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Infrared Spectroscopic Studies of Molecular Structure, Ordering, and Interactions in Enzyme-Containing Langmuir-Blodgett Films H. Ancelin,t D. G. Zhu,$ M. C. Petty,$ and J. Yarwood*vt Department of Chemistry, University of Durham, Durham DH1 3LE, U.K., and Molecular Electronics Group, School of Engineering and Applied Science, University of Durham, D H l 3LE, U.K. Received May 23, 1989 Fourier transform infrared studies on Langmuir-Blodgett Films of glucose oxidase (GOD) incorporated into fatty acid multilayers have been used to study molecular order and interactions as a function of dipping pressure on the subphase. The data show that GOD is incorporated principally by electrostatic (as opposed to hydrogen-bonded) forces and that the enzyme does not penetrate the acid head group region. Changes of conformation as a function of surface pressure may easily be monitored.
Introduction that biological membrane order and function are determined to a large extent by the subtle details of t h e structure a n d dynamics of t h e membrane components-mainly phospholipid bilayers with incorporated protein molecules. The lipid-protein molecular interactions are t h o ~ g h t l -to~ have a controlling influence on properties such as fluidity, which must in turn influence membrane transport (and, maybe, enzyme catalytic activity) at the molecular level. Thus, lipid "fluidity" in the absence and presence of protein material has been extensively studied by both infrared1-4v6 and Raman2*5*7*s spectroscopies, mainly through the detection of phase transition changes due to protein incorporation. Relatively little has been p ~ b l i s h e d ~on s ~ the ,~ structural details of the lipid-protein molecular interactions, although in principle vibrational spectroscopy should provide a wealth of information on the nature and extent of the interactions through frequency shifts and intensity c h a n g e ~ . ~ . ~ The , ~ - ~work - ' ~ which has been reported on such interactions has led to a certain amount of con-
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* Molecular Electronics Group.
(1) Chapman, D. In Biological Membranes; Chapman, D., Ed.; Academic Press: New York, 1982; Vol. 4, Chapter 4 and references therein. (2) Levine, I. R. In Adu. in Infrared and Raman Spectroscopy; Clark and Hester, Eds.; Wiley: New York, 1984; Vol. 11, Chapter 1 and references therein. (3) Mendelsohn, R.; Mantach, H. H. In Progress in Protein-Lipid Interactions; Elsevier: Amsterdam, 1986; Vol. 2, p 103. (4)Amey, R. L.; Chapman, D. In Biomembrane Structure and Functron; Chapman, D., Ed.; Macmillan: New York, 1983; Chapter 4, p 199. (5) Verma, S. P.; Wallace, D. F. H. Reference 4, Chapter 3.
(6) Mantsch, H. H.; Casal, H. L.; Jones, R. N. In Spectroscopy of Biological Systems; Clark and Hester, Eds.; Wiley: New York, 1986; Vol 13, Chapter 1. (7) Knoll, W. Biochim. Biophys. Acta 1986, 329, 863. (8) Weidekamm, E.: Bambere. E.: Janko, K.; Weber, R. Arch. Biochem. Biophys. 1978,187,339. (9) Muschayakarara, E.; Albon, N.; Levine, I. R.Biochim. Biophys. Acta 1982,153,686. Muschayakarara, E.; Levine, I. R. J. Phys. Chem. 1986,86, 2324. (10) Shimida, I.; Ishida, H.; Ishitani, A.; Kunitaka, T. J. Colloid Interface Sci. 1987, 120, 535. (11) Mantsch, H. H. Biochemistry 1987, 26, 2706. (12) Parker, F. S. Applications of Infrared, Raman and Resonance Raman Spectroscopy in Biochemistry; Plenum Press: New York, 1983 and references therein. (13) Jakobsen, R. J.; Wasacz, F. H. In ACS Symp. Ser. 1987, 343, 339.
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troversy, especially with regard to the degree of incorporation of protein into model membrane (lipid) struct u r e ~ .Certainly, ~ such processes are poorly understood at the present time, and it seems clear that much more work on model systems is required. One well understood method of producing a model membrane involves using the Langmuir-Blodgett (LB) technique.14J5 It has already been d e m ~ n s t r a t e d ' ~that J ~ the water-soluble enzyme glucose oxidase (GOD) may be incorporated into phospholipid multilayers by the method originally developed by Fromherz.'* In this paper, we report the results of a detailed study, using infrared techniques, of the interactions of GOD with multilayers of 22-tricosenoic acid (22-TA).
Experimental Section Most of the experimental details have been published previously.16-18 For these particular LB bilayers, the dipping pressure was between 10 and 40 mN m-1 on a pure water subphase at pH 5.6. All the spectra shown were recorded on a Mattson Sirius 100, FTIR spectrometer at a resolution of 4 cm-1. No spectral subtractions were performed; thus, the spectra contain both 22-TA and GOD components. Fourier deconvolution'3J9.20 was carried out by using the standard software in the Mattson package. The data in Figure 6 were generated with a Lorentzian filter function with a width of 20 em-1 and with triangular apodization. Enhancement factors of about 2 were found to give the most sensible deconvolution. Results and Discussion In our previous paper,17 we have demonstrated that GOD may be effectively incorporated into either a phospholipid bilayer or a bilayer of 22-TA. Figure 1 shows the FTIR-ATR spectra of two monolayers of 22-TA with incorporated GOD, deposited on a hydrophobic silicon surface. Two of the bands of interest are due to side(14) Blcdgett, K. B. J . Am. Chem. SOC.1935,57, 1007. (15) Barraud, A.; Rosilio, C.; Ruaudel-Teixier, A. J. Vac. Sci. Technol. 1979, 16, 2003. (16) Moriizumi, T. Thin Solid Films 1988,160,413. (17) Zhu, D. G.; Petty, M. C.; Ancelin, H.; Yarwood, J. Thin Solid Films 1989, 176, 151. (18) Fromherz, P. Biochim. Biophys. Acta 1971,225, 382. (19) Kauppinen, J. K.; Moffat, D. J.; Mantsch, H. H.; Cameron, D. G. Appl. Spectrosc. 1981, 35,271. (20) Yang, W. J.; Griffiths, P. R.; Byler, P. R.; Susi, H. Appl. Spectrosc. 1985, 39, 282.
0 1990 American Chemical Society
IR Study of Enzyme-Containing LB Films
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Table I. Band Assignments Used in This Work w , cm-l assignment 2850 u.(CHZ)of 22-TA 2935 u.(CH,) of GOD 2925-2918 u,(CHZ)of 22-TA 1739 u(C=O) of “free” acid of 22-TA 1723 u(C=O) of sideways dimers of 22-TA 1650 amide I of GOD 1540 amide I1 of GOD 1468 6(CHz) of 22-TA
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ways acid dimers at 1723 cm-l and “free” acid at approximately 1739 cm-’ (see Table I). In our work, we also showed that the GOD is effectively immobilized up to and including a subphase pH of about 6. At more alkaline pHs, the negatively charged GOD is not easily absorbed, presumably because of an increasing degree of ionization of the 22-TA head group. This result hinted a t a mode of surfactant-enzyme “binding” which is electrostatic in nature, since mutually repelling negatively charged groups prevent incorportion on the LB trough. Another way of studying the nature of this incorporation is to follow the structure and ordering of the 22-TA and the enzyme molecule as a function of dipping pressure. A series of spectra for two monolayers of 22-TA with incorporated GOD is shown in Figure 2. At low pressure (10-20 mN m-I), the 22-TA spectra show definite signs of extreme disordering, despite the presence of GOD in the “bilayer”. The us and u, (CH2) bands of the hydrocarbon chain are shifted to higher frequency and are significantly broadened. Both features are attributable to chain disorder23 of the 22-TA molecule. The corresponding arrangement on the subphase is shown in Figure 3A. As the pressure on the 22-TA monolayer a t the subphase interface is increased, the u,(CHz) band moves to lower frequency (from 2925 to 2918 cm-l) and becomes very much narrower (compare Figure 2A, 2B, and 2C). It is quite clear that reordering of the alkyl chains occurs (as expected) a t higher pressures (Figure 3B). At pressures near 35 mN m-1 there is a sudden change in u,(CH2), band shape, and width (see Figure 4), and the band halfwidth at 40 mN m-1 approaches that for pure 22-TA LB films.22 This change of width may be associated with the reduction in intensity of a band near 2935 cm-1 (see Figure 2). This band must presumably belong to the protein molecule, and the intensity change may be associ-
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Figure 1. FTIR-ATR spectra of two monolayers of 22-TA (with incorporated GOD) on hydrophobic silicon: (A) dipped a t 20 mN m-1; (B)dipped a t 40 m N m-l. The two bands of interest are sideways acid dimers a t 1723 cm-I and “free” acid a t 1739 cm-1.
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Figure 3. Schematic representation of molecular arrangements of 22-TA monolayer (with adsorbed GOD) on the water subphase a t different pressures.
ated with changing conformations of the protein,l1-13 which we show later (Figure 6) to be dependent on the dipping pressure. At the same pressures, the u(C=O) band near 1723 cm-I is accompanied by another band near 1739
1070 Langmuir, Vol. 6, No. 6, 1990
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Figure 4. Variation of u.(CH2) bandwidth as a function of dipping pressure: (1) 22-TA/GOD bilayer; (2) data for pure 22-TA LB films.
Figure 6. Fourier deconvoluted spectra of the protein (enzyme) amide I group region at two different pressures: (A) 10 mN m-1; (B) 40 mN m-1.
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Figure 5. FTIR-ATR spectrum of a bilayer of 22-TA (with incorporated GOD) dipped at 35 mN m-l. The band at 1739 cm-* is due to non-hydrogen-bonded acid carbonyl groups. cm-' (Figure 5), which is caused by non-hydrogenbonded CO groups on the 22-TA. This can only arise by the removal of GOD from the interfacial region on the LB trough creating regions of the bilayer (on Si/SiOz) over which the 22-TA molecules do not interact (Figure 3). The GOD is thus compressed outz4 of the subphase monolayer a t higher surface pressures leaving "holes" in the 22-TA packing on the substrate when dipping occurs (Figure 3C). The GOD spectrum is still present-see the broad amide I band at 1650 cm-' in Figure 5-but some enzyme molecules have been "squeezed out". Thus the evidence is that the 22-TA molecules are "ordered" as expected by an increase in surface pressure. This ordering is partly, a t least, associated with the acid-enzyme interactions at the air-water interface, since it is impossible to dip 22-TA at 10 or 20 mN m-l without enzyme incorporation. The protein molecule, in turn, has a molecular conformation which changes with surface pressure (see below). Information about the protein molecules may be deduced by use of the u(C=O) band a t -1650 cm-l, which is k n ~ w n l l - ' to ~ be highly sensitive to protein secondary (conformational) structure. In Figure 6, we show the Fourier deconvoluted spectra in this region a t 20 and 40 mN m-l surface pressures. There are clear differences, espe-
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cially in the 1630-1660-cm-' region-usually associated with a-helix and P-pleated sheat conf~rmations.~l-~~ However, it is obvious that the enzyme molecules still occupy a region of the bilayer associated with the COOH head groups. If penetration down into the alkyl chain region occurred, one might expect the interactions to include acid cyclic dimers.21t22 These are not observed to any great extent (see Figure 5). Furthermore, our spectra show no evidence for hydrogen-bonded interactions between protein and 22-TA, as is claimed for DDPC-peptide interactions.1° The amide I and amide I1 bands in the 1650and 1550-cm-' regions may be attributed to conformational variations. But more importantly, the v(C=O) band of 22-TA shows no evidence of "extra" species attributable to such effects. Thus, we are able to confirm the work reported previously17which showed that the enzyme is incorporated into these 22-TA bilayers mainly by electrostatic (Coulombic) forces.
Conclusion We have shown how the details of molecular order, conformation, and interactions of a water-soluble enzymemodel substrate system may be probed by using FTIR. The data indicate clearly that the incorporation is driven principally by Coulombic forces rather than by hydrogen bonding and that the enzyme does not penetrate out of the acid head group regon of the LB multilayers. We cannot eliminate the possibility that the protein "straddles" both hydrophobic and hydrophilic regions of the model membrane.1° However, this seems unlikely since the 22-TA chain packing appears very similar (at 40 mN m-l) to that observed for pure 22-TA multilayers (Fig~J~ ure 4). It would appear from the l i t e r a t ~ r e ~ J 7that the behavior of a particular system in this respect depends very much on the specific protein/lipid combination used. This would be consistent with the known biological specificity of particular enzyme/substrate systems. Registry No. GOD, 9001-37-0; 22-TA, 65119-95-1. (21) Bellamy, L. J. Infrared Spectra of Complex Molecules; Chapman and Hall: London, 1975. (22) Davis, G. H.; Yarwood, J. Spectrochim. Acta 1987,43A, 1619. (23) Dluhy, R.A.; Moffat, D.; Cameron, D. G.; Mendelsohn, R.; Mantsch, H. H. Can. J. Chem. 1985,63, 1925. (24) Dawson, R. M. C.; Quinn, P. J. Advances in Experimental Medicine and Biology; Plenum Press: New York, 1971; Vol. 14, pp 1-17.
