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J. Phys. Chem. 1991, 95, 8434-8431
Superexchange-Pathway Model for Long-Dlstance Electronic Couplings Conrad A. Naleway? Larry A. Curtiss,*'t and John R. Miller**+ Chemistry Division and Chemical Technology and Materials Science Divisions, Argonne National Laboratory, Argonne, Illinois 60439 (Received: August 2, 1991; In Final Form: September 4, 1991)
Long-distance electronic couplings for electron-transfer reactions are calculated in a method which makes use of McConnell's superexchange model. The necessary matrix elements come from ab initio molecular orbital calculations by transforming the UHF wave function to localized bond orbitals using Weinhold's natural bond orbital method. The result is a series of visualizable pathways between the donor (D) and acceptor (A) groups. When the material between the donor and acceptor is a saturated hydrocarbon, it is found that (1) the bulk of the interaction comes from pathways which "skip" over some bonds in contrast to the more usual "tight binding" picture, (2) C-C bonds provide most of the interaction, and (3) pathways containing C-H bonds give smaller contributions but cannot be neglected because they give rise to a large number of pathways. The method appears to have excellent potential for developing an understanding of how electronic coupling is transmitted through molecular material between donor and acceptor groups.
Introduction Long-distance electron-transfer (ET) rates, kET,are understood1" as a product of the square of an electronic coupling energy, V(r),and a Franck-Condon weighted density of states, FCWD:
kET = 2(a/h)lV(r)12(FCWD) The electronic coupling, which contains most of the distance and orientation dependence of the rates, has been the subject of theoretical and experimental investigations but still remains mysterious. It is known that rates decrease exponentially9 with distance, r, at long distances [ P a exp(-ar)] for both inter- and intramolecular reactions, but it is not clear what controls the distance dependence. Rates have been found to exhibit a similar dependence on distance for electron transfer from anions to neutrals (D- + A D A-),I"-I4 hole transfer from cations to neutrals (D+ + A D A+),15-18or charge separation and recombination (D* + A ==D+ A-).'"' A large body of data62 suggests that the distance-dependence parameter, a,is similar for many systems, but some reports of small values of CY have not been clearly e ~ p l a i n e d . ~ ~ - ~ ~ A decrease of ET rates by =lo2 due to a change of 45O in the inter-ring angle of biphenyl for biphenyl-coupled porphyrins26is qualitatively understood.2629 But effects of angles on ET rates for isomers of biphenyl-naphthyl decalins3"and porphyrinquinone compounds" are not understood on even a qualitative basis. Insight into the effects of distance and angles requires consideration of the molecular material between the electron donor and acceptor. The notion that the material between the donor and acceptor has a major role in promoting the electronic coupling interaction is well e s t a b l i ~ h e d . ~Important ~ J ~ ~ ~steps ~ ~ have ~ ~ ~been taken using both semiempirical324a,5w27"2 and ab initi02245-5254.58.73-79 molecular orbital theory. But the theoretical approaches have not yet allowed us to clearly understand the effects of distances and orientation on coupling interactions. There is a need to find ways to apply the calculations, especially those performed at the ab initio level, to the large and usually asymmetric molecules in which effects of distance and orientation are often observed. For such large molecules there is also a need to translate the results of the calculations into an intuitive notion of how the coupling passes from the donor to the acceptor through the material between them. It is our goal to develop a reliable method for computing long distance coupling interactions, which provides such an intuitive understanding of the mechanism of the coupling. It may also be possible to develop a simple semiempirical procedure optimized for computing long-distance electronic coupling in-
-- ++ +
''Chemical Chemistry Division. Technology and Materials Science Division. 0022-365419 1/2095-8434S02.50/0
teractions. This communication describes an initial step toward these goals. (1) Levich, V. 0. Adu. Electrochem. Electrochem. Eng. 1966, 4, 249.
(2) Dogonadze, R. R. In Reactions of Molecules at Electrodes; Hush, N. S., Ed.; Wiley-Interscience: New York, 1971; pp 135. (3) Kestner, N. R.; Logan, J.; Jortner, J. J. Phys. Chem. 1974, 78, 2 148-66. (4) Ulstrup, J.; Jortner, J. J . Chem. Phys. 1975, 63, 4358-68. ( 5 ) Buhh, E.; Bixon, M.; Jortner, J. Chem. Phys. 1981, 55, 41-8. (6) Marcus, R. A.; Sutin,N. Biochim. Biophys. Acta 1985,811,265-322. (7) Ulstrup, J. Charge Transfer Processes in Condensed Media; Springer-Verlag: Berlin, 1979; pp 419. (8) Siders, P.; Marcus, R. A. J . Am. Chem. Soc. 1981, 103, 748-52. (9) The exponential dependence of rates on distance is approximate. Deviations from purely exponential behavior may vary from system to system. (10) Miller, J. R. Science 1975, 189, 221-2. (11) Miller, J. R. J . Phys. Chem. 1975, 79, 1070-8. (12) Beitz, J. V.; Miller, J. R. J. Chem. Phys. 1979, 71, 4579. (13) Miller, J. R.; Beitz, J. V.; Huddleston, R. K. J . Am. Chem. Soc. 1984, 106, 5057-68. (14) Penfield, K. W.; Miller, J. R.; Paddon-Row, M. N.; Cotsaris, E.; Oliver. A. M.; Hush, N. S. J. Am. Chem. Soc. 1987, 109, 5061-5. (15) Miller, J. R.; Beitz, J. V. J . Chem. Phys. 1981, 74, 6746-57. (16) Kira, A.; Imamura, M. J . Phys. Chem. 1984,88, 1865-71. (17) Johnson, M. D.; Miller, J. R.; Green,N. S.; Closs, G. L. J . Phys. Chem. 1989, 93, 1173-6. (18) Closs, G. L.; Johnson, M. D.; Miller, J. R.; Piotrowiak. P. J . Am. Chem. Soc. 1989, 111, 3751-3. (19) Hush, N. S.;Paddon-Row, M. N.; Cotsaris, E.; Oevering, H.; Verhocven, J. W.;Heppener, M. Chem. Phys. Lett. 1985, I17 , 8-1 1. (20) Wasielewski, M. R.; Niemczyk, M. P. ACS Symp. Ser. 32l(Porphyrins, Excited States and Dynamics) 1986, 154-65. (21) Oevering, H.; Vcrhwen, J. W.; Paddon-Row, M. N.; Cotsaris, E.; Hush, N. S . Chem. Phys. Lett. 1988. 150, 179-80. (22) Newton, M. D.; Sutin, N. Annu. Reo. Phys. Chem. 1984,35,437-80. (23) Moebius, D. Acc. Chem. Res. 1981, 14, 63-8. (24) Isied, S.S.; Vassilian, A.; Wishart, J. F.; Creutz, C.; Schwarz, H. A,; Sutin, N. J. Am. Chem. Soc. 1988,110,635-7. (25) Vassilian, A.; Wishart, J. F.; van Hemclyryck, B.; Schwarz, H.; Isied, S. S . J. Am. Chem. Soc. 1990, 112,7278-86. ( 2 6 ) Helms, A,; Heiler, D.;McLendon, G. J . Am. Chem. Soc. 1991, 113. 4325-7. (27) Closs, G. L., personal communication. (28) Cave, R. J.; Siders, P.; Marcus, R. A. J . Phys. Chem. 1986, 90, 1436-44. (29) Siders, P.; Cave, R. J.; Marcus, R. A. J . Chem. Phys. 1984, 81, 56 13-24. (30) Closs, G. L.; Calcaterra, L. T.; Green,N. J.; Penfield, K. W.; Miller, J. R. J. Phys. Chem. 1986,90, 3673-83. (31) Sakata. Y.;Nakashima, S.; Goto. Y.;Tatemitsu, H.; Misumi, S. J . Am. Chem. Soc. 1989,111, 8978. (32) Halpern, J.; Orgel, L. Discuss. Faraday Soc. 1960, 29, 32. (33) McConnell, H. M. J. Chem. Phys. 1%1,35, 508, 515. (34) Hoffman, R. Acc. Chem. Res. 1971,4, 1. (35) Hoffman, R.; Imamura, A.; Hehre, W. J. J . Am. Chem. Soc. 1968, 90, 1499. (36) Gleiter, R. Angew. Chem., Int. Ed. Engl. 1974, 13, 696. (37) Pasman, P. Ph.D. Thesis, University of Amsterdam, 1980.
0 1991 American Chemical Society
The Journal of Physical Chemistry, Vol. 95, No. 22, 1991 8435
Letters
TABLE I: Electronic Couplings V (millihartrees) for Anions of Mndlcals
D A Figure 1. A superexchange pathway producing an interaction between the donor state D and the acceptor state A via high-energy intermediate are represented by state. Interactions between the ith and j t h states, curved lines. B, is the energy difference between the ith state and states D and A.
@,,
Methods The present model is based on the idea of superexchange, the indirect coupling of donor to acceptor wave functions through a (38) Martin, H.-D.; Mayer, B. Angew. Chem., In?. Ed. Engl. 1983?22, 283. (39) Laruon, S. J . Am. Chem. Soc. 1981, 103, 4034-4040. (40) Larsson, S.;Matos, J. M. 0. J . Mol. Srruct. 1981, 120, 35-40. (41) Larsson, S.J. Phys. Chem. 1984,88, 1321-3. (42) Larsson, S.; Volosov, A. J. Chem. Phys. 1986. 85, 2548-54. (43) Larsson, S.;Volosov, A. J . Chem. Phys. 1987,87, 6623-5. (44) Broo, A.; Larsson, S.Chem. Phys. 1990, 148, 103-15. (45) Ohta. K.; Closs, 0. L.; Morokuma, K.; Green, N. J. Am. Chem. Soc. 1986, 108. 1319. (46) Newton, M. D. In Chemical Reactivity in Liquids. Fundamen?al
Aspecrs, Proc. Forty-Second Inr. Meeting, Paris, France, 7-1 I Sep 1987; Moreau, M., Turq,P., Belloni, J., Prud'homme, R., Troyanowsky, C., Eds.; Plenum Prcss: New York, 1988; pp 157-74. (47) Newton, M. D.; Ohta, K.; Zhong, E. J . Phys. Chem. 1991, 95, 23 17-26. (48) Newton, M. D. J. Phys. Chem. 1988,92,3049-56. (49) Newton, M. D. Jerusalem Symp. Quanrum Chem. Biochem. 1986, 19, 305-14. (50) Newton, M. D. J. Phys. Chem. 1986,90, 3734-9. (51) Newton, M. D. In?. J . Quantum Chem. 1980, 17, 363-91. (52) Tembe, B. L.; Friedman, H. L.; Newton, M. D. J. Chem. Phys. 1982, 76, 1490-507. (53) Paddon-Row, M. N.; Jordan, K. D. In Modern Models of Bonding and Delocalizarion;Liebman, J. F., Greenberg, A., Eds.;VCH Publishers: New York, 1988; Vol. 6, pp 115-94. (54) Balaji, V.; Jordan, K. D.; Burrow, P. D.; Paddon-Row, M. N.; Patney, H. K. J . Am. Chem. Soc. 1982,104,6849-51. ( 5 5 ) Paddon-Row, M. N. Ace. Chem. Res. 1982. 15.245. (56) Balaji, V.; Ng, L.; Jordan, K. D.; Paddon-Row, M. N.;Patney, H. K. J. Am. Chem. Soc. 1987, 109, 6957-69. (57) Paddon-Row, M. N.; Jordan, K. D. Mol. Srrucf. Energ. 1988, 6, 1 15-94. (58) Falcctta, M. F.; Jordan, K. D.; McMurry, J. E.; Paddon-Row, M. N. J. Am. Chem. Soc. 1990,112, 579-86. (59) Beratan, D. N. J . Am. Chem. Soc. 1986, 108,4321-6. (60) Onuchic, J. N.; Beratan, D. N. J. Am. Chem. Soc. 1987,109,6771-8. (61) Onuchic. J. N.; Beratan, D. N. J. Chem. Phys. 1990, 92. 722-33. (62) Bixon, M.; Jortner, J.; Michel-Beyerle, M. E.; Ogrodnik, A. Biochim. Biophys. Acta 1989, 977, 273-86. (63) Wasielewski, M. R.; Niemnyk, M. P.; Johnson, D. G.; Svec, W. A.; Minsek, D. W . Terrahedron 1989, 45, 4785-806. (64) Lawson, J. M.; Craig, D. C.; Paddon-Row, M. N.; Kroon, J.; Verhoeven, J. W . Chem. Phys. Le??.1989, 164, 120-5. (65) Oevering. H.; Paddon-Row, M. N.; Heppener, M.;Oliver, A. M.; Cotsatis, E.;Verhoeven, J. W.; Hush, N. S . J . Am. Chem. Soc. 1987, 109, 3258-69. (66) Oevering. H.; Verhoeven. J. W.;Paddon-Row, M. N.; Warman, J. M. Tetrahedron 1989, 45,4751-66. (67) Pasman, P.; Verhoeven, J. W.; de Boer, T. J. Chem. Phys. Lerr. 1978, 59, 381-5. (68) Pasman. P.; Rob, F.;Verhoeven, J. W. J . Am. Chem. Soc. 1982,104, 5 127-33. (69) Verhoeven, J. W.; Paddon-Row, M. N.; Hush, N. S.;Oevering, H.; Heppcner, M. Pure Appl. Chem. 1986.58, 1285-90. (70) Brunck, T. K.; Weinhold, F. J . Am. Chem. Soc. 1976, 98,4392-3. (71) Kuki, A. In Long-Range Elecrron Transfer in Biology; Palmer, G., Ed.; Springer-Verlag: Berlin, 1991; Vol. 75, pp 49-83. (72) Bertrand, P. In Long-Range Electron Transfer in Biology; Palmer, G., EM.;Springer-Verlag: Berlin, 1991; Vol. 75, pp 1-47. (73) Newton, M. D. Mech. Aspecrs Inorg. Reac?. 1982, 198, 255-79. (74) Newton, M. D.; Friedman, H. L. J. Chem. Phys. 1988,88,4460-72. (75) Newton, M. D. Perspeciives on Photosynthesis 1990, 22, 157-70. (76) Balaji, V.; Jordan, K. D.; Gleitn, R.; Jaehne, G.; Mueller, G. J. Am. Chem. Soe. 1985, 107, 7321-3. (77) Paddon-Row, M. N.; Jordan, K. D.J. Chem. Soc., Chem. Commun. 1988, 1988, 1508-10. (78) Paddon-Row, M. N.; Wong. S. S.;Jordan, K. D. J. Chem. Soc., Perkin Trans. 1990, 2,425-30. (79) Newton, M. D. Chem. Rev. 1991, 91, 767-92.
Koopmans' theorema UHFb pathway total direct through bonds through antibonds
butane-I .4-divl 12.42c
+
+13.5oe +11.37 -2.68 +5.95 +8.10
dimethylenecvclohexane 6.97d 10.85d
5.10
0.05 2.56 2.48
a From the difference in eigenvalues of the two highest occupied alpha orbitals of the anion: V = E b E,, where E b is the energy of the bonding combination and E, is the energy of the antibonding combination of the p orbitals on the CHI donor and acceptor groups. bFrom the difference of UHF energies for the ground and first excited states of the anion, V = E b - Ea, where E b is the energy of the state for which the bonding orbital is singly occupied and E, is the energy of the state for which the antibonding orbital is singly occupied. CTheground state is 2A,. The highest and second highest occupied orbitals are a, and b,. dThe ground state is 2A,. The highest and second highest occupied orbitals are a, and b,.
-
chain of high-energy intermediate states as pictured in Figure 1. In a molecular ET reaction there will be several such chains. From perturbation theory, the coupling of the donor and acceptor by one such (the kth) is3'
v, = -n(-a,)/na, n+ 1 n The total coupling is the algebraic sum over all chains V = &Vb This superexchange picture can be effectively implemented if the ratio BIB of the coupling elements to the energy denominators is small. Unfortunately @/Bis large if the representation is made up of atomic orbitals. A more useful representation is one in which the intermediate states of the saturated hydrocarbon bridge are localized u and u* bonds. Localized natural bonding orbitals (NBOs) are used in a procedure described below. Ab initio molecular orbital (MO) theofl was used to calculate wave functions for two model systems: the anion of butane-1,4diyl, -CH2CH2CH2CH2', and the anion of 1,Cdimethylenecyclohexane, -CHZC6H&H2*. The latter has also been studied at the ab initio level by Ohta.45 These molecules have identical donor and acceptor groups, which are the simple one-carbon r systems, 'CH2, so we are computing couplings relevant to electron transfer from CH2- to *CH2. The canonical (Le., delocalized) self-consistent field (SCF) MOs were transformed into a set of orthonormal bond orbitals by Weinhold's natural bond orbital (NBO) These NBOs were then used in the calculation of the electronic coupling interactions by eq 2. The coupling elements Bij and the energy denominators, B,, used were off-diagonal and diagonal elements of the Fock matrix in the NBO representation of the unrestricted, Hartree-Fock, U H F wave function. The geometries of the two anions were determined from MM2 optimizationsa3with the (0,O)conformation of the (t-(e,e)) isomer of the dimethylenecyclohexane anion being used. All calculations were performed with the split-valence 3-21G basis set." We have found that the 3-21G basis set gives a reasonable account of the electronic splittings of these anions from comparison to higher level calculations including larger basis sets and inclusion of correlation effectsa5 (80) Frisch, M. J.; Head-Gordon, M.; Schlegel, H. B.; Raghavachari, K.; Binkley, J. S.;Gonzalez, C.; Defrees, D. J.; Fox, D. J.; Whitesides, R. A.; Sceger, R.; Melius, C. F.; Baker, R. L.; Martin, R. L.; Kahn, L. R.; Stewart, J. J. P.; Fluder, E. M.; Topiol, S.;Pople, J. A. GAUSSIAN 88; Gaussian, Inc.: Pittsburgh, PA, 1988. (81) Reed, A. E.;Weinstock, R. B.; Weinhold, F . J . Chem. Phys. 1985. 83, 735-46. (82) Foster, J. P.; Weinhold. F. J . Am. Chem. Soc. 1980, 102. 7211-18. (83) PCMODEL for the Macintosh 11, Serena Software, Bloomington, IN. (84) Binkley, J. S.;Pople, J. A.; Hehre, W. J. J . Am. Chem. Soc. 1972, 102, 939. (85) Curtiss, L. A.; Naleway, C. A.; Miller, J. R., to be published.
Letters
0436 The Journal of Physical Chemistry, Vol. 95, No. 22, 1991
KEY 0 (C-C)
a*(c-c) V= -0.820 mH [2]
Figure 2. The six most important pathways shown for the anions formed by electron addition to the butane-1,Cdiyl diradical, -CH2CH2CH2CH2'. The interactions V (millihartrees) are for one path. The number of equivalent paths (related by a symmetry operation) is given in brackets. Pathways are ordered in terms of their importance ( V X number of identical paths).
n
V
= 1.500 mH [2]
V= 0.442 mH A
[4]
n A V=0.479mH [2
w
a V=
0.142 m H [4]
Figure 3. The six most important pathways shown for the anions formed by electron addition to the dimethylenecyclohexane diradical, -CH2C6HIOCH2' (see Figure 2 caption).
Results and Discussion Table I summarizes the comparison of electronic coupling for the butane- 1,4-diyl and dimethylenecyclohexane radical anions calculated by (1) SCF calculations on both the ground-state anion and first lowest excited state of the anion, (2) the difference of the eigenvalues from the highest two occupied cy orbitals (a Koopman's theorem approximation), and (3) a superexchange perturbation method (eq 2). As can be noted from Table I, all three estimates were in good agreement for butane- 1,4-diyl, but the agreement is only fair for dimethylenecyclohexane. The superexchange model includes all pathways through the bonding and antibonding manifolds of orbitals, except those which cross between the two manifolds. The contributions from each manifold are reported in Table I along with the direct ("through space") path. Because of the very large number of possible pathways, a search was made for all possible pathways from donor to acceptor which contribute a coupling larger than a defined threshold (lo4 hartree), and include the same orbital only once. The most important pathways from the butane- 1,4-diyl and dimethylenecyclohexane radical anions are illustrated in Figures 2 and 3. Important results emerge from these calculations. First we can see that, for both the butane-I ,4-diyl and dimethylenecyclohexane cases, the dominant pathways involve C-C u bonds of the bridge. Pathways involving C-H bonds make smaller contributions, but they cannot be neglected because of their large number. The direct interaction between the donor and acceptor groups is relatively unimportant, as was previously found.45 For the two saturated hydrocarbon bridges studied, the pathways through the u bonding orbitals and the pathways through the u* antibonding orbitals make contributions of similar magnitude and the same sign. This point has been recognized at the Huckel Within one type of pathway, such as those making use only of C-C cr bonding
orbitals, there are pathways which make positive contributions to the coupling and others which make negative contributions, so that both constructive and destructive interference occur between pathways. Although only a few pathways are responsible for the bulk of the interaction, it was found that calculation of the total coupling interaction requires consideration of a large number of pathways, many of which make small contributions. Nonbonded interactions (interactions between nonadjacent bonds) are important and even dominant in contrast to common notions of through-bond interactions. In the original McConnell model33 a step along a path always went to the next nearest neighbor. Beratan and Onuchic's Huckel model makes a similar "tight-binding" approximation.61 We find, on the contrary, that most pathways skip over one or more bonds en route from the donor to the acceptor. The two most important pathways in the dimethylenecyclohexane contain steps which skip over two C-C bonds! While the simple McConnell or tight-binding path makes a contribution, it is only one among a number of comparable paths contributing to the total interaction, and it might not even have the same sign as the total interaction from all pathways through a chain of bonds. It is the fourth pathway in importance for the dimethylenecyclohexanecase. We also note that the importance of vicinal (or "1,3") interactions of bond and antibond type has been previously pointed out by Weinhold and B r u n ~ k in~ studies ~.~~ of internal rotational barriers. There are a number of potential problems associated with our approach. These studies have not proven that standard basis sets, such as 3-21G, adequately describe the tails of wave functions, which are so critical to long-distance electronic coupling. Also, Hartree-Fock wave functions are well-known to provide a poor (86) Brunck, T. K.; Weinhold, F. J . Am. Chem. SOC.1979,101, 1700-9. (87) Weinhold, F.; Brunck, T. K. J . Am. Chem. SOC.1976, 98, 3745-9.
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, thermodynamicallystable 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
