. (4) Dubrovsky, T. B.; Demcheva, M. V.; Savitsky, A. P.; Mantrova, E. Yu.; Yaropolov, A. I.; Savransky, V. V.; Belovolova, L. V. Biosens. Bioelectron. 1993, 8, 377. (5) Erokhin, V.; Facci, P.; Nicolini, C. Biosens. Bioelectron. 1995, 10, 25. (6) Facci, P.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 243, 403. (7) Dubrovsky, T.; Vakula, S.; Nicolini, C. Sens. Actuators, B 1994, 22, 69. (8) Tronin, A.; Dubrovsky, T.; De Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127.
S0743-7463(95)00879-1 CCC: $12.00
subphase and those on the surface is established. The duration of the transition period and the final ratio of surface/volume molecules depend on many factors such as trough configuration, temperature, subphase composition, type of protein, etc. Some typical estimations for the former are 2-5 h10 and 1:100 (1 molecule on the surface per 100 molecules in the solution) for the latter.4 Despite certain progress in protein LB film fabrication and even application, little understanding of the monolayer formation has been achieved yet. What forces govern this process, what are the state and structure of the resulting layer, how stable is it, what is the degree of protein denaturation, etc.sall these questions remain open so far. In this paper we address the problem of volume-surface equilibrium. We have undertaken a study of the molecular exchange kinetics between the solution and the surface layer and have not observed any inclusion of dissolved molecules in the previously formed monolayer. We suppose that this stability of the monolayer originates from partial unfolding of protein globules. The essential point is that it is possible to achieve a state of partial denaturation with preserved specific activity. 2. Materials and Methods Abbreviations Used. BSA, bovine serum albumin; MAR antibodies, polyclonal mouse anti-rat IgG; TBS, Tris-buffered saline, 30 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl, 0.05% NaN3, pH 7.4; blocking buffer, 1% BSA, 0.05% Tween-20 in TBS; binding buffer, 0.3% BSA in TBS; wash buffer TBST, 0.05% Tween-20 in TBS; GOPTS, 3-(glycidoxypropyl)trimethoxysilane; FITC, fluorescein isothiocyanate; DMF, dimethylformamide; HRP, horseradish peroxidase; ABTS, 2,2′-azinobis(3ethylbenzthiazolinesulphonate) diammonium salt; GuHCl, guanidine hydrochloride. 2.1. Materials. MAR affinity-purified polyclonal antibodies, whole molecule, specified to rat IgG (H+L); rat affinity-purified (9) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 125. (10) Kayushina, R.; Khurgin, Yu.; Sukhorukov, G.; Dubrovsky, T. Physica B 1994, 198, 131.
© 1996 American Chemical Society
Role of Protein Unfolding in Monolayer Formation polyclonal antibodies, whole molecule; HRP ImmunoPure; GuHCl 8 M solution, sequenal grade; and Blocker BSA were purchased from Pierce. GOPTS, reagent grade; sodium hydrogen carbonate, sodium chloride, sodium acetate, A.C.S. reagent grade; hydrogen peroxide, 30 wt % solution in water, A.C.S. reagent grade; sodium azide, FITC, Tween-20, reagent grade; DMF, HPLC grade; and tris(hydroxymethyl)aminomethane, ultrapure grade were purchased from Aldrich. Sephadex G-50, G-100, and Sepharose 6B were purchased from Pharmacia Biotechnology. All reagents were used as received. Water was Milli-Q purified with a resistance of 18.2 MΩ cm. 2.2. Modification of the Supports. Quartz disks with polished surfaces were carefully cleaned by soaking in fresh chromic acid, rinsed in water, dried under nitrogen flux, and silanized with GOPTS under vacuum in an apparatus4 according to the method described by Malmqvist.11 Prepared in this way, the surface remains hydrophilic and has exposed active epoxy groups which react readily with lysine -amino groups. This surface preparation method has been successfully used for previous immobilization of antibody and enzyme LB films.3,4 2.3. Deposition of the Films. Protein monolayers were formed in a Langmuir trough (KSV 5000 LB, Finland) which had been equipped with a home-made Teflon trough with dimensions of 55 × 200 × 7 mm3 and a volume of 95 mL. The trough was treated with methylene chloride before the monolayer formation and then dried with nitrogen. Carbonate buffer (50 mM) containing 150 mM NaCl, pH 8.9, was used as the subphase. This pH value is necessary for protein covalent binding. Careful surface cleaning was carried out before the monolayer spreading. IgG solution in TBS (100 µL)with a concentration of 1-4 mg/mL and 400 µL of HRP solution in TBS with a concentration of 2 mg/mL were spread on the subphase with a Hamilton rheodyne syringe (final concentration was in the range 30-100 pM). The barrier speed during compression was 2-3 mm/s. Transfer of LB films from the subphase surface onto the activated supports was performed by “touching” the silanized support in parallel mode to the subphase surface (analogous to the LangmuirSchaefer method). This technique had been successfully used for various protein film depositions.3-8 The supports were lifted after the touching with a vertical speed of 1cm/s. After film deposition, the samples were dried under nitrogen flux, incubated for 10 h at 4 °C, washed with TBST and distilled water, and dried under nitrogen flux. Each measurement was averaged over four depositions. 2.4. Measurements of Specific Activity of the Films. Antigen-antibody binding was monitored by a fluorescence technique. Rat antibodies, used as antigen, were conjugated with FITC. After antibody film deposition the supports were incubated in blocking buffer for 1 h at 37 °C with gentle stirring for blocking the excess binding sites. The immunological binding was performed from the binding buffer with gentle stirring at 37 °C for 1 h. Washing twice with TBST and water and drying in nitrogen flux were performed before the fluorescence measurement. HRP enzymatic activity was measured as oxidation of ABTS. Accumulation of oxidized product was registered at 410 nm spectrophotometrically (Jasco spectrophotometer, JS 7800). ABTS (2 mM final concentration) was dissolved in 100 mM sodium acetate buffer, pH 4.2, containing 2.5 mM H2O2 and 0.1% Tween-20. 2.5. Labeling of Antibody with FITC. Antibodies were labeled by direct coupling to FITC in accordance with the method in ref 12. Sephadex G-100 with column size 1.5/30 cm was used for separation of labeled IgG from free label. A precolumn of Sepharose 6B (1.5/15 cm) was used for removing possible protein aggregates. The labeling degree was determined spectrophotometrically, using the molar extinction coefficient for FITC, 494 ) 62 000 M-1 cm-1. For mouse anti-rat polyclonal antibodies, labeling degree was calculated as 8 mol of FITC/1 mol IgG; for rat polyclonal antibodies, labeling degree was calculated as 6.2 mol of FITC/1 mol of IgG. 2.6. Fluorescence Measurements. Fluorescence of the films was measured by a Zeiss Axioplan microscope (Zeiss Co., Germany) equipped with a mercury lamp, a filter set consisting (11) Malmqvist, M.; Olofsson, G. U.S. Patent 4,833,093, 1989. (12) Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory: Plainview, NY, 1988.
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Figure 1. Fluorescence intensity of immobilized IgG film as a function of concentration of FITC-labeled molecules in the film. Straight line was used as a calibration curve for labeled IgG concentration determination. Table 1. Fluorescence (in Arbitrary Units) of IgG Film Deposited in Certain Time Intervalsa surface pressure, mN/m
without exposure
5 min exposure
2h exposure
6h exposure
25 15 10
190 194 191
195 191 193
200 194 195
200 216 195
a Accuracy of measurements is about 10 au; noise level is in the range 180-200 au.
of BP 450-490, FT 510, and BP 515-565, and a 40× objective. Images were acquired by CCD camera CH260 (Photometrix Co., Germany) cooled at -35 °C. The exposure time was 2800 ms. The area of acquisition was 87 × 87 µm2. The integrated fluorescence intensity was calculated averaging over all pixels. Five images were acquired for every sample, and the integrated intensity was averaged over the acquisitions. The integrated intensity dispersion of acquisitions of one sample was less than 5%; the dispersion over different samples deposited under equal conditions was less than 3%, which shows good reproducibility of the monolayer formation and deposition. The images were uniform, revealing normally no visible inhomogeneites. In order to calibrate the fluorimetry measurements, films composed of various ratios of labeled and unlabeled IgG were deposited and their fluorescence intensity was measured. The dependence of the intensity on the labeled IgG content in the mixture is shown in Figure 1. One can see the linear nature of the dependence. The intensity was stable in time; it did not change after a 2 weeks storage of the samples at 4 °C.
3. Results and Discussion To study the exchange of the protein in the monolayer and subphase, we applied fluorescent labeling of mouse IgG molecules. Unlabeled IgG molecules were deposited on the water surface, exposed for 5 min, and compressed up to a certain surface pressure which was held constant during the whole experiment. After the compression of the monolayer, the labeled IgG molecules were carefully injected into the subphase in the same amount as for the unlabeled ones. After a certain period of time the monolayer was deposited onto the silanized quartz support and its fluorescence was measured. A new film was prepared for each measurement. Such a procedure prevents inclusion of the dissolved molecules in the monolayer caused by its disturbance. The time dependence of the fluorescence intensity with respect to the surface pressure of the monolayer is presented in Table 1. One can see that no matter what pressure is applied the intensity remains at the background level even after 6 h of exposure. The overall accuracy of the measurements allows us to state that the concentration of the labeled
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Tronin et al.
Table 2. Time Dependence of IgG Monolayer Area at Fixed Surface Pressuresa surface pressure, mN/m
initial film area, cm2
film area after 6 h exposure, cm2
25 15
79 123
70 184
a Note that at 15 mN/m film expands, while at 25 mN/m it slightly compresses.
IgG in the monolayer is less than 7%. This result shows that due to some reason the exchange is very restricted if it exists at all. In fact, in the case of free exchange the film would consist of 100% “new” molecules. (One should remember that almost all of the protein spread on the surface goes into the solution and only some 1% remains on the surface. Thus the number of molecules which initially form the monolayer is negligible in comparison with the total amount of molecules in the bulk.4) Since half of the volume molecules are labeled, in the case of a free exchange their concentration in the film would have been 50%. The absence of the exchange shows that the adsorption energy is rather high. However, this contradicts the general view of the proteins having a very weak surface activity. Experimental data also exclude such a conclusion. At the pressure of 10 mN/m the monolayer is very rarefied. The corresponding area per molecule is 800 nm2 (ref 4), which is much greater than a molecular cross section (160 nm2 (ref 13)). After the injection of labeled protein into the subphase, the IgG volume concentration is increased two times. The surface-volume equilibrium is shifted; thus, the appearance of the extra molecules at the surface can be expected but does not occur in fact. Thus, the reason for the absence of the exchange is not the high adsorption energy. It is more likely that the state or conformation of the molecules in the formed monolayer differs from those in the solution; this difference irreversibly stabilizes them at the surface. We find a clue for the explanation of the nature of the protein surface stabilization in the film area behavior. These data are given in Table 2. The film expands under low pressure (15 mN/m). At the same time no extra molecules appear in the film. It means that the effective molecular cross section grows with the exposure time, which most likely is the result of the surface molecules unfolding. The unfolding also results in the inner hydrophobic parts of the globule being exposed to the airwater interface. At a higher pressure, that is at a lower surface tension, the surface force cannot unfold the globule; thus, the film does not expand at 25 mN/m. The increase of the hydrophobic properties of the unfolded molecules is revealed by compression isotherms. To illustrate this process, we have registered pressurearea dependencies under the influence of various denaturing factors (Figure 2). Curve 1 is a basic isotherm obtained immediately upon spreading the protein on the subphase. Curve 2 was obtained under the same conditions as the first one but with a 15 min delay between spreading and compression. Curve 3 was obtained immediately after spreading, but the subphase contained 100 mM GuHCl, which is a well-known chaotropic agent.14 To obtain curve 4 we denatured the IgG sample completely in a 3 M solution of GuHCl and spread it on the subphase. One can see that the effects of exposing the film and adding GuHCl in a minor concentration are qualitatively the same. To understand how the exposure affects the (13) Deisenhofer, J. Protein Data Bank; Brookhaven National Laboratory: 1982; Ident. Code 1FC1, 1FC2. (14) Franks, F. Characterization of Proteins; Humana Press Inc.: Totowa, NJ, 1988.
Figure 2. IgG monolayer compression isotherms obtained under various conditions. 100 µL of a 1 mg/mL solution of IgG was spread on the buffer surface. 1, basic isotherm obtained immediately upon spreading of the protein on the subphase; 2, 15 min delay between spreading and compression; 3, immediately after spreading on 100 mM GuHCl containing subphase; 4, spread protein completely denatured in a 3 M solution of GuHCl.
Figure 3. Circular dichroism spectra for deposited films (a) and solutions (b) of IgG. (a) Solid line, film deposited immediately upon formation of monolayer; dashed line, deposition made after 15 min of exposure at the water surface. (b) Solid line, native protein; dashed line, denatured protein.
molecular structure, we measured the circular dichroism of the films and solutions of IgG subjected to the influence of different factors. Figure 3a shows CD spectra of the films, deposited on the quartz supports almost immediately after the monolayer formation (solid line) and after 15 min of exposure at the water surface (dashed line). One can see a pronounced decrease of the molar ellipticity in the second case. The same decrease is observed when the dissolved IgG molecules were denatured by GuHCl. The corresponding spectra for native and denatured molecules are shown in Figure 3b by solid and dashed lines, respectively. In order to check whether the molecules are completely unfolded or not, we measured the immunological activity of the films. The deposition was made a surface pressure of 25 mN/m. In order to achieve such pressure without
Role of Protein Unfolding in Monolayer Formation
Figure 4. Fluorescence intensity of labeled antigen (rat IgGFITC) bound by MAR IgG films deposited under the affect of various denaturing factors: control, nonspecific binding to the surface.
exposure and in the absence of GuHCl (curve 1) we spread a fourfold amount of protein. Unlabeled MAR IgG was used for the film deposition. Immunological binding with FITC-labeled rat IgG was measured using a fluorescent method. The results are presented in Figure 4. Binding by MAR IgG, which was completely denatured in 3 M GuHCl solution, was at a nonspecific binding level (control). One can see that the films deposited after a 30 min interval or from the subphase containing 100 mM GuHCl remain active; the slight decrease of the activity is almost within the experimental accuracy. Since the immunological activity of a molecule is related to the tertiary structure, we can conclude that the unfolding of the molecules is partial; e.g., either the whole form of the globule is preserved with only some part of the peptide chain being unfolded or the layer consists of some completely unfolded molecules and an essential amount of native ones. In the second case the native molecules are incorporated into but not adsorbed to the layer of unfolded chains. The unfolded chains serve like a matrix for the native molecules, stabilizing the film as a whole. Deposition onto the silanized substrate and successive washing with detergent (TBST) ensures the deposition of the superficial molecules exclusively. Only those molecules that are covalently bound stay on the support after washing; otherwise we would have seen the labeled “volume” molecules at the supports in our experiments on the monolayer stability. (In fact we detected them when we had not used a detergent for washing.) We cannot make a choice between these two models of partial denaturation; moreover, a combined situation can be imagined as well. In any case, the unfolding seems to have two stages with different time scales. The first one, rather rapid, takes place upon protein spreading. After some minutes the unfolding stabilizes the monolayer, making it unpenetrable for the volume molecules. At the second stage the surface molecules continue to unfold but much more slowly. Unfolding can be stopped by increasing the surface pressure. At a surface pressure of 25 mN/m the monolayer does not expand and the molecules remain
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Figure 5. HRP monolayer compression isotherms obtained with and without delay between spreading and compression: 1, without delay; 2, 15 min delay. The values indicate the relative enzymatic activity of the corresponding films (spectrophotometrical measurements).
active even after 6 h of exposure, while at the pressure of 10 mN/m we registered the whole loss of immunological activity after the same exposure time. In order to generalize the consideration given above, we have undertaken a similar study with horseradish peroxidase. It is a globular protein, but its structure is very different from that of IgG. The main differences are that the HRP molecule does not have S-S bonds and contains a molecule of protoheme IX as a prosthetic group. The isotherms of the HRP monolayer obtained after different exposure times are shown in Figure 5. The relative enzymatic activity of the films is indicated near each curve. In this case we also see that the exposure makes molecules more surface active (the isotherm 2 goes much higher), not affecting the enzymatic activity essentially. 4. Conclusions We believe that the results of the study undertaken with IgG and HRP allow us to make the following general conclusions: 1. The protein molecules on the water-air interface are partially denatured. It means that either some part of the molecules is completely unfolded or each molecule is unfolded to some extent. In any case the native or “quasi native” molecules are incorporated in but not adsorbed to the monolayer. 2. The partial denaturation does not affect significantly the specific activity of the molecules in the monolayer. 3. The partial denaturation stabilizes protein molecules on the air-water interface. 4. One can control the process of the denaturation by changing the time of the molecule exposure on the water surface and use it for the formation of the soluble protein monolayer. Acknowledgment. This work was supported by ELBA Foundation. LA950879+
