J. Phys. Chem. 1991, 95, 8437-8440 description of the virtual orbitals, which we used to compute the pathways through u* bonds. It is also necessary to address the issue of convergence of the high order of perturbation procedure inherent in the superexchange calculation. Initial inquiries have been made into these and a number of other questions, which will effect the accuracy of this procedure. These will be discussed later.*s On the basis of these initial inquiries, we believe the superexchange procedure proposed here provides a qualitatively valid
8437
pictorial understanding of how electronic couplings are transmitted from the donor to the acceptor, through the material between them.
Acknowledgment. We thank Ken Jordan, Marshall Newton, and Mike Falcetta for insightful and enjoyable discussions. Work was performed under the auspices of the Office of Basic Energy Sciences, Division of Chemical Science, US-DOE under Contract W-3 1-109-ENG-38.
Spectroscopic and Rheological Studies of Enzymes in Rigid Lipidic Matrices: The Case of a-Chymotrypsin in a Lysoiecithin/Water Cubic Phase Michael Portmann, Ehud M. Landau, and Pier Luigi Luisi* Institut f u r Polymere, ETH Zentrum, Universitatstrasse 6, CH-8092 Zurich, Switzerland (Received: August 5, 1991; In Final Form: September 16, 1991)
Transparent, thermodynamically stable lipidic cubic phases were used as membrane mimetic matrices for direct spectroscopic studies of immobilized enzymes. We present here the case of a-chymotrypsin immobilized in a cubic phase composed of 1-palmitoyl-sn-glycere3-phosphocholine and water. UV/vis and circular dichroic studies indicate that the enzyme's conformation in the rigid lipidic environment is very similar to that in water. Rheological studies on enzyme-containing and enzyme-free cubic phases show that incorporation of the macromolecule does not alter the viscoelastic properties of the gel. The a-chymotrypsin-catalyzed hydrolysis of succinyl-Ala-Ala-Phe-p-nitroanilidehas been directly monitored in the immobilized phase by UV/vis absorption.
Introduction Many enzymes are hosted in biological membranes and perform their activity in this immobilized state. Information about the kinetics, mechanism, and conformation of enzymes under those conditions is scarce; it would become more readily available if one were able to apply directly to the bound system electronic spectroscopic techniques which are commonly used for the characterization of enzymes in aqueous solutions, such as UV absorption, fluorescence, circular dichroism (CD), and infrared spectroscopy. In order to accomplish this, one needs to develop an in vitro system with the following properties: (i) the system should be a stable and manageable lipidic matrix which resembles the biological membrane, i.e., constituted basically by the bilayer structure; (ii) it should be transparent and thus suited for spectroscopic analyses; (iii) it should be able to host large enough amounts of protein to perform these structural studies, without losing transparency and thermodynamic stability. Having set these conditions, one recognizes that gels obtained from lipids or phospholipids could be suitable materials for this kind of approach. We have recently developed transparent, thermoreversible, and thermodynamically stable lecithin gels in our However, only catalytic amounts of enzymes, e.g. lipase, could be solubilized in such gels; this was enough to perform some simple kinetic studies but not to study the structure of the enzyme.' In comparison, Ericsson et al.5 have shown that relatively large amounts of lysozyme and various other globular proteins could be solubilized in a cubic phase obtained from (1) Schurtenkrger, P.; Scartauini, R.; Magid, L. J.; Leser, M. E.; Luisi, P. L. J . Phys. Chem. 1990,94, 3695. (2) Schurtenbcrger, P.; Scartazzini, R.; Luisi, P. L. Rheol. Acta 1989,28,
-172 . -.
(3) Scartazzini, R.; Luisi, P. L. J . Phys. Chem. 1988, 92, 829. (4) Scartazzini, R.; Luisi, P. L. Biocurulysis 1990, 3, 377. ( 5 ) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acfa 1983, 729, 23.
monoolein in water; cubic phases containing casein and gliadin were also de~cribed.~,' The cubic phase, first described by Luzzati et a1.: is one of the many aggregation forms (in addition to the micellar, hexagonal, and lamellar phases among others) which appear in lipid/water systems and owes its name to the particular long-range threedimensional liquid crystalline order. The cubic phase is thermodynamically stable, and although its overall spacial organization may be very complex? the basic structure is that of a lipid bilayer. These lipidic structures are studied as models for the biological organization of lipids,'O and it has been suggested that cubic phases may possibly occur in biomembranes during the process of fusion." In this work, we will describe the basic spectroscopic properties of a-chymotrypsin immobilized in a cubic phase composed of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PLPC) and water and some results obtained in a monoolein-based cubic phase as well as rheological measurements on a PLPC cubic phase with and without the enzyme. a-Chymotrypsin was chosen for this first investigation because it is a readily available enzyme, whose spectroscopic properties in aqueous solution were well-known and sensitive to the conformation. In fact, the intensity of the small dichroic band at 230 nm, which arises from the moderate helical content of the protein, can be related to the activity of the protein and depends, among other factors, on PH.'**'~Furthermore, the absorption and dichroic properties of a-chymotrypsin in the region (6) Buchheim, W.; Larsson, K. J . Colloid Inrerfuce Sci. 1987. I 1 7. 582. (7) Larsson, K.; Lindblom, G. J . Dispersion Sci. Technol. 1982, 3, 61. (8) Luzzati, V.; Mustacchi, M.; Skoulios, A. Discuss.Furuduy Soc. 1958,
25, 43. (9) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acru 1989, 988. 221. (10) Seddon, J. M.; Hogan, J. L.; Warrender, N. A.; Pebay-Peyroula, E. Prog. Colloid Polym. Sci. 1990, 81, 189. (1 1) Arvidson, G.; Brentel, I.; Khan, A.; Lindblom. G.; Fontell, K. Eur. J . Biochem. 1985, 152, 753. (12) Fasman, G . D.; Foster, R. J.; Beychock, S. J. J . Mol. Biol. 1966, 19, 240. (13) McConn, J.; Fasman, D.; Hess, G. P. J . Mol. Biol. 1969, 39. 551.
0022-3654/91/2095-8437$02.50/00 1991 American Chemical Society
Letters
0438 The Journal of Physical Chemistry, Vol. 95, No. 22, 1991 of the aromatic chromophores is rather sensitive to the environment and the aromatic side chain conf~rmation.'~Preliminary results obtained with bacteriorhodopsin, a typical membrane protein, will also be presented.
N"
Experimental Section Reagents. Suc-Ala-Ala-Pro-Phe-NH-Np, Suc-Ala-Ala-PheNH-Np, and Gr-Phe-NH-Np (Bachem), L-a-PLPC (Avanti Polar), D,L-~-PLPC (Fluka), and monoolein, a-chymotrypsin, and bacteriorhodopsin (Sigma) were all of highest purity grade and were used as received. (Here SUC= succinyl, NH-Np = p nitroanilide, and Gr = glutaryl.) Cubic Phases. PLPC-based samples were prepared by adding buffer solutions (with or without the protein) to PLPC in 1-cm, I-mL UV cuvettes (1 14-QS, Hellma) and centrifuging for 1-2 days (2900g) at 22 OC. The samples were subsequently allowed to stand for 2 days, during which the UV scattering was decreased markedly. Complete formation of cubic phases was determined by appearance of highly stiff and transparent gels. For enzymatic studies a small volume (ca. 2.5%of the cubic phase) of highly concentrated substrate solution in phosphate buffer was injected into the bulk of the cubic phase, rigorously stirred with a needle, centrifuged for 1 h, and allowed to equilibrate for 1 h. Monoolein-based cubic phases were prepared by slow addition of phosphate buffer (with or without enzyme) to the appropriate amount of molten monoolein at 40 OC. The gel was equilibrated for a few hours at 40 OC and then a few days at room temperature under N2. Aqueous Stock Solutions. All aqueous solutions for PLPC experiments, as well as bacteriorhodopsin suspensions, were prepared in 18 mM phosphate buffer, pH 8.0. The concentrations of enzyme and substrate stock solutions were determined spectrophotometrically, using t280= 51 400 M-'cm-' and zgls = 14000 M-I cm-' for a-chymotrypsin and Suc-Ala-Ala-Phe-NH-Np, respectively. Aqueous solutions for monoolein experiments were prepared in phosphate buffer, pH 6.0. Spectroscopic Measurements. Generally, the cubic phase samples were prepared directly in the spectroscopic cells. However, not all commercially available cells can withstand the centrifugal field needed for the preparation of homogeneous, transparent samples, which set some limits to the experiments we could perform. UV. All concentration determinations of stock solutions and cubic phases were carried out in a Beckman DU-68 spectrophotometer. Enzymatic activity of a-chymotrypsin hydrolysis in the cubic phase was followed in a Hewlett-Packard 8452A diode array spectrophotometer using e380 = 13000 M-' an-'for pnitroaniline. CD. CD measurements were carried out in a Jasco 5-600 instrument. The samples were measured as films pressed between quartz plates (1 24-QS, Hellma). Rheological Measurements. Dynamic shear viscosity measurements of the complex viscosity q*(w), the storage modulus G'(w), and the loss modulus C"(w) were carried out in a Rheometrics RDS-7700 instrument a t the EMPA, Diibendorf, Switzerland, using parallel plates measurement configuration.
b 2
b
-.-
1E6 1E5
Q
.-$0
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n U
$ 4
1 0 0 0 ~ : : : : m + l : : : : w: : ' ~ l + m t : : : I + + + 1E-2 1E-1 1 10 100
::::J 1000
frequency [rad/secs]
Figure 1. Frequency dependence of the magnitude of the complex vis-
cosity ?* (0,m), the storage modulus G'(0,@), and the loss modulus G" (A, A) for a 44% (w/w) PLPC cubic phase in 10 mM phosphate 0,A) pure cubic phase; buffer, pH 8.0, at 27 "C,under 2% strain: (0, (W, @, A) cubic phase with 9.7 fiM a-chymotrypsin.
846.
of buffer solution and lipid. Only at the end of this operation is the sample homogeneous, optically clear, devoid of air bubbles, and has attained its final stiffness. In order to prepare proteincontaining cubic phases, the protein-containing buffer solution is added to the lipid. In the case of a-chymotrypsin, we could prepare samples which remained stable for a t least 4 weeks at room temperature up to a protein concentration of 13 pM, as judged from the constancy of the spectra given in the following. They seem to remain indefinitely stable in sealed vials. No rheological studies have been reported thus far for these cubic phases. Figure 1 shows the frequency dependence of the magnitude of the complex viscosity q*(o),the storage modulus G'(w), and the loss modulus C"(w) for 44% PLPC cubic phases, pure and incorporating 9.7 pM a-chymotrypsin. The plots for the enzyme-containing and enzyme-free gels are practically identical, showing that incorporation of the macromolecule does not alter the viscoelastic properties of the gel. This may indicate that the structure of the cubic phase remains intact upon incorporation of the protein. The rheological features of these 44%PLPC cubic phases are distinctly different from those obtained with lecithin g e k 2 Whereas the lecithin gels exhibit typical features found for entanglement networks in polymer solutions, the cubic phases behave like solids: G'and G"are nearly frequency independent over a large frequency range, and no appearance of plateau of q*, Le. no zero shear viscosity, can be observed at low frequencies. Systematic rheological investigations of these systems are currently in progress. Although a-chymotrypsin and bacteriorhodopsin exhibit markedly different solubility properties, the former being a hydrophilic and the latter a membrane protein, both were easily incorporated into the PLPC cubic phase. Figure 2 shows the UV/vis absorption spectra of a-chymotrypsin and bacteriorhodopsin in the PLPC cubic phase a t different concentrations. This experiment shows that the absorption properties of the protein are essentially the same as in aqueous solution, that the Lambert-Beer law is obeyed in the gel, at least within the investigated concentration range, and that a protein concentration up to 30 pM can be obtained without impairing the optical transparency and stability of the gels. Suc-Ala-Ala-Phe-NH-Np has been used in the literature as a water-soluble chromophoric substrate for a-chymotrypsin.'* Products and substrate of the reaction differ in an interesting and complicated way in their absorption properties, which lends itself to a test for the spectroscopic resolution power of the lipidic gel matrix as a medium for enzymatic reactions. Figure 3 shows the time course of the a-chymotrypsin-catalyzed hydrolysis of SucAla-Ala-Phe-NH-Np in the PLPC cubic phase: for this reaction,
(16) Erikuon, P.-0.; Lindblom, G.; Arvidson, G. J . Phys. Chem. 1985,89, 1050. (17) Tardieu, A.; Luzzati, V. Blochim. Eiophys. Acta 1970, 219, 1 1 .
(18) Mao, Q.;Waldc, P. Biochem. Biophys. Res. Commun. 1991, 178, 1105.
Results and Discussion PLPC at a concentration of 39-45s (w/w) in water forms a cubic phase which is located between the micellar solution L1 and the hexagonal liquid crystalline phase E,'0*15*16 whose space group was shown to be P43n or Pm3n.I' These optically transparent gels (we will use the term "gel" to describe the general physical appearance of these materials regardless of the actual microscopic structure) were prepared as described in the literat~re:'~-'~.'~ the process involves ca. 2 days of centrifugation after the initial mixing ~~
(14) Jirgensons, B. Optical Acriviry ofProteins and other Macromolecules, 2nd ed.;Springer-Verlag: Berlin, 1973; p 104. (15) Eriksson, P.-0.;Lindblom, G.; Anidson, G. J . Phys. Chem. 1987,91,
The Journal of Physical Chemistry, Vol. 95, No. 22, 1991 8439
Letters 1.81
a
..190
wavelength (nm)
\\I
I
I
0.2
Figure 2. UV/vis spectra of a-chymotrypsin (a) and bacteriorhodopsin (b) in a cubic phase: (a) 41% (w/w) PLPC in 10 mM phosphate buffer, pH 8.0, equilibrated for 4 days at room temperature. A = 1 pM, B = 3 pM, C = IO pM, and D = 30 pM a-chymotrypsin. (b) 44% (w/w) PLPC in IO mM phosphate buffer, pH 8.0, equilibrated for 1 day at room temperature. E = 7.3 pM and F = 14.5 pM bacteriorhodopsin.
t 0.20
a
1s
U d c
f0.1 0 M
i
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0.00
300
I
'
250
t
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wavelength Cnm)
300 300 460 540 620 700 wavelength ( n m )
5.E.
210 230 wavelength Cnm)
300
0
-
a
350
400
Wavm I m n g t h C n m )
450
580
Figure 3. a-Chymotrypsin-catalyzed hydrolysis of Suc-Ala-Ala-PheNH-Np in a PLPC cubic phase. Absorption spectra measured 3,6,9, 12, 15, 18, and 21 h after substrate injection. Conditions: cubic phase (40% (w/w) PLPC in 10 mM phosphate buffer, pH 8.0) equilibrated for 4 weeks at room temperature with 1 pM a-chymotrypsin. Substrate initial concentration [SI, = 31 pM. Path length = 1 cm.
the substrate was added as a highly concentrated supernatant aqueous solution to the stiff cubic phase; the entire volume was stirred, centrifuged, and allowed to reequilibrate. The disappearance of the substrate and formation of the product are neatly detected, and the two isosbestic points at 278 and 331 nm are well-resolved. It is apparent that the enzyme kinetics in the gel are much slower than in aqueous solution. The determination of the initial velocities and the corresponding kinetic parameters in the gel is not possible as an equilibration time of ca. 2 h is needed after injecting the substrate solution into the cubic phase. Experiments with two other substrates, Gr-Phe-NH-Np and SucAla-Ala-Pro-Phe-NH-Np, did not provide additional kinetic information. The former was found to react too slowly, and even 18 h after gel formation, no product was detected. The latter reacted too rapidly, and the reaction was over within the first 2 h, before complete reequilibration of the cubic phase.
Figure 4. Circular dichroism spectra of a-chymotrypsin in solution and PLPC (a) and monoolein (b) cubic phases at room temperature. (a) 1: 12.4 pM a-chymotrypsin in 10 mM phosphate buffer, pH 8.0 (0.02-cm cell); 2: 30.4 pM a-chymotrypsin in a 44% (w/w) ruc-PLPC cubic phase in 10 mM phosphate buffer, pH 8.0 (0.01-cm plate). (b) 195 pM achymotrypsin in (1) 50 mM phosphate buffer, pH 6.0 (0.1-cm cell), and (2) a 65% (w/w) monoolein cubic phase in 17 mM phosphate buffer, pH 6.0 (0.1-cm plate). [e] is the mean residue ellipticity in [deg cm2dmol-'1.
In order to ass= whether the conformation of a-chymotrypsin was mqdified in the lipidic environment with respect to the original aqueous solution, we performed C D studies in the near- and far-UV region. Cubic phases of racemic PLPC were prepared, since optically active PLPC exhibited a large C D contribution in the far-UV region. Results are shown in Figure 4a: it is apparent that in the 200-250-nm region the spectra in water and in the cubic phase are practically the same, which suggests that the main chain conformation is with all probability not much affected by the lipidic matrix. The spectrum of the cubic phase could not be recorded below 200 nm, due to the large absorption of PLPC. It was possible to obtain reliable CD spectra of achymotrypsin in the region of aromatic chromophores by using cubic phases obtained from monoolein (Figure 4b). It is apparent that also in this case the spectra in the gel and in the aqueous solution are very similar, indicating that the aromatic side chains have not undergone significant conformational changes. In conclusion, it is possible to perform electronic spectroscopy directly on immobilized proteins and to study the conformation and possibly the enzyme mechanism in a membranelike environment. The observation that most likely the conformation of a-chymotrypsin is not modified by the lipidic entrapment is significant: it may suggest that a bilayer lipid structure with a water composition approaching 50% does not affect the rigid native folding. Obviously, the generality of this observation should be tested with other proteins. Moreover, addition of a-chymotrypsin to the cubic phase does not affect the rheological properties thereof, suggesting that the gel's structure is unchanged upon incorporation of the enzyme. The results with bacteriorhodopsin, a more relevant protein from the point of view of membrane biochemistry, are encouraging, and there is no reason to foresee problems with other proteins as well. We believe in fact that this approach is quite general. Studying enzymology in the cubic phase has a major difficulty: diffusion of substrates is slow, so that classic enzymology may not be applicable. One may resort to a biphasic system, in
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J. Phys. Chem. 1991, 95, 8440-8441
which the substrates are present in an aqueous system supernatant to the lipidic phase. In this way, kinetic data could be obtained for a reaction at the interface between water and the lipidic gel, by following the transformation of the substrate in the aqueous phase, as catalyzed by enzymes embedded in a lipid phase. Such a system would be closer to the biological situation. Studies in this direction are in progress in our group.
Acknowledgment. We are indebted to A. Gandolfi, who performed in the group a "Diplomarbeit" ETH Abt.X IFP Nr.60 (1991) about the immobilization of enzymes in monoolein cubic phases, from which Figure 4b is taken. We thank H. Kramer of the EMPA for help with the rheological measurements. Discussions with Drs. P. Walde and P. Schurtenberger have been very useful.
Appllcablllty of SmoluchowskCType Klnetlcs to Eley-Rldeal Reactions between Gaseous and Adsorbed Reactants In Porous Solids J. Samuel, M. Ottolenghi,* and D. Avnir* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91 904, Israel (Received: August 5, 1991)
An analysis is carried out considering the applicability of Smoluchowski-type kinetics to gas-phase target-annihilation (Eley-Rideal) reactions in porous solids. Previous reports have argued that, within the Knudsen regime, the reaction rate should be governed by Knudsen's diffusion which, coupled to Smoluchowski's expression, leads to a linear dependence of the rate constant on the pore diameter. We show that, for any feasible target-annihilation reaction in a porous material, concentration gradients associated with Smoluchowski-type kinetics cannot be generated. Consequently, the reaction rate is not controlled by Knudsen's diffusion. Thus, such reaction rates should simply depend on the reactant's pressure but not on pore diameter.
Bimolecular reactions in confined environments of complex, including fractal geometries, have been the focus of intensive research in the past decade,' with particular emphasis on lightinduced processes.'V2 A key issue in these studies is the effect of the detailed structure of the surrounding matrix on the reaction kinetics, which obviously differs from those carried out in homogeneous media. Bimolecular reactions with rates governed by the relative diffusion of the reactants have been extensively investigated in liquid solutions. The classical theoretical approach initiated by Smoluchowski3 is to treat the liquid as a continuum, in which the reactants undergo diffusion, leading to encounter and reaction. For fast reactions in homogeneous liquids, the familiar expression for the observed bimolecular rate constant, k', k' = 4 ~ r D (1) is derived (where r is the reaction radius and D is a relative, macroscopic, diffusion coefficient). In ordinary liquids eq 1 is found to be in general agreement with the observed long-time (steady-state) rate constant. However, when dealing with complex environments, a major question is the extent to which a bimolecular reaction can be described by a Smoluchowski-type behavior. In other words, can such (fast) reactions be governed by the rates of macroscopic diffusion of the reactants? This problem was recently addressed in this Journal for the particular case of quenching of the triplet state of benzophenone by molecular oxygen a t the solid-gas interface of porous silica. Silicas with average pore diameter 2R ranging between 68 and 572 A were employed.4 It was reported that within the Knudsen regime (Le., when X >> R, where X is the mean free path of oxygen in the gas phase) the observed quenching rate constants increased (1) For example: Havlin, S.; Ben-Avraham, D. Adu. Phys. 1987,36,695. (2) Photochemistry on Solid Surfaces; Ano, M., Matsuura, T., Eds.; Elsevier: Amsterdam, 1989. Photochemistry in Organized and Constrained Media; Ramamurthy, V., Ed.;VCH: New York, 1991. Turro, N . J. Pure Appl. Chem. 1986,58, 1269. Kopelman, R. In Fractal Approach to Heterogeneous Chemistry, Surfaces, Colloids, Polymers; Avnir, D., Ed.;Wiley: Chichater. 1989. Gafney, H. D. Coord. Chem. Rev. 1990,104,113. Samuel, J.; Ottolenghi, M.; Avnir, D. J . Phys. Chem. 1991, 95, 1890. (3) Noyes, R. M. frog. React. Kiner. 1961, 1, 129. (4) Drake, J. M.; Levitz, P.; Klafter, J.; Turro, N . J.; Nitsche, K. S.; Cassidy, K. F.Phys. Rev. Lett. 1988, 61, 865.
linearly with the average pore radius. These observations were rationalized by a theoretical model based on the assumption that the reaction is "target-annihilation" (Le., Eley-Rideal) in nature, and by arguing that, within the Knudsen regime, the system obeys a Smoluchowski-type behavior. Here we reexamine the applicability of the Smoluchowski approach to target annihilation reactions in porous media. In variance with the conclusions of Drake et aL4J we show that it is essentially impossible to construct a feasible experimental system in which the reaction rate is controlled by the Knudsen diffusion rates, within the framework of Smoluchowski's approach. Consequently, we predict that the reaction rate should depend on gas pressure but not on pore size. The classical approach of Smoluchowski for the treatment of diffusion-controlled reactions is based on the assumption of a continuous medium. Accordingly, the rate of reaction between a pair of molecules, A and B, is governed by the flux of B molecules toward A. For intrinsically fast reactions and relatively low diffusion coefficients such a flux results in the creation of a stationary concentration gradient (of B around A) which determines the reaction rate. The general expression for the observed molecular rate constant is
where D is the relative diffusion coefficient of A and B, r is the reaction radius per molecule, and k i s a constant. ( k is defined as #/C, where # is a pseudefirst-order reaction rate constant and C is the average concentration of B molecules that are in contact with an A molecule.) In solution, for relatively slow reactions and relatively nonviscous media k
