J. Phys. Chem. 1995,99, 4837-4841
4837
Self-Assembled Monolayers as Novel Biomembrane Mimetics. 1. Characterization of Cytochrome c Bound to Self-Assembled Monolayers on Silver by Surface-Enhanced Resonance Raman Spectroscopyt Yasushi Maeda, Hiroyuki Yamamoto, and Hiromi Kitano" Department of Chemical and Biochemical Engineering, Toyama University, Toyama, Japan Received: September 14, 1994; In Final Form: December 21, 1994@
Characterization of cytochrome c (Cyt C) bound to self-assembled monolayers (SAMs) of mercaptoalkanoic acids (C,COOH) on colloidal silver was carried out by means of surface-enhanced resonance Raman spectroscopy. Cyt C selectively adsorbed on negatively charged surfaces of SAMs on Ag, and a clear surfaceenhanced resonance Raman scattering (SERRS) from Cyt C superimposed on surface-enhanced Raman scattering (SERS) from SAMs was observed under 514.5 nm excitation (in resonance with the electronic absorption of Cyt C @ band)). Whereas, in the spectrum of the same sample under 488.0 nm excitation (off resonance with the electronic absorption of Cyt C), any signals attributable to Cyt C were not observed, but only those attributable to C,COOH were detectable. These observations strongly suggest that Cyt C makes a second layer on a S A M which is directly attached to Ag. Comparing the surface-enhanced resonance Raman spectra with the resonance Raman spectra in solution revealed that the native structure of Cyt C was fully preserved after adsorption on a S A M , while adsorption-induced structural change took place in Cyt C which was directly adsorbed on Ag. It was concluded that Ag modified with a SAM is a suitable SERSactive substrate to analyze the structure of proteins on biomimetic membrane surfaces without denaturation.
Introduction In many biological phenomena including cellular recognition, immunological protection, and transportation of information in tissues and organs, interactions between proteins and cell surfaces are quite essential.' To reveal details of these interactions by physicochemical techniques, simple biomembrane mimetic systems such as liposomes2 and Langmuir-Blodgett (LB) films3 composed of natural lipids or synthetic lipid analogues have been used. Organosulfur compounds such as alkyl and aromatic thiols are known to form close-packed ordered monolayers, so-called self-assembled monolayers (SAMs), on gold or silver surfaces via chemisorptive S-Au or S-Ag bonds.4 Recently, SAMs of alkanethiol derivatives are considered interesting as novel cell mimetic membranes because of their structural analogy to biomembranes, ease of preparation, and apparent ~tability.~ Among various methods to study the structure and interaction at interfaces, surface-enhanced resonance Raman scattering (SERRS) is a sensitive and chemically specific analytical technique for chromophores.6 It may also provide information about the local environment around molecules and molecular orientation at surfaces. In particular, SERRS has a great advantage in the analysis of biological molecules because of its ability to quench fluorescence, which frequently causes trouble in normal Raman spectroscopy.' However, when biomolecules directly adsorbed on a surface-enhanced Raman scattering (SERS)-active substrate are analyzed, the possibility of structural alternation induced by electrostatic interaction between adsorbed molecules and the metal surface remains a concern that must be addressed in each individual case. In practice, adsorption-induced denaturation has been reported in some heme-8 and flavo- protein^.^ Consequently, SAWAg
* To whom all correspondence should be addressed. +Presented at the 43rd Annual Meeting of the Society of Polymer Science, Japan, at Nagoya, Japan, in May 1994. Abstract published in Advance ACS Abstracts, March 1, 1995. @
0022-365419512099-4837$09.00/0
composite systems which possess biomimetic surfaces are believed to be a quite suitable system to clarify the interaction between proteins and biomimetic membranes by SER(R)S, because they can be expected to avoid the unfavorable conformational change in adsorbed proteins. In this regard, we carried out characterization of cytochrome c (Cyt C), which is one of the most extensively studied proteins by Raman spectroscopy,'0 adsorbed to a SAM of alkanethiol derivatives on colloidal Ag by SERRS.
Experimental Section Materials. 1-Alkanethiols (CH3(CH2),SH (abbreviation: C,CH3), n = 3, 5, 7, 11) were purchased from Wako Pure Chemicals, Osaka, Japan, and used as supplied. w-Mercaptoalkanoic acids (HS(CH2),COOH (abbreviation: C,COOH), n = 2, 5 , 7, 10, 11) and w-mercaptoalkanols (HS(CH2),0H (abbreviation: C,OH), n = 10, 11) were synthesized from corresponding w-bromoalkanoic acids (Br(CH2),COOH, n = 2,5,7, 10, 11) or w-bromoalkanols (Br(CH2),0H, n = 10, 11) and purified according to established procedures.' Cytochrome c (horse heart) was purchased from Wako Pure Chemicals. The oxidized form of Cyt C (Cyt C3+)was prepared by oxidation of Cyt C (62 mg/5 mL of 33 mM phosphate buffer, pH 7.0) with K3Fe(CN)6 (1.65 mg). The reduced form of Cyt C (Cyt C2+)was prepared by reduction of Cyt C (62 mgl5 mL of phosphate buffer) with sodium hydrosulfite (2.2 mg). Both Cyt C3+ and Cyt C2+ were purified by dialysis against the phosphte buffer (pH 7.0) for 48 h at 4 "C. Ag colloids were prepared by reduction of AgN03 with NaBH4 at 0 "C, and finally the pH of the suspension was adjusted to 7.0. The final concentration was 2.5 x lov4mol of A g L The absorption maximum of the colloids was 393 nm. Using a dynamic light scattering method (DLS-7000DL, Otsuka Electronics, Hirakata, Japan; light source, Ar laser 488.0 nm), the average hydrodynamic diameter of the Ag particles was estimated to be 50 nm. 0 1995 American Chemical Society
Maeda et al.
4838 J. Phys. Chem., Vol. 99, No. 13, 1995 c-s T
c-c
1200 1200
1000
800
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Wavenumber (cm-')
Figure 1. Raman spectra in the v(C-C) and v(C-S) regions (488 nm excitation) of the dodecanethiol (a) SAM adsorbed on colloidal Ag (25 "C), (b) bulk liquid (25 "C), and (c) bulk solid (-20 "C). Incident laser power: 200 mW. Other reagents were commercially available. Milli-Q grade water was used for the sample preparation. Determination of Concentration of Cyt C. The concentration of cytochrome c was spectroscopically determined at 340 nm ( E ~ M= 21.4, for both reduced and oxidized states). The percentage of oxidized cytochrome c was determined at 550 nm by using molar extinction coefficient ~ m h ?= 28 for reduced cytochrome c and c m =~ 8 for the oxidized form at pH 7.0.12 Preparation of SAM on Colloidal Ag. Thiol derivatives dissolved in 1% acetonitrile/water were added to colloidal Ag and incubated at ambient temperature. After 1 h or more, the solution of Cyt C (100 pM) was added. Raman Spectroscopy. The Raman spectra were recorded on a NR-1100 spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan; light source, Argon laser 457.9, 488.0, or 514.5 nm) with a band resolution of 5 cm-I or less in a 300 p L quartz cell (liquid sample) or a sealed glass capillary (solid sample). The observation cells were thermostated by a Peltier device (RTIC, Japan Spectroscopic Co.).
Results and Discussion Characterization of SAMs of Alkanethiolate on Ag. The SERS spectrum of C I I C Hon ~ colloidal Ag is shown in Figure 1 together with the Raman spectra in solid and liquid states. Key features of the SERS spectrum are (1) the about 25 cm-' shift of the band (Figure la, 711 cm-I) attributable to a stretching vibration of the C-S bond in the trans conformation compared with the positions in the spectra for the liquid state (Figure lb, 734 cm-I) and solid state (Figure IC, 736 cm-I) and (2) the absence of the S-H stretching band found at 2580 cm-I in the solid and liquid spectra (spectra are not shown). These features are reported to be characteristic of cleavage of S-H bonds followed by formation of S-Ag bands.I3 The v(C-S) region (600-750 cm-I) contains conformational information about C-C bonds adjacent to the C-S bond, and information about the conformation of the alkyl chain can be obtained from the v(C-C) region (1050-1150 cm-I). The relative intensities of SERS bands attributable to trans (T) and gauche (G) conformations in both the v(C-C) and v(C-S) regions suggest that the torsional angles of the C-C and C-S bonds in the trans conformation are dominant in the SAM of C I I C H ~Previously, . SERS of SAMs of 1-alkanethiols on Ag or Au was extensively studied by Bryant et al.13a3bThe features of the SERS spectrum observed here are consistent with their results.
1000
800
600
Wavenumber (cm-')
Figure 2. SERS spectra in the v(C-C) and v(C-S) regions (488 nm excitation) of the SAM from o-mercaptoalkanoic acids on colloial Ag: (a)CllCOOH;(b)C~~COOH;(c)C~COOH;(d)C~COOH;(e)C~COOH. Incident laser power: 200 mW. TABLE 1: Raman Vibrational Assignments and Peak Frequencies (cm-') in the v(C-S) and v(C-C) Regions of CH3(CH&SH and HOOC(CH2)"SHAdsorbed on Ag assignments n = 2 n = 3 n = 5 n = 7 n = 10 n = 11 CH~(CHZ),SH on Ag 635" 630b 636b 702" 699b 707b 1067b 1O8lb 1096b 1097b HOOC(CH&SH on Ag 652 701 710 721 712 1033 1071 1090 1105 1105 Results of Bryant et al.I3" Results of Joo et (I
The SERS spectra of the S A M formed from C,COOH ( n = 2, 5 , 7, 10, 11) are shown in Figure 2, and the frequencies of the bands in the v(C-C) and v(C-S) regions are given in Table 1. As for the conformation of the C-S bonds, the trans conformer was dominant with the exception of the case of CZCOOH, in which the gauche conformer was recognized. Comparing the relative intensities due to the T and G conformers, trans conformer with respect to C-C bonds is also dominant when YZ > 7. After all, both C,CH3 and C,COOH with a relatively longer alkyl chain form well-ordered SAMs on colloidal Ag, the structures of which resemble those of lipid bilayers in the gel phase. Details of the structural characterization of the S A M formed from C,COOH will be reported e1~ewhere.l~ SERRS Spectra of Cyt C Adsorbed on SAMs. By addition of Cyt C to colloidal Ag modified with CllCOOH, a clear Raman spectrum of Cyt C superimposed on the signals of C I I COOH was obtained under 514.5 nm excitation (Figure 3b). In this concentration region the resonance Raman (RR) spectra of Cyt C could not be observed under our experimental conditions, and therefore, the contribution of Raman scattering from free Cyt C molecules was negligible. The spectrum shown in Figure 3b was obtained for the same sample but under 488.0 nm excitation. Any signals attributable to Cyt C were not observed, and only those attributable to CllCOOH were detectable. Since the 514.5 nm exciting line was in resonance with the electronic absorption of Cyt C @ band), a strong SERRS of Cyt C detached from the Ag surface was observed with SERS of CIICOOH directly attached to Ag. On the other hand, since the sensitivity of SERS is limited at shorter distances from the Ag surface than that of SERRS,I5 signals attributable to Cyt C were not observed under 488.0 nm excitation. As reported by Aroca et al., the spectroscopic tuning was possible due to the fact that
Cytochrome c Bound to Self-Assembled Monolayers
J. Phys. Chem., Vol. 99, No. 13, 1995 4839
n v)
V
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'
- 5--. A
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1200 1000 800 Wavenumber (cm-')
1400
.
I
600
Figure 3. RR and SERRS spectra of Cyt C2+: (a) RR, 100 pM aqueous solution excited at 514.5 nm; (b) SERRS, adsorbed on a SAM from CllCOOH excited at 514.5 nm; (c) same as part b but excited at 488
nm.
10
9
8
PH Figure 5. Relative intensities of of Cyt C to v(C-S) of cysteine plotted as a function of pH of solutions in the Cyt Ckysteine SAM/Ag system. Excited at 514.5 nm with 200 mW.
Electronic absorption
IKTnm 457.9 nm
n I/)
-" A
W
1.0'
n
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A W I
//
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6'a
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Incident laser beam Raman scattering
Figure 4. Schematic drawing of a hypothetical structure of Cyt
CISAMIAg. The relationship between the wavelength of the incident laser beam (either on or off resonance to electronic absorption of Cyt C) and the relative intensity of the Raman scattering from Cyt C (SERS or SERRS) and the SAM (SERS) is also schematically explained. SERRS at a distance is stronger than the SERS of the monolayer which is directly attached to Ag s u r f a ~ e . 'These ~ results c o n f i i that Cyt C molecules formed an upper layer on the CIICOOH monolayer which was directly attached to the Ag surface, as schematically illustrated in Figure 4. Contrary to the cases of SAMs formed from o-mercaptoalkanoic acids, attempts to observe SERRS spectra of Cyt C with Ag colloids modified with 1-alkanethiols or 2-mercaptoethylamine, which have hydrophobic or positively charged surfaces, respectively, yielded negative results. To c o n f i i that Cyt C was adsorbed on negatively charged surfaces selectively, we evaluated the relative intensity of SERRS from Cyt C adsorbed on the Ag colloid modified with a SAM of cysteine at various pH's. The relative intensity of ~ 1 (mode 3 numbering as in ref 16, in-plane bending vibration) of Cyt-C to that of v(C-S) of cysteine abruptly increased around pH 9 (Figure 5). The SAM formed from cysteine has no net charge at neutral pH, but the negative charge on the SAM may increase suddenly around the pK, of the amino group (=10.8 in free amino acid).
Figure 6. Relative intensities of
VI3 of Cyt C to v(C-S) of C,COOH plotted as a function of number of methylene groups (n) in Cyt C/C,COOH SAM/Ag system. Excited at 5 14.5 nm with 200 mW.
The abrupt increase in SERRS intensity strongly insists that Cyt C was adsorbed on negatively charged surfaces selectively. It is well-known that protonated lysine residues located in the exposed heme edge region of Cyt C are available to interact with the carboxylate-rich surface domain on its protein reaction partner such as cytochrome oxidase." Moreover, Cyt C is adsorbed on negatively charged surfaces such as liposomes,2b.'8 SAMs,I9and polyanions.'Od Our finding is consistent with those results. Effects of Chain Length of the SAM on the Intensity of the SERRS of Cyt C. The effects of chain length of C,COOH on the intensity of the SERRS from Cyt C adsorbed on the SAM of C,COOH were evaluated. In Figure 6, the relative intensities of V I 3 of Cyt C to v(C-S) of C,COOH excited at 514.5 nm are plotted as a function of number of methylene groups (n)in Cyt C/C,COOH S M A g system. The relative intensity of vl3/v(C-S) increased with an increase of chain length when n < 7 and decreased when n > 7. The intensity of SERRS from Cyt C is dependent on both the amount of Cyt C adsorbed and the enhancement factor, which is determined by the distance from the surface and the orientation of the heme chromophore with respect to the surface. Using an ultracentrifugation technique, the amount of Cyt C adsorbed on the S M A g composite surface was found to increase gradually with an increase of R (42%of the added Cyt C was adsorbed when n = 2, and 65% was adsorbed when n = 11). Consequently, the phenomenon in Figure 6 is partially due to the difference in the amounts of adsorbed Cyt C, but the difference in the relative intensities is much larger than that in the amounts of adsorption. As for the distance between the heme and the Ag surface, the tilt angle of the mercaptoalkanoic acid on the Ag surface
4840 J. Phys. Chem., Vol. 99, No. 13, 1995 was reported to be 30" to the normal,20 and therefore, the distance from Ag surface to the SAM surface increases 1.1 8, per methylene group on average. If we consider the shortest possible distance of the protein-bound porphyrin to the surface of the SAM, the center of the tetrapyrrole ring is 15 8, away from the surface.Iof Since adsorbed Cyt C molecules are detached from the Ag surface, the strong S E W S of Cyt C is due to electromagnetic enhancement. The electromagnetic theory of SERS and SERRS assumes that the electric fields of both the exciting and scattered radiation are enhanced by coupling with surface plasmons.21 The intensity of SERRS largely depends on the intensity of light absorbed by the chromophore, and therefore, the orientation of the chromophore with respect to the surface of the Ag is also important for the magnitude of the enhancement factor. As for the heme chromophore, the strongest enhancement factor is obtained when the plane of the heme is perpendicular to the surface of the Ag.Iof Considering the most stable orientation of the Cyt C which is adsorbed on the negatively charged surface, protonated lysine residues located in the exposed heme edge region of Cyt C are attached to the surface.I7 In this state, the heme chromophore is almost the nearest to the surface and the heme plane is nearly perpendicular to the surface of the Ag, which means that the most stable orientation of Cyt C on a S M A g composite is the most effective orientation for SERRS. As mentioned above, the structure of the SAM is partially irregular when n < 7. It is highly probable that the orientation of the Cyt C molecules adsorbed on an irregular surface is more dispersed than that of Cyt C's adsorbed on ordered and flat surfaces which are formed when n > 7. The possible reasons for the increase of SERRS intensity with n below 7 are the increase in the amount of adsorbed Cyt C and the degree of orientational regularity. The gradual decrease in SERRS intensity above 7 can be accounted for by the increase of the distance from the surface. Characterization of Cyt C Adsorbed on SAMs. SERRS spectra of Cyt C adsorbed on Ag modified with SAMs were similar to resonance Raman spectra in solution (Figure 3a,b), whereas SERRS spectra of Cyt C directly adsorbed on the Ag surface were different from the solution spectra. As pointed out by Hildebrandt et al., Cyt C adsorbed on charged surfaces exists in two different conformational states.'& In state I, the native structure is fully preserved and the heme Fe exists in a six-coordinate low-spin configuration. In state 11, the heme cleft assumes an open conformation compared to the closed structure in state I and the heme Fe exists in mixture of five-coordinate high-spin (HS) and six-coordinate low-spin (LS) configurations. In SERRS spectra of Cyt C which is directly adsorbed on the metal surface, an intensity decrease of depolarized and anomalously polarized bands (v16, v22, v30, vl3, v21) with respect to the polarized band v4 is recognized. These phenomena are consistent with the results by Hildebrandt et a1.,Iof and the intensity decrease of those bands is at least partially due to the population of the HS state. The transition from LS to HS is accompanied by a blue shift of the electronic absorption, and therefore, in the HS state the @ band moves out of resonance with the 514.5 nm line. Since only the depolarized and anomalously polarized bands have sharp resonances in their excitation profiles at the ,L? band position, they lose the intensity in the HS state while the strength of the polarized v4 band is little affected. The position of the v4 band is known to be altered depending on the oxidation state of the heme.'." By comparing the position of the oxidation-state marker bands of Cyt C in resonance Raman spectra in solution and SERRS spectra
Maeda et al.
TABLE 2: Peak Positions of the Oxidation-StateMarker Band (vd of Cvt C ~~~
excited at 457.9 nm
excited at 514.5 nm
Cyt c
RR"
Agb
SAMc
RR"
Agb
SAM'
Cyt c*+ Cyt C2+ SHSd Cyt c3+
1361
1369 1364 1372
1362 1363 1373
1360
1367 1361 1369
1360 1361 1368
+
1373
1370
a Resonance Raman spectra of 100 p M solution. SERRS spectra of Cyt C directly adsorbed on colloidal Ag. SERRS spectra of Cyt C adsorbed on the C1 lCOOWAg system. Observed in the presence of sodium hydrosulfite.
adsorbed on a SAM, it is concluded that the oxidation states of both Cyt C2+ and Cyt C3+ are preserved by adsorption on a SAM. On the other hand, adsorption-induced oxidation was observed in Cyt C2+ which was directly adsorbed on a Ag surface. By addition of sodium hydrosulfite as a reductant, oxidized Cyt C on Ag was reduced to Cyt C2+,as is shown in Table 2 . In conclusion the biomembrane mimetic system examined here provides abundant information about the interaction between proteins and membranes with a very small amount of sample. As for the interaction between Cyt C and SAMs, it is concluded that Cyt C is selectively adsorbed on the negatively charged surface of SAMs, and the oxidation and spin states of Cyt C are preserved after the adsorption on the SAMs.
Acknowledgment. This work was supported by Grants-inAid (06453153, 06750922) from the Ministry of Education, Science and Culture, Japan. References and Notes (1) Watts, A,, Ed New Comprehensive Biochemistry, Vol. 25, ProteinLipid Interactions; Elsevier: Amsterdam, 1993. (2) (a) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 113-158. (b) Kakinoki, K.; Maeda, Y.; Hasegawa, K.; Kitano, H. J. Colloid Interface Sci., in press. (c) Bergers, J. J.; Vingerhoeds, M. H.; Bllois, L.; Herron, J. N.; Janssen, L. H. M.; Fischer, M. J. E.; Crommelin, D. J. A. Biochemistry 1993, 32, 4641-4649. (d) Takeuchi, H.; Ohtsuka, Y.; Harada, I. J . Am. Chem. SOC. 1992, 114, 5321-5328. (3) Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E. Langmuir 1993, 9, 136-140. (4) (a) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. SOC.1983,105,44814483. (b) Hill, W.; Wehling, B. J. Phys. Chem. 1993, 97, 9451-9455. (5) (a) Spinke, J.; Liley, M.; Gunder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993,9, 1821-1825. (b) Prime, K. L.; Whitesides, G. M. J. Am. Chem. SOC. 1993, 115, 10714-10721. (c) Lopez, G. P.; Albers, M. W.; Schreiber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J . Am. Chem. SOC. 1993, 115, 5877-5878. (6) (a) Broderick, J. B.; Natan, M. J.; O'Halloran, T. V.; Van Duyne, R. P. Biochemistry 1993, 32, 13771-13776. (b) Rohr, T. H.; Cotton, T.; Fan, N.; Tarcha, P. J. Anal. Biochem. 1989,182, 388-398. (c) Caron, K.; Peltersen, L.; Lewis, M. Environ. Sci. Technol. 1992, 26, 1950-1954. (7) Carey, P. R. Biological Applications of Raman and Resonance Raman Spectroscopies; Academic Press: New York, 1982. (8) Smulevich, G.; Spiro, T. G. J. Phys. Chem. 1985, 89, 5168-5173. (9) Lee, N.-S.; Hsieh, Y.-Z.;Moms; M. D.; Schopfer, L. M. J. Am. Chem. SOC.1987, 109, 1358-1363. (10) (a) Hildebrandt, P.; Vanhecke, F.; Buse, G.; Soulimane, T.; Mauk, A. G. Biochemistry 1993, 32, 10912-10922. (b) Hu, S.; Moms, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. SOC. 1993, 115, 12446-12458. (c) Walker, D. S.; Hellinga, H. W.; Saavedra, S. S.; Reichert, W. M. J . Phys. Chem. 1993,97, 10217-10222. (d) Hildebrandt, P. Biochim. Biophys. Acta 1990, 1040, 175- 186. (e) Hildebrandt, P.; Stockburger, M. Biochemistry 1989.28, 6010-6021. (0 Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986, 90, 6017-6024. (11) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. SOC.1989, 111, 321-335. (12) Moore, G. R.; Pettigrew, G. W., Eds. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects; Springer Verlag: Berlin, 1990; pp 1-361. (13) (a) Bryant, M. A.; Pemberton, J. E. J . Am. Chem. SOC. 1991,113, 3629-3637. (b) Bryant, M. A,; Pemberton, J. E. J . Am. Chem. SOC.1991, 113, 8284-8293. (c) Joo, T. H.; Kim, K.; Kim, M. S. J . Phys. Chem. 1986, 90, 5816-5819.
Cytochrome c Bound to Self-Assembled Monolayers (14) Maeda, Y.; Yamamoto, H.; Kitano, H. Publication in preparation. (15) Aroca, R.; Guhathakurta-Ghosh, U. J . Am. Chem. SOC.1989, 111, 768 1-7687. (16) Abe, M.; Kitagawa, T.; Kyogoku, Y. J . Chem. Phys. 1978, 69, 4526-4534. (17) (a) Salemme, R. J . Mol. Biol. 1976, 102, 563-568. (b) Wendolowski, J. J.; Matthew, J. B.; Weber, P. C.; Salemme, F. R. Science 1987, 238, 794-797. (c) Mauk, M. R.; Mauk, A. G.; Weber, p. C.; Matthew, J. B. Biochemistry 1986,25,7085-7091. (d) Poulos, T. L.; Kraut, J. J . Biol. Chem. 1980,255, 10322-10330. (e) Satterlee, J. D.; Moench, S. J.; Eman, J. E. Biochim. Biophys. Acta 1987, 912, 87-97.
J. Phys. Chem., Vol. 99, No. 13, 1995 4841 (18) Spooner, P. J. R.; Watts, A. Biochemistry 1992,3I, 10129-10138. (19) Song,S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J . Phys. Chem. 1993, 97, 6564-6572. (20) (a) Ulman, A. An Introducfion fo Ultrathin Organic Films from Langmuir-Blodgett to SelfAssembly; Academic Press: San Diego, CA, 1991. (b) Laibinis, P. E.; Fox, M. A,; Folkers, J. P.; Whitesides, G. M. hngmuir 1 9 1 , 7, 3167-3173. (21) Garrell, R. L. Anal. Chem. 1989, 61, 401A-411A. JP9424724
