Langmuir 1994,10, 2382-2388
2382
Specific Protein Attachment to Artificial Membranes via Coordination to Lipid-Bound Copper(I1) D. R. Shnek, D. W. Pack, D. Y. Sasaki, and F. H. Arnold* Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, California 91125 Received February 17,1994. In Final Form: April 11, 1994@ A versatile and convenient method for targeting proteins to lipid assemblies using metal ion coordination is described. Mixed lipid bilayers and Langmuir monolayers containing a metal-chelatinglipid and divalent copper ions are shown to bind protein via surface-accessible histidine residues. Cu2+ chelated by iminodiacetate (IDA) in the headgroup serves as an affinity ligand to target the protein to the interface. The compact,uncharged CuZ+-IDA headgroup can be incorporated into lipid assemblies without disrupting the lipid packing. Surface pressure-area isotherms of DSPC monolayers containing 5 mol % of IDA-lipid show that Cu2+enhances the rate and extent of myoglobin association with the interface. Myoglobin binds to small unilamellar vesicles containing 2% Cu2+-IDA lipid (48% DSPC and 50% cholesterol)at least an order of magnitude more tightly than to vesicles without metal or loaded with Ca2+. The Cu2+-IDA lipid more than doubles the amount of protein targeted to the interface. Cu2+ESR parameters gll and Ail, measured for liposomeswith native and DEPC-modifiedmyoglobin, support coordination of surface histidine side chains to Cu2+ as the binding interaction.
Introduction The specific targeting of proteins to interfaces is important for applications in biomedicine, structure determination, sensors and devices, synthesis of materials with defined, highly-ordered molecular architectures, as well as for model studies of protein interactions in biological membranes. The organization of proteins on artificial lipid membranes can be mediated by interactions with specific lipid-bound receptors or affinity ligands, a widely cited example of which is the orientation and twodimensional crystallization of streptavidin on biotinfunctionalized membranes.l Although the biotin-streptavidin system exhibits very strong and specific binding, the range of proteins that can be bound to the membrane surface and the types of molecular architectures produced are limited. Other affinity ligands that have been used to target proteins to model membranes and surfactant assemblies include haptens for antibodieq2 antibodies for specific protein^,^ inhibitorsleffectors for enzyme^,^ and the heme prosthetic group for ap~myoglobin.~ Unfortunately, no convenient natural affinity ligands have been identified for many proteins with interesting biological, optical, electrical, or catalytic properties. A versatile, generally-applicable affinity system for targeting and orienting functional proteins a t membranes would be extremely useful. We are also interested in the template-directed patterning of metal ions in lipid assemblies. Using the
* To whom correspondence should be addressed:
-
Tel, 818-395-
4162,FAX: 818-568-8743. Abstract published in Advance ACS Abstracts, May 15,1994. (1)(a) Ahlers, M.; Muller, W.; Reichert, A,, Ringsdorf, H.; Venzmer, J. Angew. Chem., Znt. Ed. Engl. 1990,29,1269.(b) Blankenburg, R.; Meller, P.; Ringsdorf, H; Salesse, C. Biochemistry 1989,28,8214. (2)(a) Uzgiris, E. E.; Kornberg, R. D. Nature 1983,301, 125.(b) Ahlers, M;Grainger, D. W.; Herron, J. N.; Lim, IC;Ringsdorf, H.; Salesse, C. Biophys. J. 1992,63,823.(c) McCloskey, M. A.; Poo, M. J. Cell Biol. 1986,102,2185. (d)Tamm, L. KBiochemistry 1988,27,1450. (0Lewis, J. T.; Hafeman, D. G.; McConnell, H. M. Biochemistry 1980,19,5376. (3)(a)Ribi, H.0.; Reichard, P.; Kornberg, R. D. Biochemistry 1987, 26,7974.(b)Lebeau,L., Regnier,E., Schultz, P., Wang, J. C.;Mioskowski, C.; Oudet, P. FEBS Lett. 1890,267,38. (4)Grainger, D. W.; Reichart, A.; Ringsdorf, H.; Salesse, C. FEBS Lett. 1989,252,73. (5)Hamachi, I.; Nakamura, K.; Fujita, A.; Kunitake, T. J.Am. Chem. SOC.1993,115,4966. @
technique of template polymerization, or “molecular imprinting”, we have prepared macroporous polymers capable of recognizing bis(imidazo1e)“protein analogsn.6s7 These polymers were synthesized using the desired target compound as a template to position metal-containing monomers such t h a t they complement the template’s metal-coordinating groups. The goal is to create a binding cavity in the final material with a specific arrangement of metal ions that matches the template. We are currently extending this template polymerization concept to mixed lipid assemblies in order to prepare metal-chelating lipid monolayers and bilayers patterned a t the nanometer scale.8 The metal coordination interaction should be sufficiently strong to direct the placement of metalchelating lipids in monolayer or bilayer assemblies; this pattern could be “fixed” by polymerization and crosslinking of the lipid tails.g The imidazole moieties of histidyl residues on the surfaces of proteins readily coordinate to divalent transition-metal ions such as Cu2+, Ni2+, and Zn2+;10when immobilized onto solid supports via appropriate chelating agents, metal ions can serve as affinity ligands for protein purification by metal-affinity chromatographyll and for protein immobilization.12 The chelating group should bind the metal ion tightly yet leave coordination site(s) available for formation of a ternary complex with the protein. A tridentate chelator widely used in metal-affinity chromatography, iminodiacetate (IDA), binds Cu2+ with a n association constant of loll M-l.13 The Cu2+-IDAcomplex in turn binds imidazole with moderate affinity (K 103.5 (6)Dhal, P.; Arnold, F. H. J. Am. Chem. SOC.1991,113, 7417. (7)Dhal, P.; Arnold, F.H. Macromolecules 1992,25,7051. (8)Mallik, S.;Plunkett, S. D.; Dhal, P. K; Johnson, R. D.; Pack, D. W.; Shnek, D. R.; Arnold, F. H. New J. Chem. 1994,18,299. (9)Polymerizable diacetylenic IDA-lipids have been prepared for this purpose by our collaborators a t the Naval Research Laboratory, Markowitz, M. A.; Tsao, Li-I., Singh, A. Unpublished results. (10)Sundberg, R. J.; Martin, R. B. Chem. Rev. 1974,74,471. (11)(a) Porath, J.;Carlsson, J.; Olsson, I.; Belfrage, G.Nature 1975, 258,598.(b) Arnold, F.H. BiolTechnology 1991,9,151. (12)(a)Ill, C. R.; Keivens, V. M.; Hale, J . E.; Nakamura, K. K.; Jue, R. A.; Cheng, S.; Melcher, E. D.; Drake, B.; Smith, M. C. Biophys. J. 1993,64, 919.(b) Piesecki, S.; Teng, W.-Y.; Hochuli, E. Biotechnol. Bioeng. l99S,42, 178.(c) Smith, M. C.; Cook, J. A,; Smanik, P. A.; Wakulchik, M.; Kasher, M. S. Methods 1992,4,73. (13)Martel1,A. E.;Smith, P. M. CriticaZStabiZity Constants;Plenum Press: New York, 1974;Vol. 6.
Q743-7463/94/241Q-2382$04.50/0 0 1994 American Chemical Society
Protein Attachment to Artificial Membranes
M-l) in s ~ l u t i o nand ' ~ at the surface of a chromatographic support.15 While proteins with a single accessible histidine bind with similar affinities to surfaces densely derivatized with Cu2+-IDA, those with multiple surface histidines can adsorb much more strongly by forming multiple, simultaneous histidyl-Cu2+ coordination bonds. Studies with engineered protein variants containing as few as two surface-accessible histidines have shown that multipoint attachment results in apparent protein binding constants greater than lo6M-'.15 Alternatively, proteins can be engineered to display high-affinity surface metalbinding siteslSor metal-binding peptides a t their N- or C-termini," either ofwhich can be used for specificprotein immobilization on metal-derivatized surfaces.12 The metal-protein interaction is reversible under mild conditions: competitors such as imidazole, small quantities of acid, or strong chelating agents all effectively disrupt the protein-Cu2+-IDA complex, releasing the bound protein." Chelating amphiphiles have been prepared for studies of metal ion binding to monolayers and artificial bilayer membranes. Porphyrins,18 dithio~arbamate,'~ cyc1am,20 iminodiacetatq21 crown ethers,22 and noncyclic crown analogsmhave been incorporated into the polar headgroup regions of these materials to study the ion-binding properties and the anisotropic organization of metal chelates in monolayers and bilayers,2O to make metal ion sensors,1g and to control the orientation and packing of membrane component^.^^ For the purpose of targeting proteins to monolayer and bilayer assemblies, we have synthesized a lipid with an IDA moiety in the headgroup (1in Scheme 1).When loaded with Cu2+,small quantities ofthis IDA-lipid in monolayers and liposomes of distearoyl phosphatidylcholine (DSPC) effectively bind a small, histidine-rich protein, myoglobin. Horse heart myoglobin (M, = 17641) contains 11 histidines, at least four of which can coordinate to Cu2+IDA." We report herein on myoglobin binding to monolayers and vesicles of DSPC and IDA-lipid 1 containing Cu2+and Ca2+. Because imidazole is not a good ligand for Ca2+ (K,= 1.2 M-1),26 this divalent ion provides a noncoordinating surface for comparison. Monolayer surface pressure-area (JC-A)isotherms, measurements of protein binding to liposomes, and ESR analyses of Cu2+containing liposomes in the presence of unmodified and diethyl pyrocarbonate (DEPC)-modified protein indicate (14)Sinha, P.C.; Saxena, V. K; Nigam, N. B.; Sriastava, M. N. Indian J. Chem. 1989,28A,335. The reported binding constant of 2.2 x los M-1 at 35 "C waa extrapolated to 25 "C using AH = -7.6 kcdmol (ref 13). (15)Todd, R.J.; Johnson, R. D.; Amold, F. H. J. Chromutogruphy 1994, 662,13. (16)(a)Amold, F. H.; Haymore, B. L. Science 1991,252,1796. (b) Haymore, B. L.; Bild, G. S.; Salsgiver, W. J.;Staten, N. R.; Krivi, G. G. MGhods 1992,4,25. (17)(a) Smith, M. C.; Furman, T. C.; Ingolia, T. D.; F'idgeon, C. J. Biol. Chem. 1988.263.7211. (b) Hochuli. E.:Bannwarth., W.:, Dobeli. H.; Gentz, R.; S&ber, D.BiolTechnolo& 1988,6,1321. (18)Tsuchida, E.;Komatau, T.; Arai,K. J. Am. Chem. SOC.1993,9, 730. (19)(a) Budach, W.;Ahqja, R. C.; Mtibius, D. Langmuir 1993,9, 3093.(b) Budach, W.; Ahqja, R. C.; Mtibius, D.; Schrepp, W. Thin Solid Films 1998,210,434. (20)(a) Kunitake, T.; Iehikawa, Y.; Shimoumura, M.; Okawa, H. J. Am. Chem. Soc. 1986, 108,327. (b) Ishikawa, Y.;Kunitake, T. J. Macromol. Sci.-Chem. 1990,A27,1157. (21)Furhop, J.-H.; Koesling, V.; Schonberger, G. Justus L i e b i g s h n . Chem. 1984.10., 1634. (22)Munoz, S.;Mallen, J.;Nakano, A; Chen, Z.; Gay, I.; Echegoyen, L.; Gokel, G. W. J. Am. Chem. Soc. 1995,115,1705. (23)Yanagi,M.; Tamamura, H.; Kurihara, K; Kunitake, T.Langmuir 1991,7, 167: (24)Wuenschell, G. E.;Wen, E.; Todd, R.; Shnek, D.; Amold, F. H. J . Chromutogr. 1991,543,345. (25)Schubert, J. J. Am. Chem. Soc. 1954,76,3442. - r -
1
>On
I
(a)NaH, 1-(methanesulfonyl)-lO-(tziphenylcarbinyl)-l,4,7,lOtetraoxadecane, THF, reflux, 14 h. (b)p-Toluenesulfonic acid, MeOH/THF, room temperature, 5 h, 88%(two steps). (c)CBr4, PPh, THF, 0 "C room temperature, 4 h, 79%. (d) Diethyl iminodiacetate, triethylamine, THF, reflux, 2 days, 34%. (e) NaOH, THFNeOH, H2O (7:7:1),reflux, 15 h, 51%.
-
a no CU" b 0.20pMCu2' C
0.30pMCu"
d 0.50 pM Cu"
z
E 30
e 10pMCu"
k!
20
40
20
60 80 100 120 140 160 MMA (A'/molecule)
Figure 1. Surface pressure-area isotherms of pure lipid 1 on
buffered subphase (20mM MOPS, pH 7.5,24 "C) containing 0-10 pM CuC12. t h a t myoglobin binding to these artificial membrane assemblies is significantly enhanced by coordination of surface histidines to Cu2+ions immobilized a t the membrane surface. '
Results and Discussion Amphiphile 1 was designed to have a structure similar to the phosphatidylcholine lipids to optimize its miscibility in DSPC. To eliminate the possibility of tail cleavage, however, 1 features ether linkages from the 2 and 3 positions of the glycerol backbone to the octadecyl tails. A triethylene glycol unit serves as a spacer between the IDA headgroup and the glycerol backbone to enhance the accessibility ofthe metal ion and minimize steric hindrance to protein binding. The synthesis of 1 is outlined in Scheme 1(see Experimental Section for details). Metal-Chelating Monolayers. Surface pressurearea (n-A) isotherms are sensitive indicators of the packing and arrangement of molecular assemblies at the air-water interface. The n-A isotherm of pure 1 t h a t has been fully loaded with Cu2+ exhibits only a solid condensed phase (Figure 1,curve e). With 100 mM NaCl in the buffered subphase, the n-A curve condenses further and exhibits a limiting mean molecular area of 41 &I molecule, which corresponds closely to the area of the two alkyl tails. The n-A isotherms of a pure DSPC monolayer and a monolayer of DSPC containing 5 mol % 1 and loaded with Cu2+ in Figure 2 show t h a t small amounts of the metal-chelating lipid have a minimal effect on the lipid packing, particularly at high surface pressures. This lipid, with its compact, charge-neutral Cu2+-IDA headgroup,
2384 Langmuir, Vol. 10, No. 7, 1994
Shnek et al. 70
6ol a
I
I
50
30 20
t
10 01
20
I
I
30
40
50
60
I
I
70
80
90
20
50 60 70 MMA (A2/molecule)
40
30
80
90
I
,
80
90
MMA (A*/molecule)
Figure 2. Surface pressure-area isotherms of DSPC and mixed DSPC/1-Cu2+ (19:l)monolayers on a buffered subphase (20 mM MOPS, 100 mM NaC1, pH 7.5), 24 "C. The DSPC/lCu2+subphase contained 10 pM CuClz + 1 mM CaC12. can be assembled into compact monolayers and incorporated into phosphatidylcholine monolayers with little effect on the film structure. Changes in the isotherms can provide useful information on interactions between monolayer components and molecules added to the subphase. n-A isotherms of monolayers of pure lipid 1titrated with varying amounts of CuClz (0-10 pM) in the buffered subphase are shown in Figure 1. The n-A curve for the unmetalated monolayer (curve a ) shows biphasic behavior with a liquid expanded region, a take-off point of 140 A2Aipid molecule and an extended phase transition region at 3 mN/m, and a solid phase with a limiting mean molecular area of 49 AVmolecule. The n-A curve is little affected by concentrations of Cu2+below 0.2 pM. As more metal is added, however, the liquid expanded phase is diminished, leaving the solid phase region with a take-off point of 60 A2/ molecule and a limiting area of 50 &/molecule a t only 0.5 pM Cu2+. The isotherm remains essentially unchanged between 0.5 and 10 pM, and it is reasonable to assume that the surface chelating sites are saturated. The loss of the liquid expanded phase of the monolayer upon metal binding can be rationalized by considering the influence of the metal on the IDA headgroup. At pH 7.5, chelation of a divalent metal ion by the negativelycharged IDA results in a compact, charge-neutral headgroup. The charge and the mobility of the acetate groups are most likely responsible for the build-up of pressure a t high molecular area. Both expansionz3and c o n d e n ~ a t i o n ~ ~ upon metal binding have been observed for n-A isotherms of other chelating monolayers. Lipid 1 differs from previously studied metal-chelating amphiphiles, however, in that metal binding to IDA neutralizes the charge in addition to changing the headgroup structure. Similar behavior of the monolayer can be effected with calcium, although this requires much higher concentrations of metal ion. The n-A curve of lipid 1 on the same buffered subphase containing 1mM CaClz is identical to the 1-Cu2+ n-A curve in Figure 1. The behavior of the monolayer over the range of calcium concentrations from 0 to 1mM mirrors the behavior in the presence of 1-10 pM Cu2+. That saturation of the monolayer requires several orders of magnitude more Ca2+than Cu2+is to be expected, given the relative binding constants of IDAwith the two metal ions (&-IDA = 1010.6 versus KC~-IDA = 102,6 M-1).133 Further, the n-A isotherm of lipid 1 on a buffered subphase containing both 10 ,LAM Cu2+and 1mM (26) The high local concentration of IDA and the presence of a charge double layer can cause the stability constants for metal complexation at the interface to differ from those in bulk solution. However,the relative affinities for different ions should not change greatly.
::I1 I
$0
L
,
I
50 60 70 MMA (A*/molecule)
40
30
Figure 3. (A, top) Surface pressure-area isotherms of pure lipid 1on buffered subphase (20 mM MOPS, 100 mMNaC1, pH 7.5) containingdivalent metal ions and protein(a and b): curve a, 10 pM CuClz 1 mM CaClz and 10 pM hMb (5min after injection of protein); curve b, 1mM CaClz and 10 pM hMb (15 min after injection of protein); curve c, either 1mM CaC12, or 10 pM CuClz 1mM CaClz (curves are identical), no protein. (B,bottom) Surface pressure-area isotherms of mixed monolayers of DSPC/1 on buffered subphase (20 mM MOPS, 100 mM NaCl, pH 7.5) containing divalent metal ions and protein (a and b): curve a, 10pM CuClz + 1mM CaClz and 10pM hMb (9 min after injection of protein); curve b, 1 mM CaClz and 10 pM hMb (23 min after injection of protein); curve c, either 1 mM CaClz or 10pM CuClz 1mM CaClz (curves are identical), no protein.
+
+
+
Ca2+is identical to the isotherms in the presence of either metal alone. Although the n-A curve does not indicate which metal is bound to the lipid under these conditions, we can reasonably expect the monolayer to consist almost exclusively of 1-Cu2+, given the relative metal concentrations and their affinities for IDA (~CU-IDA[C~~+] = 105.6, while ~ & - I D A [ C ~ ~ +1). ] Qualitative features of protein binding to metal-chelating lipids can be discerned from changes in the pressurearea (n-A) isotherms of monolayers of the lipids spread a t the air-water interface. Monolayers of pure lipid 1 spread on a buffered subphase containing the metal were compressed to 5 mN/m, and a concentrated solution of myoglobin was injected into the subphase behind the barriers to give a final protein concentration of -10 pM. The monolayers began to expand immediately after injection. n-A isotherms for pure 1-Cu2+ and 1-Ca2+ (Figure 3A, curves a and b) were measured after the monolayers were allowed to expand to 80 A2/moleculea t constant pressure.27 In these experiments, the isotherm measurements were initiated at the same surface pressure (5 mN/m), with the same area available to each lipid molecule (80 A2) and equal amounts of lipid in each monolayer. Thus, the increase in area per lipid molecule relative to the monolayer in the absence of protein (curve c) can be attributed to protein insertion (vide infra), and the two metal-containing monolayers presumably contain the same amount of myoglobin when compression begins.
-
(27) The area of 80 AVmolecule for the beginning of the isotherm is arbitraly. When allowed to, the monolayers continued to expand until the trough area reached its maximum. At this point, the surface pressure began to rise due to further insertion of protein.
Lungmuir, Vol.10, No. 7,1994 2385
Protein Attachment to Artificial Membranes
The F A isotherm of 1-Cu2+ in the presence of myoglobin (curve a) shows a steep increase in surface pressure at high molecular areas, followed by a region of high compressibility at 25-30 mN/m and then a highly condensed region with a n area of 41 &/molecule at 48 mN/m. In contrast, the 1-Ca2+ isotherm exhibits greater compressibility a t high areas and a less pronounced transition region. At low molecular areas this monolayer is much more compressible than the Cu2+-containing monolayer and exhibits a n area of 38 A2/molecule a t 48 mN/m. The isotherms of mixed monolayers (19:l) of DSPCI 1-Ca2+ and DSPC/l-Cu2+ in the presence of myoglobin, shown in Figure 3B, are similar to those of the corresponding monolayers of pure, metalated lipid 1. As for the 1-Cu2+ monolayer, the DSPC/1-Cu2+ monolayer on the protein-containing subphase (curve a)is significantly expanded at high pressure relative to the same monolayer in the absence of protein (curve c). Addition of myoglobin increases the molecular area at 50 mN/m from 39 to 45 &/molecule. The DSPC/l-Ca2+ monolayer (curve b) is also expanded at high pressure, but to a lesser degree (42 fiVmolecule at 50 mN/m). Because monolayers of 1-Cu2+ and 1-Ca2+ produce identical n-A behavior in the absence of protein, differences in the isotherms of the Cu2+-and Ca2+-containing monolayers highlight the effects of specificprotein-copper interactions. I n the presence of myoglobin, monolayers of both 1-Cu2+ and 1-Ca2+ are highly expanded at low pressure relative to the same monolayers in the absence of protein. This expansion can be attributed to nonspecific insertion of protein into the lipid film. Nonspecific protein insertion into lipid monolayers at low surface pressure has been observed for a variety of protein-lipid s y ~ t e m s . ~ The regions ofhigh compressibility a t large areas indicate t h a t the protein is being "squeezed out" of the lipid monolayer.28 A large fraction of the protein appears to be ejected from the films at -30 mN/m. Above this pressure the monolayer of pure 1-Ca2+ remains more expanded with respect to the same monolayer in the absence of protein. Because the strong coordination interactions are not available in the film containing calcium, we believe this residual expansion to reflect a small fraction of myoglobin t h a t remains nonspecifically at the interface. Above 30 mN/m the 1-Cu2+ film exhibits stillhigh& areas a t a given pressure, with a compressibility similar to that of the film without protein, indicating a solidlike phase has been reached. The enhanced protein binding can be attributed to specific interactions with the lipid-bound Cu2+. This further expansion could result from repulsion between protein molecules bound at the film surface. Alternatively, some of the protein molecules nonspecifically incorporated at low pressure could remain irreversibly bound via specific metal-protein interactions within the Cu2+-containing monolayer at high pressures. The metals also have different effects on the kinetics of protein association with the monolayers. Figure 4 shows isotherms of pure 1-Cu2+ (curve a) and 1-Ca2+ (curve b) monolayers, begun 5 min after protein injection. The isotherm of pure, metalated lipid 1in the absence of protein (curve c) exhibits an area of 45 AVmolecule at 5 mN/m. After 5 min a t 5 mNIm, the 1-Cu2+ monolayer expanded by 35Az/molecule,while the l-Ca2+ monolayer expanded (28) (a) Peschke, J.; Mohwald, H. Colloids Surf. 1987,27, 305. (b) Saint-Pierre-Chazalet, M.; FressignO, C.; Billoudet, F.; Piled, M. P. Thin Solid Films 1992,210l211, 743. (29) (a) Colacicco, G. Lipids 1969,5,636.(b)Bougis,P.; Rochat, H.; Pieroni, G.; Verger, R. Biochemistry 1981,20,122. (c) Phillips,M. C.; Sparks, C. E. Ann. N.Y. h a d . Sci. 1980,122.
MMA (A*/molecule)
F'igure 4. Surface pressure-area isotherms of pure lipid 1 (solid lines) and mixed monolayers of DSPC/l (brokenlines)on buffered subphase (20 mM MOPS, 100 mM NaCI, pH 7.5) containing divalent metal ions and protein: curve a, 10 p M CuClz 1mM CaC12 and 10 pM hMb (5min after injection of protein); curve b, 1mM CaClz and 10 pM hMb (5 min after injection of protein);curve c, either 1mMCaClz or 10pM CuCl2 1mM CaClz (curvesare identical),no protein; curve d, 10pM CuClz 1mM CaClz and 10 pM hMb (9min after injection of protein); curve e, 1 mM CaClz and 10 p M hMb (9 min after injection ofprotein);curve f, either 1mM CaClz or 10pM CuCl2 1mM CaC12 (curves are identical), no protein.
+
+
+
+
by only 10 &/molecule. (Full expansion of the l-Ca2+ monolayer to 80 A2/molecule required -15 min (Figure 3A)J Expansion of the mixed DSPC/1 monolayers to the same area was somewhat slower. Nine minutes after addition ofmyoglobin, the DSPC/1-Cu2+ monolayer (curve d) expanded by 32 &/molecule, while the molecular area of the DSPC/l-Ca2+ monolayer (curve e) increased by only 14 &/molecule, relative to the metalated DSPC/l monolayer in the absence of protein (48 .&molecule, curve (Full expansion of the DSPC/1-Ca2+ monolayer to 80 Vmolecule required -23 min (Figure 3BI.l The constant pressure expansion upon protein injection is most likely due to insertion of myoglobin into the lipid films. Inclusion of Cu2+in the lipid monolayers, however, significantly increases the rate of protein insertion. Because the identity of the metal has no effect on the films in the absence of protein, the rate enhancement in the Cu2+-chelating systems may be attributed to an increase in local protein concentration near the interface due to interactions with the bound copper ions. Metal-ChelatingLiposomes. Metal-chelating small unilamellar vesicles (SUVs) (mean size 35-48 nm, distributionwidth 15-30 nm) were made from DSPC and cholesterol incorporating a small amount of the chelating lipid 1(2 mol % lipid 1, 48 mol % DSPC, and 50 mol % cholesterol). Assuming cholesterol is buried in the hydrophobic region of the bilayer,s0 the chelating IDA function represents 4% of the headgroups at the membrane surface. The liposomes were metalated with the appropriate metal chloride, followed by dialysis to remove free metal (for Cu2+). Quasi-elastic light scattering (QELS) measurements showed t h a t the mean vesicle size was not altered by metalation. Since some lipid was lost during filtration to remove titanium particles after sonication, the liposome samples were analyzed for total phosphate to establish lipid c o n c e n t r a t i ~ n .The ~ ~ average phosphate concentrations were 2.82 f0.25 mhlfor liposomes without metal and 2.49 f 0.18 mM for the metalated liposomes after dialysis. Cu2+concentrations measured for selected batches of liposomes by ICP-MS were found to be 105 f 5 pM. This value agrees well with the expected metal concentration calculated from the phosphate concentration and the known composition of chelating lipid 1. Therefore,
2
(30)Liposomes: A Practical Approach; New, R. R. C., Ed.; Oxford University Press: New York,1990; p 19. (31) Morrison, W. R. Anal. Bwchem. 1964, 7 , 218.
2386 Langmuir, Vol. 10, No. 7, 1994
Shnek et al. Table 1. ESR Spectral Parameters for Complexes of Cu2+-lDA-imidazole and 2%Lipid-1-Cu2+ Mixed Liposomes (48%DSPC and 50%Cholesterol)Loaded with Native and DEPC-Modified Horse Myoglobin (hM’b)l
T
z
)ol
4’0 Cu2+-IDAimidazo1e
l:o
1
1:l 1:2
3a
2.0
0 e.
0.0 0.0
s
158
2.285 2.268 2.265
157 170
2.287 2.288 2.264
160 152 177
CuZ+-liposomes no protein
DEPC-modified hMb
0.50
1.0
1.5
2.0
2.5
3.0
3.5
hMb
4.0
a
free hMb [x 10’’ M] Figure 6. Binding of horse myoglobin t o mixed liposomes containing 2%1-Cu2+(48% DSPC and 50%cholesterol) (m), to mixed liposomes containing 2% 1-Ca2+ (0) and to mixed liposomes containing lipid 1with no metal (A) at 25 “C in 20 mM MOPS, 100 mM NaCl, pH 7.5. Solid lines represent best fits t o a Langmuir isotherm. the liposomes can be assumed to be fully loaded with Cu2+ under the experimental conditions. In Figure 5, the binding ofhorse myoglobin (normalized by the total phospholipid content of the sample) to coppercontaining liposomes is compared to liposomes with no bound metal and ones that contain Ca2+. Nonspecific binding of myoglobin to pure DSPC liposomes is similar to the unmetalated DSPC/l liposomes (data not shown). Although myoglobin has been reported to exhibit negligible adsorption to zwitterionic phosphatidylcholine liposomes32 and monolayer^^*^^ a t neutral pH, we do observe nonspecific binding to DSPC lipsomes and to the mixed DSPC/ lipid 1 liposomes that is not altered by the presence of Ca2+. The monolayer studies also indicate that myoglobin interacts nonspecificallywith lipid 1, even in the presence of Ca2+. When loaded with Cu2+,the 19:l DSPCAipid 1liposomes interact strongly with myoglobin. The Cu2+-liposomes exhibit a maximum loading of 0.0075 mol of protein per mol phospholipid (0.18 mol ofproteidmol oflipid 11, while the unmetalated liposomes and those loaded with Ca2+ bind less than half that. Apparent association constants for myoglobin binding to the metalated and unmetalated liposomes were obtained by fitting the data in Figure 5 to a Langmuir isotherm. Myoglobin binding to the coppercontaining liposomes is so strong that only a lower limit on the association constant could be obtained from these measurements: K, > 2.9 x lo6 M-l. The association is weaker by a t least a factor of 10 for binding to liposomes without copper: K,= (2.5 f1.5) x 105M-l. Adding copper greatly enhances both the amount of myoglobin targeted to the interface and the strength of binding. Assuming that the phosphatidylcholine lipid headgroups occupy 70 A2/molecule a t the liposome surface,34 the surface area of a Cu2+-chelating liposome t h a t is occupied by a myoglobin molecule is -70/0.0075 = 9300 &,an area significantly larger than the largest dimensions of the globular protein32(44 x 44 x 25 The maximum protein loading therefore corresponds to less than mono-
A).
(32) Bergers, J.J.; Vingerhoeds, M. H.; van Bloois, L.; Herron, J.N.; Janssen, L. H. M.; Fischer, M. J.E.; Crommelin, D. J. A.Biochemistry 1993,32,4641. (33)Hamachi, I.;Honda, T.;Noda, S.; Kunitake, T. Chem.Lett. 1991, 1121. (34)A typical surface area for DMPC S U V s is -70 A2/molecule (Fendler, J. H. Membrane Mimetic Chemistry;John Wiley & Sons: New York, 1982, p 137). This area will of course be further increased by the incorporation of cholesterol.
22-fold molar excess of protein to total copper.
layer coverage, assuming that myoglobin does not undergo a significant conformational change upon interaction with the liposomes. The myoglobin loading observed for these liposomes corresponds to five 1-Cu2+ per protein molecule, which equals the number of histidines accessible to Cu2+IDA. It is possible that all the 1-Cu2+ is bound to surface histidines, which could result from myoglobin-induced cross-linking and aggregation of the liposomes upon standing or during sedimentation. Precipitation of copperloaded liposomes is in fact induced by myoglobin. (Liposomes lacking copper remain suspended over the same time periods in the presence ofthe protein.) Similar effects have been reported for biotin-containing S W s and streptavidin, a protein with four biotin binding sites.35 Myoglobin, with five binding sites, is readily precipitated by Cu2+-IDA-derivatized polyethylene glycols; the resulting precipitate contains a stoichiometric amount of metal to surface-accessible histidines, indicating that histidine-to-copper coordination is the dominant crosslinking i n t e r a ~ t i o n .Precipitation, ~~ if it poses a problem, could be avoided by using proteins with engineered highaffinity metal chelating sites or by blocking “excess” histidines after adsorption to the liposomes. Cu2+-loadedliposomes precipitate when allowed to stand overnight, even in the absence of protein. Since the IDA moiety is tethered on a spacer arm, chelating groups on two separate liposomes may be sufficiently flexible to form cross-links by coordinating a single copper ion. This precipitation can be minimized by introducing a slight excess of copper. ESR Analyses of Cu2+-Protein Interactions. Changes in the ligand environment of the chelated copper ions upon protein binding were investigated using electron spin resonance (ESR) spectroscopy. Substitution of a n oxygen ligand (HzO) by a nitrogen-containing ligand (histidine or imidazole) often increases the hyperfine coupling constant (All) and decreases g,~.~’,’ Spectral parameters for complexes of Cu2+-IDA and imidazole measured in the buffer used for liposome-protein binding show the same trend (Table 1). Thus, ESR is a convenient spectroscopic method for monitoring histidine coordination to IDA-bound copper ions. As shown in Figure 6 and Table 1, the ESR spectrum of the Cu2+-loadedvesicles (curve a) is characteristic of Cu2+chelated by iminodiacetate. Some superhyperfine features are visible in the liposome spectrum lacking protein, although the number of superhyperfine lines cannot be clearly discerned. In the presence of myoglobin (35) Tortorella, D.; Ulbrandt, N. D.; London, E. Biochemistry 1993, 32, 9181. (36) Van Dam, M.; Wuenschell, G. W.; Arnold, F. H. Biotechnol.Appl. Biochem. 1989,11,492. (37) (a)Plesch, G.;Friebel, C.; Svajlenova, 0.;Kratsmlr-Smogrovic, J . Polyhedron 1991, 10, 893. (b) Bonomo, R. P.; Cucinotta, V.; D’Alessandro,R.; Impellizzeri, G.; Maccarrone, G.;Vecchio,G.;Rizzarelli, E. Inorg. Chem. 1991,30, 2708.
Langmuir, Vol. 10, No. 7, 1994 2387
Protein Attachment to Artificial Membranes
l a
2400
2600
2800
3000
3200
3400
field (Gauss)
Figure 6. ESR spectra (77K)of (a)preformed mixed liposomes 2% 1-Cu2+ (48% DSPC and 50% cholesterol), (b) liposomes and DEPC modified horse myoglobin, and (c) liposomes and unmodified horse myoglobin in 20 mM MOPS, 100 mM NaC1, pH 7.5. Arrows correspond to field position used to calculategtl values listed in Table 1. from 2.287 to 2.264, similar to the values for Cu2+-IDA as it forms a complex with imidazole. The accompanying increase in All also mirrors the behavior of Cu2+-IDA-imidazole. The experiment was repeated using horse myoglobin that had been chemically modified with diethyl pyrocarbonate (DEPC) in order to block the surface histidines. The protein was found to be modified with DEPC a t five histidines (see Experimental Section). The liposome ESR spectrum in the presence of excess DEPC-myoglobin is very similar to that of the liposomes without any protein (Figure 6 and Table 1). It is reasonable to conclude that the changes in the spectral line positions of the liposome-bound Cu2+in the presence of unmodified myoglobin are caused by histidine coordination, rather than, for example, interactions with the heme. Metal ion coordination is a promising strategy for targeting proteins to artificial membranes, monolayers a t a gas-water interface, and molecular assemblies deposited or self-assembled on solid substrates. Small, stable, inexpensive metal-chelate affinity ligands can be incorporated into membrane components with relative ease by a variety of chemical routes. Especially when metalated, the compact IDA headgroup fits easily into phosphatidylcholine monolayers and bilayers with little structure disruption. The strength and kinetics of the interactions with proteins or other biological materials can be tailored through the choice of the metal ion and the chelating ligand.8 A wide range of natural proteins can be targeted in this way, while more specific protein targeting and orientation a t the interface could be achieved by engineering specific metal-binding sit&) into the protein surface.
gll decreases
Experimental Section DSPC was obtained from Avanti Polar Lipids. Cholesterol, horse heart myoglobin ( =- 95%pure), and morpholinosulfonicacid (MOPS) were obtained from Sigma. HPLC grade chloroform, DEPC, and stearyl bromide were obtained from Aldrich. All other solvents are reagent grade unless specified. Synthesis ofIDA-Lipid 1. All reactions were conductedin oven-dried (160 "C) glassware under positive Nz atmosphere. Tetrahydrofuran (THF)was distilled from Na-benzophenone and dimethylformamide (DMF) dried over 4-A molecular sieves. Diethyl iminodiacetate (Kodak) was distilled prior to use. All other reagents were used as received from Aldrich. Compound
2 was prepared as reported in the 1iterature.w 1H and l3C NMR were performedin CDCbon a Nicolet QE-300NMR spectrometer, and infrared (IR) spectra were obtained on a Perkin-Elmer 1600 Series FTIR spectrometer. Chemical analyses were performed by Desert Analytics (Tucson, AZ). lO-("riphenylcarbinyl~-l-~2,3-bis~octadecylosy~propyll~ 1,4,7,lO-tetraoxadecane(3).To a suspensionof sodium hydride (440mg of 60% oil dispersion, 11.1"01) in THF (10 mL) was added 2 (1.78 g, 2.99 "01) in THF (20 mL). After 30 min of stirring, 1-(methanesulfonyl)-lO-~triphenylcarbiny)-1,4,7,l0-tetraoxadecane (1.40 g, 2.98 mmol) was added as a solid and the solution was brought up to reflux. After 14 h the reaction was cooled to room temperature, quenched with HzO (70 mL), and diluted with ethyl acetate (70 mL). The mixture was shaken, layers were separated, and the aqueous layer was extractedwith fresh ethyl acetate (2 x 70 mL). The organiclayers were combined and washed with aqueous saturated sodium chloride (50 mL). The organic layers were further dried over anhydrous MgS04. The solutionwas filtered and concentrated in uacuo. The residual material were taken on to the next reaction. ~[2~-Bis(octade~1o~yloxy)propy11-3,g,9.Mo(4). p-Toluenesulfonicacid (100mg,0.53 mmol)and 3 (fromprevious reaction) were dissolved in a mixture of MeOH (10 mL) and THF (10 mL) and allowed to stir for 5 h. The mixture was then concentrated in uacuo and flash column chromatographed on silica gel with ethyl acetamexanes (40% (v/v);Rf = 0.20). A white solid, waxy material, 4,was collected (1.91 g, 88%from 2: mp 46-47 "C; 1H NMR 6 3.54 (m, 21H, -OC(H)-), 2.05 (br d, lH, -OH), 1.55 (m, 4H, -OCHaCH2-),1.25 (m, 60H),0.88 (t,J = 6.5 Hz, 6H, -CHs). IR (KBr)3462,2920, 1466,1350, 1265, 1116, 738 cm-1. Anal. Calcd for CaHe206: C, 74.12; H, 12.72. Found: C, 74.30; H, 12.87. 9-[2,3-Bis(octadecylosy)propyll-1-bromo-3,6,9~trioxadecane (6). Compound 4 (1.16 g, 1.59"01) was dissolved into THF (30 mL) and cooled to 0 "C. To the stirred solution was added carbon tetrabromide (0.80 g, 2.4 mmol) and triphenylphosphine (0.63 g, 2.4 "01). After 14 h the solution was concentrated in uacuo and flash column chromatographed on silica gel with diethyl etherhexanes (25% (v/v); Rf = 0.24). Product S was isolated at 1.01 g of a white wax (79%). lH NMR 6 3.81 (t, J = 6.3 Hz,2 H, -CHar), 3.53 (m, 19H, -OC(H)-), 1.55 (m, 4H, -OCHzCHz-), 1.25 (m, 60H), 0.88 (t, J = 6.5 Hz, 6H, -CH& IR(KBr) 2921,2851,1686,1467,1351,1276,1116, 909, 734 cm-l. DiethylN-(9-[2,~-Bis(octadecyloxy~propyll-3,6,9-~oxanony1)iminodiacetate (6). Diethyl iminoacetate (0.71 g, 3.8 mmol), S(l.01 g, 1.25 mmol), and triethylamine (0.34 mL, 0.25 g, 2.5 "01) were placed in THF (15 mL) and allowed to stir at reflux for 2 days. After the reaction mixture was allowed to cool to roam temperature, H20 (50mL) and ethyl acetate (50 mL) were added and the mixture was shaken. The layers were separated and the aqueous layer was extracted with fresh ethyl acetate (2 x 50 mL). The organic layers were combined and washed with HzO (2 x 50 mL) and once with aqueous saturated NaCl(50 mL). The organic layers were dried over anhydrous MgSO4, filtered, and concentrated in uacuo. The residual oil was flash column chromatographed on silica gel with ethyl acetatehexanes (33%(v/v);R f = 0.21). Compound 6was isolated as a white wax at 0.379 g yield (34%). lH NMR 6 4.16 (9, J = 7.09 Hz, 4H, -COOCH&H3), 3.63-3.40 (m, 23H, -OC(H)-, N-CHz-C(--O)), 2.99 (br t, 2H, -NCH2), 1.54 (m, 4H, -0CH2CHz-), 1.27 (m, 66 H, aliphatic tails, C(-O)CH&Hs), 0.88 (t,J = 6.5 Hz, 6H, -CH3). 13C NMR 6 171.41, 78.06, 71.67, 71.36, 71,24,71.13,70.82,70.56,70.36,70.12,79.01,60.43,56.26,55.89, 31.91, 30.22,29.70,26.11,25.86,22.72, 14.36, 14.15. IR(neat)
2917,2850,1743,1467,1135,1032,911,722 cm-l. Anal. Calcd for CasHlo,NOlo: C, 69.31; H, 11.74;N, 1.53. Found: C, 69.70; H, 11.60; N, 1.68. ~[2p.Bis(octad~losy)propyll-S,~~~o~onyl~ 1-iminodiacetic Acid (1). Into a solution of THF (7 mL)/MeOH (7 mL)/H20 (1mL) was placed 6 (0.379 g, 0.422 mmol) and NaOH (100 mg). The solution was stirred at reflux for 15 h, allowed to cool to room temperature, and acidified to pH 1-2. The solids were collectedby filtration and recrystallized 3 times in hot EtOW ~~~~
~
(38) Nuhn, P.; Rtiger, H. J.; Kertscher, P.; Gawrisch, K.;Amold, K. Phannazie 1978,33,181.
Shnek et al.
2388 Langmuir, Vol. 10,No. 7, 1994 THF (20:l). Amphiphile 1 was obtained as a white powder (183 mg, 51%): mp 124-127.5 "C; lH NMR 6 8.91(br s, 2H, -CO2H), 3.80 (br s, 4H, N-CHz-C(=O)), 3.71-3.41 (m, 19H, -C(H)-O), 3.28 (br s, 2H, N-CHz), 1.55 (m, 4H, CH2CH20), 1.25 (m, 60H, aliphatictails),0.88(t,J=6.4Hz,6H,-Cff3). 13CNMR6171.16, 71.76,71.30,71.02,70.59,70.39,70.32,67.90,57.66,55.35,31.95,
30.06, 29.74, 29.55, 29.39, 26.14, 26.10, 22.71, 14.14. IR (KBr) 3480,2917,2850,1719,1636,1467,1364,1120,721 cm-'. Anal. Calcd for C4gHggN010: C, 68.25; H, 11.57; N, 1.62. Found: C, 68.07; H, 11.33; N, 1.48. Monolayer Studies. Pressure-area (n-A)isotherms were obtained on a computer-controlled Langmuir instrument (KSV Instruments, Model M1200) with a surface area of 250 x 75 mm2. The surface pressure was measured using a platinum Wilhelmy plate. For all experiments the subphase was buffered with 20 mM MOPS, 100mM NaCl at pH 7.5 and the temperature was maintained at 24 f 0.5 "C. Lipids were spread from CHC13 (0.75 pmol/mL) as the free acid. Monolayers of lipid-metal complexes were prepared on subphase containing the appropriate divalent metal ion as the chloride salt. Approximately 40 pL of the lipid solution was spread with a Hamilton syringe and, after allowing 15 min for solvent eva oration, the monolayer was compressed at aconstant rate of 3 V(mo1eculemin)to a pressure of 5 mN/m. After a period of equilibration of -15 min, 1mL of 1 mM myoglobin solution was injected into the subphase at several points behind each barrier. The protein solution was distributed evenly throughout the subphase by circulation of buffer from one end of the trough to the other by means of a peristaltic pump. With the instrument operating in constantpressure mode, the monolayer was allowed to expand to the desired area at which point compression was begun at a rate of 3 AZ/(molecule*min)until film collapse was observed. Between each experiment the trough and barriers were cleaned by two washings with a decontaminating solution (Extran 1000)followed by thorough rinses with EtOH and CHC13. In addition, the Wilhelmy plate was cleaned by sonication for 90 min at 50 "C in Extran 1000 solution. Liposome Preparation. Lipids were dissolvedin chloroform (48 mol % DSPC, 50 mol % cholesterol and 2 mol % lipid 1). Twelve-milliliter glass centrifuge tubes were coated with 10pmol of total lipid under a stream of argon gas. The coated tubes were dried for 12 h under vacuum and hydrated with 3 mL of 20 mM MOPS, 100 mM NaC1, pH 7.5 buffer. The liposomes were vortexed at 50 "C and then probe tip sonicated (Heat Systems Model 375)at 4 "C for 15min at 15-25% power output. Titanium fragments were removed by filtration on a 0.2-pm syringe filter. A 2-fold molar excess of CuCl2 (5 mM CuCl2 in 100 mM NaC1) was added, and excess copper was removed by dialysis against MOPS buffer (12 mL of liposomes/l L) using tubing with a MW cutoff of 3500. Cu2+concentrations were determined by inductively-coupledplasma mass spectrometry (ICP-MS)on a PerkinElmer Elan 5000 ICP-MS using copper standards in 1%HN03 (Sigma). A 0.2-mL portion of liposomes was hydrolyzed in 0.4 mL of perchloric acid at 190 "C in heating blocks to make homogeneous solutions prior to ICP-MS. The resulting hydrolyzate was diluted with distilled, deioized H2O to 2 mL. Liposome samples were prepared daily and used within 48 h, since precipitation of copper-containingliposomes was observed beyond this time. Liposome sizes were measured by quasi-elastic laser light scattering (QELS) using a Microtrac Ultrafine Particle Analyzer (Leeds & Northrop) at 25 "C, in MOPS buffer. Protein Binding to Liposomes. Horse myoglobin was dissolved (0.1-0.3mM)inMOPS bufferwithstirring andfiltered on 0.2-pm syringe filters. One milliliter of prepared liposome solution was added to a 5-mL volumetric flask, and varying
8:
amounts of concentrated protein solution and MOPSbuffer were added to achieve final protein concentrations of 4-40pM. After equilibration for 1h, the samples were ultracentrifuged at 45 000 rpm (200000g)for 4 h at 25 "C. Phosphate analysis of a liposome sample before and after centrifugation showed that 95% of the liposomes were sedimented. The liposome pellets could not be fully resuspended by mechanical dispersion. Bound protein was determined from a mass balance on the total protein added to the sample minus the free protein remaining aRer sedimentation. The supernatant was decanted without disturbing the liposome pellet and the protein concentration determined spectrophotometrically at 409 nm using the extinction coefficient of 160mM-l ~ m - l . ~The 9 total protein added was determined from identically treated protein samples containing no liposomes. For the binding studies involving Ca2+-liposomes, a concentrated solution of CaCl2 (93 mM CaC12,lOO mM NaCl) was added to the liposomeprotein mixture to achieve a final Ca2+ concentration of 1mM. DEPC Modification of Horse Myoglobin. Horse heart myoglobin was chemically modified with DEPC according to a published procedure40to block histidine nitrogens. Myoglobin was dissolved in 100 mL of buffer (10 mM phosphate, 0.1 mM EDTA, pH 6.0)t o 20 pM concentration. DEPC was added (0.12 M in anhydrous EtOH) in 6.0 molar ratio to surface histidines (assumingfive accessiblehistidines per protein), and the reaction was allowed to proceed for 1h with stirring. Histidine modification was quantified spectrophotometrically at 240 nm, using an extinction coefficient of 3.2 mM-l cm-l.*O The reaction mixture was concentrated at 4 "C by ultrafiltration with a 10 000 MW cutoff filter. A 150-mL portion of MOPS buffer (20 mM MOPS, 100 mM NaC1, pH 7.5) was added and the sample was concentrated again at 4 "C. The samples were kept refrigerated until used. ESR Studies of Liposome and Protein. Electron spin resonance (ESR)experiments were performed on ax-band Bruker ESP 300 spectrometer operating at 9.2 GHz. A Hewlett-Packard 5342A microwave frequency counter was used to measure the microwave frequency. Spectral line positions were measured with a homemade proton gaussmeter. Spectra were recorded withthe following parameters: scan range, 1000G; time constant, 0.16 s; scan time, 480 s; modulation amplitude, 6.3 G; microwave power, 4.9 mW, modulation frequency, 100 kHz; temperature, 77 K. Each spectrum was scan averaged 3 times. Concentrated (0.2mM)native and DEPC-modifiedhorse myoglobin was added to 0.3-mL metalated vesicles at ratios of protein to Cu2+above 2.0. All samples (0.5 mL) were frozen immediately in liquid nitrogen and immersed into a liquid Nz-filledESRDewar. While in the spectrometer cavity, the dewar was purged with nitrogen gas t o prevent condensation during spectral aquisition. gll, All values were calculated from the spectral line positions.41
Acknowledgment. This research is supported by the Office of Naval Research (N00014-92-5-1178) and the National Science Foundation (BCS-9108502).F.H.A. acknowledges a n NSF PYI award and a fellowship from the David and Lucile Packard Foundation. D.R.S. is supported by a predoctoral training fellowship from the National Institute of General Medical Sciences, Pharmacology Sciences Program. D.W.P. is a Landau Fellow. (39) Breslow, E. J. Biol. Chem. 1964,239,486.
(40) Konopka,K.; Waskell,L.Biochim. Biophys. Acta 1988,954,189.
(41)Bolton J. R.; Wertz, J.E. Electron Spin Resonance: Elementary Theory and Practical Applications; Chapman and Hall: New York , 1986; p iii.
