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Langmuir 1991, 7, 238-242
Spin-Labeling Study of Human Serum Albumin in Reverse Micelles P. Marzola,*tt C. Pinzino,t and C. A. Veracinit Dipartimento di Chimica e Chimica Industriale, via Risorgimento 35-56126 Pisa, Italy, and Istituto di Chimica Quantistica ed Energetica Molecolare, via Risorgimento 35-56126 Pisa, Italy Received J u n e 14, 1990 Human serum albumin (HSA), spin labeled at the sulfhydryl group by the reagent 3-maleimidoproxyl (3MAL), was studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The electron spin resonance spectra were recorded at different water contents and analyzed by computer simulations. In order to obtain agreement between experimental and calculated spectra, the use of an anisotropic model of reorientational diffusion for 3MAL-HSA in reverse micelles was necessary. An isotropic reorientational motion was suitable to simulate the 3MAL-HSA spectrum in aqueous solution. This result suggests that conformational changes of the protein which modify the label environment occur in reverse micelles. The rotational correlation times for 3MAL-HSA in reverse micelles were strongly dependent on the water content: the protein experienced a more hindered environment for rotational diffusion as the water pool size decreased.
Introduction Dissolution of surfactants in organic solvents may give rise to spherical aggregates, called reverse micelles.1~2 The polar head groups of surfactants are directed toward the interior of the aggregate, thus forming a polar core which can solubilize water (water pool). The unusual physical properties of the entrapped water, markedly different from those of bulk water, have drawn the attention of many investigators in the last few The capability of reverse micelles to host proteins and other biomolecules is now well documented, and the resulting structural and dynamical properties are investigated with increasing interest.'^^ The potential of reversed micelle systems in studying the hydration of biological molecules has been reported.3 In these systems, the absence of large pools of bulk water allows one to put in evidence the physical parameters related to the interfacial water (water adjacent to biological molecule^).^ We carried out an electron spin resonance (ESR) spinlabeling6study on the effect of the water pool size on the dynamics of a guest protein. The aim was to explore the usefulness of reverse micelles in studying the relation between hydration water and protein dynamics. Moreover, since some enzymes, when hosted in reverse micelles, show an activity which can sometimes be higher than in aqueous solution, these systems have acquired importance also in the field of biotechnology.5 The investigation of the structural and dynamical properties of the guest molecules, as well as their location in the micellar environment, is important for understanding their activity and can give information relevant for practical applications of these systems.'
The electron spin resonance (ESR) technique has been recently utilized in reverse micelles. The location of spin labels (molecules bearing a free nitroxide radical) in reverse micelles'and the water pool dynamicsa have been studied. Spin labels are also designed and synthesized in order to bind covalently at particular groups in protein^.^ The ESR spectrum of a labeled protein gives information on the protein dynamics, the local physicochemical environment experienced by the macromolecule, and the structural modifications that possibly occurred in the macromolecule itself.6 The present study was carried out on a well-characterized protein, the human serum albumin (HSA). HSA was labeled at the free sulfhydryl group by a nitroxide spin label with a maleimide attaching group, 3-maleimidoproxyl (SMAL), and introduced in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The ESR spectra were analyzed by using computer simulations for slow motions.'OJ The best simulations were obtained by using an anisotropic model of rotational reorientation. From this analysis and from the comparison with the spectrum in aqueous solution, we inferred that HSA, hosted in reverse micelles, undergoes conformational changes, which could be due to interactions with the AOT molecules. The increase of the water content resulted in an increased mobility of the protein. In particular, the rotational correlation times of the 3MAL-HSA system sharply decreased in the range of water to AOT concentration ratio (WO)between 2.2 and 7.2; at w o greater than 7.2, no further significant changes were detected.
Materials and Methods Dipartimento di Chimica e Chimica Industriale. Istituto di Chimica Quantistica ed Energetica Molecolare. (1) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (2) Luisi, P. L.; Steinmann-Hofmann, B. Methods Enzymol. 1987,236, t
*
188. (3) Gierasch, L. M.; Thompson, K. F.; Lacy, J. E.; Rockwell, A. L. In Reuerse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1986; p 265. (4) Wong, M.; Thomas, J. K.; Nowak, T. J . Am. Chem. Soc. 1977,99, 4730. ( 5 ) Luthi, P.; Luisi, P. L. J. A m . Chem. SOC.1984, 106,7285. (6) Spin Labeling: theory and applications; Berliner, L. J., Ed.; Academic: New York, 1976; Vol. 1.
0743-7463/91/2407-0238$02.50/0
Chemicals. HSA (essentially fatty acid free, A-1887) and 3-maleimidoproxyl (3MAL) were purchased from Sigma. AOT (research grade) was obtained from Serva and purified according to ref 4; its purity was checked by the procedure of Luisi et al.* Isooctane of spectroscopic grade was purchased from Merck and (7) Hawing, G.; Luisi, P. L.; Hauser, H. J. Phys. Chem. 1988,92,3574. (8) Kotake, Y.; Janzen, E. G. J. Phys. Chem. 1988,92, 6357. (9) Morrisett, J. D. In ref 6, Chapter 8. (10) Freed, J. H. In ref 6, Chapter 3. (11) Schneider, D. J.;Freed, J. H. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J., Eds.; Plenum: New York, 1989; Chapter 1.
0 1991 American Chemical Society
Langmuir, Vol. 7, No. 2, 1991 239
Spin-Labeling of Human Serum Albumin
SI
n
V
7
20 G
Figure 1. Pattern a: experimental ESR spectrum of 3MALHSA in buffer a t 20 "C; experimental settings were field set 3270 G, microwave power 5 mW. Pattern a': computer simulation of 3MAL-HSA spectrum; the parameters are reported in the test. used without further purification. Buffer (Tris: 50 mM, pH 7.4) was used for all experiments. Spin Labeling of HSA and Preparation of Micellar Solutions. Spin labeling of HSA was carried out as reported in the literature;I2 the unreacted label was removed by extensive dialysis against buffer. Micellar solutions of desired w o were obtained by adding concentrated protein solutions or plain buffer to a 100 mM AOT solution in isooctane; the parameter w o = [H*O]/[AOT] measures the water content. The samples were gently shaken until complete clarification. The HSA concentration was kept roughly constant and less than 0.02 mM, as determined by absorbance at 279 nm.l3 Spectroscopic Measurements. ESR spectra were recorded a t 9.2 GHz and 20 "C on a Varian E-112 spectrometer equipped with a Varian variable-temperature controller. The low amount of water present in reverse micelles allowed one to place samples in quartz ESR tubes with a diameter of 4 mm, thus giving a satisfactory signal to noise ratio. 2,2-Diphenyl-l-picrylhydrazyl (DPPH) powder was used as agvalue standard (g = 2.0037). The experimental settings are reported in the figure captions. The rigid-limit ESR spectrum of spin-labeled HSA was obtained a t -50 "C in 50% (v/v) glycerol-buffer to determine all elements of the nitrogen hyperfine (A) and electron Zeeman (g) tensors that are required as inputs for computing rotational correlation times. The absorbance measurements were performed on a Pye Unicam SPS-150 UV-vis spectrophotometer. Circular dichroism spectra were recorded on a Jasco 5500 spectropolarimeter. Fluorescence spectra were acquired on a Greg 200 multifrequency phase fluorometer (ISS-Italy) used in the steady-state mode of operation. Computational Methods. The programs of FreedloJ1were implemented on an Encore N P l vector computer. Computational time was about 60 s for the most complex spectra by using the last published version." Output data were received on an AT compatible personal computer for visualization and plot.
Results and Discussion HSA in Buffer. Figure 1 (pattern a) shows the ESR spectrum of the labeled and extensively dialyzed HSA in buffer solution a t 20 OC. The spectrum contains contributions from a highly immobilized label (arrows W) and a more freely rotating spin label (arrows S). The last contribution cannot be attributed to the presence of unbound label. In fact, we reproducibly obtained this composite spectrum also after gel filtration through a Sephadex G25 column as well as after ultrafiltration (12) Cornell, C. N.; Chang, R.; Kaplan, L. J. Arch. Biochem. Biophys. 1981, 209, 1. (13)Steinhardt, J.; Krijn, J.; Leidy, J. G. Biochemistry 1971,10,4005.
through a Spectrum C20K membrane. A similar spectrum was previously reported for 3MAL-labeled HSA in ref 12, where the weakly immobilized component was attributed to labels bound to a surface lysyl residue. The highly immobilized component is due to spin label bound to the free sulfhydryl group.12 The rotational correlation time of the protein-bound spin label can be obtained by computer simulation of the ESR line shape if the components of the g and A tensors are known.1° These values can be determined from the simulation of the rigid-limit spectrum."J This spectrum was obtained in 50% (v/v) glycerol-buffer at -50 OC14 and simulated by using a Brownian motion model of isotropic rotational diffusion. The best simulation was achieved by using the following values of the magnetic parameters: A,, = A, = 6.325 G , A,, = 36.1 G,g,, = 2.0084, g,, = 2.0061, and g,, = 2.0025. Line broadening equal to 3.7 G was used; the rotational correlation time was 0.98 x 10-6 s. The HSA spectrum of Figure 1was simulated by using the previously determined parameters and a superposition of two spectral components having a rotational correlation time of 11.1 and 0.21 ns, respectively. The Brownian motion model of isotropic rotational diffusion was used; the line broadening was 1.25 G. The calculated spectrum is shown in Figure 1 (pattern a'). The rotational correlation time of the weakly immobilized component (0.21 ns) indicates that the spin label is bound to the albumin molecule a t sites which allow the label to retain a high degree of rotational freedom with respect to the whole protein. The go = (1/3)(gxx+ g , + gz,) and A0 = (1/3)(A,, + A, + Az,) values of this component are equal to the go and A0 values of 3MAL in buffer (spectrum not shown); these values are consistent with a spin label bound to surfacial, solvent-exposed groups of the protein. The strongly immobilized component of the HSA spectrum is due to the label bound to the sulfhydryl group. The sulfhydryl group environment in HSA (and in bovine serum albumin (BSA) which is very similar to HSA) has been widely investigated by ESR. In particular, it was studied by the so-called "molecular dipstick" technique: spectra of the system under investigation are recorded with a series of spin labels, varying in the length of the chain between the nitroxide free radical and the attaching group, and their motion is studied as a function of chain length.12J5 Both in BSA and in HSA it was shown that the sulfhydryl group is localized in a somewhat restrictive, crevice-like region of the protein12J5 at a depth of about 10 A from the surface. However, since the rotational correlation time of the strongly immobilized component (11.1ns) is smaller than the value expected for a protein with a molecular weight of 66.000 D (about 22 ns),I6even in the above-mentioned buried location, the 3MAL molecule possesses some residual motion with respect to the whole macromolecule. The present value of rotational correlation time for the 3MAL-HSA system is in agreement with previously reported data.12 HSA in Reverse Micelles. Some representative spectra of spin-labeled HSA in reverse micelles a t varying water content are reported in Figure 2; the experimental spectra are named a, b, c, and d. The calculated spectra, reported in the same figure, are named a', b', c', and d'. As we previously observed for HSA in buffer, the ESR spectra of the labeled protein in reverse micelles contain (14) Chang, S.; Hammes, G. G. Biochemistry 1986, 25, 4661. (15) Cornell, C. N.; Kaplan, L. J. Biochemistry 1978,17, 1750. (16) Cannistraro, S.; Sacchetti, F. Phys. Reu. A 1986, 33, 745.
Marzola et al.
240 Langmuir, Vol. 7,No. 2, 1991
644
5
10
8
8
20
40
0
wo Figure 3. Separation between the outer hyperfine extrema of
3MAL-HSA spectra in reverse micelles as a function of
WO.
Figure 4. Molecular structure of 3-maleimidoproxyl. The structure of the proxy1 group is from ref 25, and the maleimide moiety was obtained by using a force field method (N. L. Allinger, University of Georgia, Athens, GA 30602). The nitroxide molecular axes (x,y,z) are labeled according to refs 10 and 11. The principal axis of the tensors A and g is assumed to be coincident and rigidly fixed to the molecular framework.
v
-
206
Figure 2. Experimental ESR spectra of 3MAL-HSA in reverse micelles: (a) wo = 2.2, (b) wo = 5.9, (c) wo = 7.2, (d) wo = 26.3. The relative simulations are reported as a’, b’, c’, and d’. The experimentalsettings were the same as reported in Figure 1.The
simulationswere obtained with the parametersreported in Table I. contributions from a weakly and a strongly immobilized component. A t low water content (patterns a, b, and c), the weakly immobilized component is practically absent, while it is observable at high wo (pattern d). The lack of motional freedom for this surfacial label a t low wo can be justified by considering that in these conditions a very thin layer of water (with respect to the label dimensions) is present around the protein. By applying to HSA the simple model proposed by Luisi et al.,17the width for the water layer around the protein can be estimated. According to this model, at wo = 2.2 and wo = 7.2 a layer of about 3 and 6 A of water, respectively, is present around the protein, thus supporting our hypothesis. A t w o> 7.2, the weakly immobilized component becomes detectable and increases in intensity with the water content. The hyperfine splitting and the go value of the weakly immobilized component were the same as in buffer, indicating that no significant change in the solvent polarity is experienced.9 This finding indicates that the weakly (17)Bonner, F.J.; Wolf,R.; Luisi, P.L.J. Solid-Phose Biochem. 1980, 5 , 255.
immobilized label is localized in the micellar water pool, at least in the range of wo in which it is detectable. Let us examine, now, the strongly immobilized component of 3MAL-HSA spectrum in reverse micelles. From Figure 2, it can be observed that the separation between the outer hyperfine extrema (generally referred to as 2A,’)lo decreases by increasing WO. A plot of 2A,‘ versus wo is reported in Figure 3: 2A,’ decreased from 65.1 to 59.7 G in the range 2.2 Iwg I 7.2 while it remained practically constant (around 59.5 G ) a t w o 2 8.5. The value of 2A,’, a t high W O ,is smaller than the value obtained for 3MALHSA in buffer (64 G). A decrease in 2A;indicates a parallel decrease in the rotational correlation time of the label; however, direct information on the rotational correlation times can be obtained, from 2A,‘, only if the motion is isotropic.10 Unlike the spectrum in buffer, the 3MAL-HSA spectra in reverse micelles could not be simulated by assuming isotropic rotational diffusion, and an anisotropic model for rotational diffusion had to be used. We first simulated the spectra by using the Freed program reported in ref 10, which includes axially symmetric rotational reorientation about a principal axis of the tensors A and g (see Figure 4). We obtained a good reproduction of the experimental spectra by assuming a rapid reorientational motion about the nitroxide x axis (with a diffusion coefficient RIIand a correlation time 711 = 1/6Rll)and a slower motion around the perpendicular plane (diffusion coefficient R l and correlation time 7 1 = 1/6R1). We then simulated the spectra by using the last published version of the Freed program,” which includes a rotational reorientation axially symmetric about an axis forming a variable angle, +, with respect to the nitroxide
Spin-Labeling of Human Serum Albumin
Langmuir, Vol. 7, No. 2, 1991 241
Table I. Parameters Used in Computer Simulations of the Strongly Immobilized Component of SMAL-HSA Spectra in Reverse Micelles. wo
71,ns
2.2 3.4 4.6 5.9 7.2 8.5 11.2 14.1 20.0 26.3 33.1 40.5 48.2
151.5 79.4 41.7 40.6 32.0 31.2 30.5 30.5 30.0 29.8 29.0 29.2 29.2
711,
ns
6.7 5.0 4.1 3.7 3.6 3.5 3.4 3.1 3.1 3.1 3.0 2.9 2.9
A,, = A,, = 6.325 G, A,, = 36.1; g,, = 2.0084, gyy = 2.0073, gZ2= 2.0030 (g,, = 2.0084,gyy= 2.0073,g~~ = 2.0025, at wo = 2.2); the line broadening was 1.75 G a t 2.2 5 wo 5 8.5, 1.25 G a t 11.2 5 wo 5 20.0, and 1.00 G at
U~O 2
26.3.
t axis.
The new simulation confirmed the previous results, since the best simulations were obtained with = 90". The simulations reported in Figure 2 were obtained by using the new version of the program.11 The magnetic parameters determined in buffer were not suitable to simulate 3MAL-HSA spectra in reverse micelles. The magnetic parameters used for the simulations are reported in Table I; they were obtained by a trial and error procedure carried out minimizing the integral of the square difference between the calculated and the simulated spectra. The components of g are slightly different from those determined in aqueous solution, the value of go being higher in reverse micelles ( A g o = 0.0005). It is well-known that the parameters A0 and go of nitroxides depend on solvent polarity;18 in general, A0 decreases and go slightly increases with decreasing solvent polarity. The increase ofgo, observed in the present case, is probably due to variations in the label environment polarity; it could be accompanied by a decrease in A0 of 1 or 2 G.18 The components of A can be considered as variable parameters in the simulation program. However, in the present work we report the data obtained by using the components of A determined in buffer, for the following reasons. Since the spectra are not well resolved, the simulations were quite insensitive to variations, by the order of 1 G, on A,, and AYy. They were only sensitive to variations on A,,. It is well-known that a decrease in A,, increases the correlation times necessary to reproduce the position of the outer hyperfine extrema.10 We carried out some simulations by changing the A,, component with respect to the value determined in solution, but a decrease in A,,, by 1 or 2 G (and a subsequent increase of TI( and 71), did not result in any improvement of the simulations (as judged by the integral of the square difference between the simulated and experimental spectra). In other words, we did not have a valid tool to monitor possible variations in the components of A. From these simulations, we calculated that a decrease in A,, by 1 G will result in a 15 7;)increase in the average rotational correlation time ( T , " ~ = [6(Rl,RL)1/2]-1). This gives an indication of the approximation possibly introduced in the rotational correlation times by fixing the A components a t the values found in water. From Table I, it can be observed that 711 and T~ monotonically decrease with WO. This trend is not dependent on the assumption on the values of the A components. (18)Griffith, 0. H.; Jost, P. C. In ref 6, Chapter 12.
+
7 I decreases from a very high value a t low wo to a value of about 29 ns (at high WO). Interestingly, this value is close to the rotational correlation time expected for a hydrated protein with a 27-A radiusl6 in water. 7 L can be interpreted as reflecting the macromolecular motion:19 at low W O ,the scarcity of water makes the HSA rotation hindered; a t high W O ,a deep layer of water surrounds the protein and its rotation is allowed: A quite similar behavior was found in studying the rotational correlation times of liver alcohol dehydrogenase in reverse micelles by fluorescence depolarization.20 The motion around the x axis (with a correlation time 711)can be due to librational motion of the label with respect to the whole macromolecule; the values assumed by T~ are characteristic for this kind of motion.21 The physical meaning of this librational motion is not obvious. If this motion was due only to rotation of the proxy1 group with respect to the maleimide moiety, then should be about 70".22 Other motions which add to this rotation could average the angle C#J to a value different from 70". The evidence that the molecular motion of 3MAL becomes strongly anisotropic when the complex 3MALHSA is hosted in reverse micelles is quite interesting. As already mentioned, in aqueous solution the 3MAL molecule is localized in a restrictive crevice-like site in HSA.'2 This crevice prevents the label from rotating with respect to the structure of the protein as fast as it does in reverse micelles (the label possesses, indeed, some residual motion also in aqueous solution). A more open structure of the crevice in which the label is bound can give this anisotropic rotation. Therefore, HSA, when hosted in reverse micelles, undergoes conformational changes having a relevant effect on its structure. Circular dichroism (CD) studies were carried out to better assess the occurrence of conformational changes. The CD spectra of HSA in reverse micelles showed a decrease (20%)of the ellipticity of the characteristic band a t 220 nm with respect to the buffer solution; the spectra were not significantly dependent on the w o value. This finding confirms that HSA undergoes conformational changes in reverse m i c e l l e ~ ~ ~ ~ ~ which are maintained also at high water content. Fluorescence studies showed that the maximum of the emission spectra, upon excitation at 280 nm, of HSA in reverse micelles is significantly blue shifted (about 10nm) a t each investigated wo. The change in the local solvent polarity experienced by the label, which is supported by the go increase and by the fluorescence blue shift, is not necessarily due, in our opinion, to interactions with the organic solvent. In fact, because of the buried location of the sulfhydryl group, it is unlikely that 3MAL is in contact with the organic solvent. In addition, the weakly immobilized component of the 3MAL-HSA spectrum has a hyperfine splitting equal to the value assumed in water. More likely a change in the local polarity could be determined by the presence of some molecules of surfactant bound to HSA. In fact, a blue shift in the fluorescence emission, similar to the one obtained in reverse micelles, was reported on binding of surfactant molecules to HSA.13 It is well-known that HSA has some specific binding sites for surfactant molecules, and interactions between albumin and ligands are often accompanied by detectable
+
(19)Mason, R. P.; Polnaszek, C. F.; Freed, J. H. J.Phys. Chem. 1974, 78, 1324. (20) Vos, K.; Laane, C.; Van Hoek, A.; Veeger, C.; Visser, A. J. W. G. Eur. J. Biochem. 1987, 169, 275. (21) Timofeev, V. P.; Dudich, I. V.; Volkenstein, M. V. Biophys. Struct. Mech. 1980, 7, 41. (22) Lajzerowicz-Bonneteau, J. In ref 6, Chapter 6. (23) Steinmann, B.; Jackle, H.; Luisi, P. L. Biopolymers 1986,25,1133.
242 Langmuir, Val. 7, No. 2, 1991
conformational changes of the protein.24 Thus, the conformational changes and the polarity effects observed by ESR in reverse micelles could be due to specific interactions (or binding) with AOT molecules. The conformational changes observed by optical spectroscopies were not dependent on the water content. On this basis, we could make the hypothesis that HSA undergoes these changes when it is introduced in reverse micelles, and then its structure does not change ulteriorly. This gives relevance to our ESR results on the protein dynamics as reflecting the water pool size. (24) Kragh-Hansen, U. Pharmacol. Reu. 1981,33, 17. (25) Ament, S. S.;Wetherington, J. B.; Moncrief, J. W.; Flohr, K.; Mochizuki, M.; Kaiser, E. T. J. Am. Chem. SOC.1973, 95, 7897.
Marzola et al.
Besides the difficulties in spectra analysis, the ESR spinlabeling technique appears to be useful in probing the environment and the dynamics of proteins in reverse micelles. Its application to enzymes whose activity is dependent on the water content could be particularly interesting, since it could allow one to elucidate the correlation between protein activity and dynamics.
Acknowledgment. We are indebted to Prof. S. Cannistraro for his help in the spin labeling of the protein and to Dr. R. Ambrosetti for the assistance in computational work. Thanks are due to Mr. A. Biagi for his technical support. P.M. is supported by a fellowship from E.N.I. (Ente Nazionale Idrocarburi).
