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Langmuir 1999, 15, 2346-2350
Circular Dichroic Properties of Phosphatidylcholine Micelles† Peter Walde,* Eveline Blo¨chliger, and Kenichi Morigaki‡ Institut fu¨ r Polymere, ETH-Zentrum, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received August 21, 1998. In Final Form: December 15, 1998 The circular dichroic (CD) properties of aqueous and methanolic solutions of a series of short-chain phosphatidylcholinessranging from 1,2-dipropionyl-sn-glycero-3-phosphocholine (diC3PC) to 1,2-dioctanoylsn-glycero-3-phosphocholine (diC8PC)shave been studied. In methanol, the CD spectrum is characterized by a negative peak centered around 210 nm. The peak intensity decreased with increasing acyl chain length. In aqueous solution, not only did the CD spectra recorded at concentrations below the critical concentration for micelle formation (cmc) show a clear dependency on the acyl chain length, but they were also slightly different from the corresponding spectra recorded above the cmc. The latter findingsat least qualitativelysconfirms earlier extensive NMR investigations [e.g., Roberts; et al. Biochemistry 1978, 17, 935; Hauser; et al. Biochemistry 1988, 27, 9166]. The experimentally determined cmc for diC6PC in water at 25 °C was 15 mM. The conformation of micellar lipids (above the cmc) was more “rigid”si.e., was characterized by a higher degree of conformational restrictions involving the glycerol backbonesthan in the case of monomers (below the cmc). Furthermore, the CD measurements indicate that monomeric phosphatidylcholines start to become more rigid if longer than diC4PC. In addition, the dependency of the conformational rigidity on the acyl chain length correlates with published data on the chain length dependency of the rate constants for pancreatic phospholipase A2 [Berg; et al. Biochemistry 1997, 36, 14512].
Introduction All naturally occurring glycerophospholipids are chiral amphiphilic molecules.1 They are derivatives of sn-glycero3-phosphoric acid.2 In the case of the phosphatidylcholines, the sn-1 and sn-2 positions of the glycerol moiety are esterified with two carboxylic acids, forming the hydrophobic part of the molecule; the hydrophilic headgroup in position sn-3 is the zwitterionic phosphocholine moiety. The chiral center of phosphatidylcholine molecules is the sn-2 carbon atom, see Figure 1. For phosphatidylcholines with two identical, nonbranched carboxylic acid chains, it is known that bilayers (closed lipid vesicles, also called liposomes) form in aqueous solution, if each of the two carboxylic acids contains nine or more carbon atoms.3 On the other hand, shorter chain phosphatidylcholines aggregate into micelles, depending on chain length and concentration.4 In our previous study, we have investigated the circular dichroism (CD) properties of different phosphatidylcholine * To whom to address correspondence. † Dedicated to Prof. Pier Luigi Luisi, on the occasion of his 60th birthday. ‡ Present address: Max-Planck-Institut fu ¨ r Polymerforschung, Ackermannweg 10, D-55128 Mainz, Germany. (1) Hauser, H.; Poupart, G. In The Structure of Biological Membranes; Yeagle, P., Ed.; CRC Press: Boca Raton, FL, 1992; pp 3-71. (2) The nomenclature of glycerophospholipids applied is based on the stereospecific numbering (sn) of glycerol derivatives.1 The following abbreviations are used: diC3PC, 1,2-dipropionyl-sn-glycero-3-phosphocholine; diC5PC, 1,2-dipentanoyl-sn-glycero-3-phosphocholine; diC6PC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine, diC7PC; 1,2-diheptanoyl-sn-glycero-3-phosphocholine; diC8PC, 1,2-dioctanoyl-sn-glycero-3-phosphocholine; diC10PC, 1,2-didecanoyl-sn-glycero-3-phosphocholine, diC12PC, 1,2-didodecanoyl-sn-glycero-3-phosphocholine; diC14PC, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine. Cmc stands for critical concentration for micelle formation. (3) Tausk, R. J. M.; Oudshoorn, C.; Overbeek, J. T. G. Biophys. Chem. 1974, 2, 53-63. (4) (a) Roberts, M. F. Methods Enzymol. 1991, 197, 95-112. (b) Jones, M. N., Chapman, D. Micelles, Monolayers, and Biomembranes; WileyLiss, Inc.: New York, 1995. (c) Bian, J.; Roberts, M. F. J. Colloid Interface Sci. 1992, 153, 420-428.
Figure 1. Chemical structure of 1,2-diacyl-sn-glycero-3phosphocholine. The chiral center is marked with an asterix (/). RCOOH: carboxylic acid.2
liposomes (e. g. made from diC14PC or diC16PC),2 and a comparison was made with the CD spectra of the corresponding monomeric, nonaggregated lipids in methanol.5 One conclusion drawn in that investigation was that the equilibrium distribution of the different phosphatidylcholine conformations in the liposome bilayers is considerably different from the corresponding equilibria in methanol.5 We have now extended the CD measurements to shortchain phosphatidylcholines (all containing two identical, straight-chain carboxylic acid moieties), some being wellknown to form micelles in aqueous solution.4 We were in particular interested in knowing whether there are differences in the CD propertiessand therefore detectable differences in the conformational equilibriumson going from phosphatidylcholine monomers to phosphatidylcholine micelles. Furthermore, a comparison was made between the CD spectra of the phospholipids recorded in methanol under conditions where no aggregation occurs.6,7 Materials and Methods DiC3PC, diC4PC, diC5PC, and diC8PC were from Avanti Polar Lipids, Inc.; Alabaster, AL; diC7PC, diC10PC, and diC12PC were (5) Walde, P.; Blo¨chliger, E. Langmuir 1997, 13, 1668-1671. (6) (a) Kellaway, I.; Saunders: L. Biochim. Biophys. Acta 1970, 210, 185-186. (b) Lee, A. G.; Birdsall, N. J. M.; Levine, Y. K.; Metcalfe, J. C. Biochim. Biophys. Acta 1972, 255, 43-56. (7) Hauser, H.; Guyer, W.; Pascher, I.; Skrabal, P.; Sundell, S. Biochemistry 1980, 19, 366-373.
10.1021/la9810814 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/06/1999
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Langmuir, Vol. 15, No. 7, 1999 2347
from Sigma, St. Louis, MO; diC6PC was from Doosan Serdary Research Laboratories Inc., Toronto, ON, Canada; and diC14PC was from Bachem, Bubendorf, Switzerland. All phosphatidylcholines were used as obtained. The purity was checked by UV spectroscopy. In some cases (in particular diC4PC), the phosphatidylcholine products as purchased from different distributors contained impurities which showed absorbance at 280 nm. In this case, the lipids were not used, since these impurities might influence the CD measurements. All lipid concentrations were determined spectrophotometrically, using ammonium molybdate and inorganic phosphate (KH2PO4) as standard.8 UV absorption spectra were recorded on a Lambda 9 spectrophotometer (from Perkin-Elmer) or on a Cary 1E spectrophotometer (from Varian) using quartz cells with a path length of 0.1 cm. If not otherwise stated, CD measurements were carried out at 25 °C with a JASCO J-600 spectrophotometer using quartz cells with a path length of 0.05 cm. All CD data are expressed as molar ellipticities ([Θ]).
Results and Discussion CD Spectrum of Phosphatidylcholines Dissolved in Methanol. From our earlier measurements it is known that the CD signal of chiral long-chain phosphatidylcholines dissolved in methanol is weak and difficult to measure.5 For diC16PC and diC14PC, the molar ellipticity in the region of the nfπ* transition of the carboxylic acid ester chromophore at 210-220 nm is very low (|[Θ]210| < 50 deg cm2 dmol-1).5 A series of CD spectra of short-chain phosphatidylcholines have now been recorded, and the following trend is rather obvious; see parts A and B of Figure 2. The shorter the chain length, the more intense the CD signal. While for diC12PC, the molar ellipticity at 210 nm is about -50 deg cm2 dmol-1, for diC3PC, [Θ]210 is about -380 deg cm2 dmol-1. For diC4PC about the same value has been obtained, although in this case, the presence of impurities was obvious (absorbance in the UV range); see Materials and Methods. |[Θ]210| linearly decreases from diC3PC to diC8PC and levels off at diC12PC (Figure 2B). To interpret this behavior, there are basically two aspects to be considered. (i) One may explain the linear decrease in |[Θ]210| with increasing chain length qualitatively on the basis of the chemical structures of these homologous lipids: the longer the carboxylic acid chains, the more similar the electronic environment of the ester chromophores, thereby decreasing the asymmetry of the moleculesand therefore the weaker the CD signal. This explanation finds support through a comparison with the optical rotatory dispersion (ORD) and CD spectra of the protonated R-alkyl amino acids alanine (side chain, -CH3), valine (-CH(CH3)2), norvaline (-(CH2)3-CH3), leucine (-CH2-CH(CH3)2), norleucine (-(CH2)3CH3), and isoleucine (-CH(CH3)CH2CH3). Although in this case, the chromophore is directly linked to the asymmetric carbon atom, the difference lies in the length and constitution of the side chain. In these earlier studies, it was found that the longer the carbon chainsthe more different from the amino groupsthe more intense the ORD or CD signal.9 On the basis of this comparison with R-alkyl amino acids, one may argue that the chemical structure per se is responsible for the differences in the intensity of the CD spectra observed within the series diC3PC f diC12PC. (ii) On the other hand, possible intramolecular interactions between the two acyl chains may lead to a shift in the conformational equilibrium on going from short-chain (8) Ames, B. N. Methods Enzymol. 1966, 8, 115-118. (9) (a) Dirkx, I. P.; Sixma, F. L. J. Recl. Trav. Chim. Pays-Bas 1964, 83, 522-534. (b) Katzin, L. I.; Gulyas, E. J. Am. Chem. Soc. 1968, 90, 247-251. (c) Fowden, L.; Scopes, P. M.; Thomas, R. N. J. Chem. Soc. C 1971, 833-840.
Figure 2. CD spectra of 1,2-diacyl-sn-glycero-3-phosphocholines (50 mM) in methanol at 25 °C. (A) The molar ellipticities are plotted as a function of the wavelength. The phosphatidylcholines used were diC3PC (most intense signal), diC5PC, diC6PC, diC7PC, diC8PC, diC10PC, diC12PC, and diC14PC (least intense signal). (B) The molar ellipticities at 210 nm are plotted as a function of n, the number of carbon atoms in the carboxylic acid chain.
Figure 3. CD spectra of 50 mM diC3PC (1) and 25 mM diC5PC (2) dissolved in water and recorded at 25 °C.
to long-chain phosphatidylcholines. Such a conformational change would also be reflected in the CD spectrum, and it is likely that its contribution to the CD spectrum is dominating over the chemical structure argument discussed above; see part i. The CD Spectrum of Phosphatidylcholines Dissolved in Water. DiC3PC. The CD spectrum of diC3PC (50 mM) dissolved in water is shown in Figure 3. Similar to the CD spectrum recorded in methanol, the CD spectrum is characterized by a negative peak centered around 210
2348 Langmuir, Vol. 15, No. 7, 1999
nm. In contrast to the spectrum recorded in methanol, the CD signal is less intense. The molar ellipticity at 210 nm is now only ∼-100 deg cm2 dmol-1 (compare with Figure 2A,B). The reason for this solvent effect is not completely clear. Since, in both solvents, diC3PC most likely exists as highly flexible monomersthe cmc in water is expected to be above 200 mM (see the cmc value for diC4PC below)sthe observed differences in the CD spectrum possibly originate from a different solvation of the ester chromophores (methanol vs water).10 DiC4PC. The CD spectrum of monomeric diC4PC (50 mM) in water is comparable with the spectrum of diC3PC (data not shown). The cmc of this lipid in water is around 280 mM4 (determined at 25 °C). The intensity of peak in the CD spectrum recorded in water is again lower if compared with the spectrum recorded in methanol. DiC5PC. The CD spectrum of diC5PC (25 mM) in water is shown in Figure 3. It is almost identical with a spectrum of a 50 mM solution (data not shown). In comparison with diC3PC (or diC4PC), the CD spectrum of diC5PC dissolved in water shows now a positive CD signal in the region of 210 nm, with [Θ]210 ∼ +120 deg cm2 dmol-1. Since the cmc of diC5PC is about 50 mM4c (determined at 25 °C), the CD spectrum of diC5PC shown in Figure 3 is therefore the spectrum of mainly monomeric lipid, and it is interesting to note that the ellipticity of monomeric diC5PC is just opposite to the ellipticity of monomeric diC3PC (or diC4PC); see Figure 3. This indicates that the extension of the acyl chain in diC4PC by one methylene group leads to a significant change in the conformational equilibrium of the monomers. Most likely, intramolecular interactions between the two hydrocarbon chains in diC5PC lead to a “rigidification” of the monomers. Such interactions are expected to be more pronounced the longer the acyl chains; and it seems as if only above a critical length of four carbon atoms, chain packing is playing a major role. (With “rigidification” of the phospholipids, we mean increased confromational restrictions involving the glycerol backbone.) Alternatively, it may also be that small premicellar aggregates, like diC5PC dimers or trimers are responsible for the change in the size of the CD signal on going from diC3PC (or diC4PC) to diC5PC. We have, however, no experimental data to support this assumption. DiC6PC. Among the short-chain phosphatidylcholines, diC6PC is probably the one which has been studied most extensively so far. From the literature it is known that nearly spherical micelles form at room temperature11 above about 15 mM12,13 with a mean aggregation number (Nagg) of 19.11 CD spectra recorded below as well as above the cmc are shown in Figure 4A for 4, 25, 50, and 80 mM diC6PC. At all concentrations measured, the spectrum is characterized by a positive peak at 208-212 nm. The peak intensity is always higher than in the case of monomeric diC5PC; compare with Figure 3. Furthermore, the peak maximum tends to shift slightly toward higher wavelength with increasing concentration, and the molar ellipticity (10) The general problems of separating conformational from solvation effects in analyzing CD spectra has been discussed before: (a) Crabbe´, P. Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry; Holden-Day: San Francisco, CA, 1965. (b) Rassat, A. In Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry; Snatzke, G., Ed.; Heyden & Sons: London, 1967; Chapter 16. (c) Moscowitz, A. In Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry; Snatzke, G., Ed.; Heyden & Sons: London, 1967; Chapter 17. (11) Schmidt, C. F.; Barenholz, Y.; Huang, C.; Thompson, T. E. Biochemistry 1977, 16, 3948-3954. (12) deHaas, G. H.; Bonsen, P. P. M.; Pieterson, W. A.; van Deenen, L. L. M. Biochim. Biophys. Acta 1971, 239, 252-266. (13) Tausk, R. J. M.; Karmiggelt, J.; Oudshoorn, C.; Overbeek, J. T. G. Biophys. Chem. 1974, 1, 175-183.
Walde et al.
Figure 4. Concentration dependence of the CD spectrum of diC6PC dissolved in water and recorded at 25 °C; (A) the whole spectrum between 260 and 200 nm for 4 mM (1), 25 mM (2), 50 mM (3), and 80 mM (4) diC6PC; (B) concentration dependence of the molar ellipticity at 210 nm. Squares: measured values. The solid line has been obtained by simulation, using [Θ]210monomer ) 295 deg cm2 dmol-1 and [Θ]210micellar ) 519 deg cm2 dmol-1 (see text for details).
nonlinearly increases with increasing concentration, as shown in Figure 4B. A clear change in [Θ]210 is obvious around 15 mM, just in the region of the cmc reported. It is therefore reasonable to conclude that micelle formation leads to conformational changes of diC6PC which are detectable by CD measurements. Although the intensity of the CD band centered around 210 nm in the spectrum recorded below the cmc is significantly different from that recorded above the cmc, the conformational equilibrium of diC6PC in the micelle is, however, certainly not drastically different from the conformational equilibrium of monomeric diC6PC in water. Although CD spectroscopy is sensitive enough to detect these small conformational differences. From earlier NMR measurements it is known that monomeric and micellar diC6PC have similar basic conformational equilibria (rotamer distribution), the two states of the lipid seem to differ only slightly.14-17 Our CD measurements are therefore in good agreement with these independent observations. If one assumes that the free monomer concentration above the cmc is approximately the cmc, then the recorded CD spectrum above the cmc is always a combination of (14) Lin, T.-L.; Chen, S.-H.; Gabriel, N. E.; Roberts, M. F. J. Am. Chem. Soc. 1986, 108, 3499-3507. (15) Roberts, M. F.; Bothner-By, A. A.; Dennis, E. A. Biochemistry 1978, 17, 935-942. (16) Burns, R. A., Jr.; Roberts, M. F.; Dluhy, R.; Mendelsohn, R. J. Am. Chem. Soc. 1982, 104, 430-438. (17) Hauser, H.; Pascher, I.; Sundell, S. Biochemistry 1988, 27, 91669174.
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Langmuir, Vol. 15, No. 7, 1999 2349 Table 1. Comparison of [Θ]λ,max with Published Values for kcat*, the Rate Constant for the Decomposition of the Pancreatic Phospholipase A2-PC Complex (T ) 25 °C) phospholipid
state of the phospholipid
[Θ]λ,max (deg cm2 dmol-1)a
diC3PC di C4PC diC5PC diC6PC diC6PC diC7PC diC8PC diC14PC diC16PC
monomeric monomeric monomeric monomeric micellar micellar micellar vesicular vesicular
-100 -100 +120 +300 +520 +700, +640b +550b +500c +400d
kcat* (s-1)e
300 700 2350 450
a λ,max ) 210-220 nm; approximate values determined in water. At T ) 50-52 °C. c From ref 5, at 25 °C. d From ref 5, at 50 °C. e From ref 25, [NaCl] ) 4 M. b
Figure 5. CD spectrum of diC7PC dissolved in water at 10 mM, 20 mM, and 50 mM and recorded at 25 °C.
the CD spectrum of the monomers and the micellar molecules. The contribution of the monomers decreases with increasing phospholipid concentration since the relative amount of monomers decreases with increasing total amount of lipid. If one assumes that the two contributions are additivesin analogy to the case of the secondary structure contributions in the far-UV region of the CD spectrum of proteins18sthe observed molar ellipticity at wavelength λ ([Θ]λobserved) is
[Θ]λobserved ) x[Θ]λmonomer + y[Θ]λmicellar
(1)
where [Θ]λmonomer and [Θ]λmicellar correspond to the molar ellipticity at λ of monomeric and micellar lipid, respectively; x and y are the mol fractions of monomeric and micellar lipid, respectively, whereby x + y ) 1. If measurements are made at a mM diC6PC, then x ) cmc/a and y ) (a - cmc)/a, for a g cmc. For diC6PC, the cmc is 15 mM (see above), and [Θ]210monomer ) 295 deg cm2 dmol-1 (Figure 4). Fitting all the measurements made above the cmc with eq 1, the mean value of [Θ]210micellar is obtained to be 519 deg cm2 dmol-1. The simulated concentration dependence of [Θ]210 using this value is shown as solid line in Figure 4B. If the CD spectra of diC6PC (Figure 4) are compared with the CD spectra of diC3PC (diC4PC) (Figure 3), clear differences are obvious. While diC6PC gives positive signals, the spectrum of diC3PC (diC4PC) is negative. One can therefore propose that the two molecules show significantly different conformations in water, possibly caused by increased intramolecular interactions between the two acyl chains on going from monomeric diC3PC to monomeric or micellar diC6PC. Such differences could apparently not be detected by 13C NMR measurements,19 CD is therefore in this case more sensitive. DiC7PC. The CD spectra of diC7PC recorded at 10, 20, and 50 mM are shown in Figure 5. These are all spectra of mainly micellar diC7PC since the cmc of this phospholipid is about 2 mM.4,13,20 Small-angle neutron scattering measurements reported in the literature indicated that around the cmc, the micelles are small and grow into long rods with increasing concentration (with Nagg > 100).21 (18) (a) Greenfield, N.; Fasman, G. D. Biochemistry 1969, 8, 41084116. (b) Chen, Y.-H.; Yang, J. T.; Martinez, H. M. Biochemistry 1972, 11, 4120-4131. (19) Burns, R. A., Jr.; Roberts, M. F. Biochemistry 1980, 19, 31003106. (20) Bonsen, P. P. M.; deHaas, G. H.; Pieterson, W. A.; Van Deenen, L. L. N. Biochim. Biophys. Acta 1972, 270, 364-382. (21) (a) Lin, T.-L.; Chen, S.-H.; Gabriel, N. E.; Roberts, M. F. J. Am. Chem. Soc. 1987, 91, 406-413. (b) Lin, T.-L.; Tseng, M.-Y.; Chen, S.-H.; Roberts, M. F. J. Phys. Chem. 1990, 94, 7239-7243.
The CD spectrum seems to be insensitive to this drastic micellar growth, since between 10 and 50 mM all CD spectra are identical, characterized by a positive peak at ∼215 nm with a molar ellipticity at peak maximum of ∼700 deg cm2 dmol-1 (Figure 5). The corresponding values ([Θ]215) obtained for a 50 mM diC7PC solution at 36 °C and 50 °C were 670 and 640 deg cm2 dmol-1, respectively. In comparison with monomeric diC5PC, monomeric diC6PC or micellar diC6PC (Figure 4), the CD signal is considerably more intense in the case of micellar diC7PC. Furthermore, the maximum is slightly red-shifted to ∼215 nm. The reason for this spectral shift is not clear. DiC8PC. The CD spectrum of diC8PC dissolved in water (20 mM) could not be recorded at 25 °C due to the wellknown phase separation properties occurring at concentrations greater than a few millimolar.13,22,23 At 52 °C, the lipid is above the upper cosolute temperature of 47 °C, and the CD spectrum again shows a positive peak, centered around 215 nm with [Θ]215 ∼ 550 deg cm2 dmol-1 (data not shown). The molar ellipticity at peak maximum is lower than for diC7PC, but not as low as in the case of long chain phosphatidylcholines in the fluid-analogue state in liposome bilayers.5 Since the cmc of diC8PC is about 0.2-0.3 mM,4 the measured CD spectrum at 20 mM predominantly reflect the conformational equilibrium of micellar phospholipid. The micelles were reported to be very large aggregates,13,23 and mean aggregation numbers of 5 × 104 at 20 mM have been reported.13,22 These are very large structures, the number of lipid molecules per aggregate being comparable to large unilamellar phosphatidylcholine vesicles. For example, the lipid shell of a vesicle made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine with a diameter of 80 nm is also composed of about 5 × 104 molecules.24 A Comparison with the Activity of Phospholipase A2. In Table 1 the molar ellipticities at peak maximum ([Θ]λ,max) are compared for the series diC5PC f diC16PC. Interestingly, the highest values are obtained for micellar diC7PC and diC8PC, indicating that, in these cases, the phospholipids have possibly the most rigid conformation, and this finding can be compared with kinetic measurements of (pancreatic) phospholipase A2, an enzyme that catalyzes the hydrolysis of phospholipids, like phosphatidylcholines, preferentially if presented as micellar or (22) Carvalho, B. L.; Briganti, G.; Chen, S.-H. J. Phys. Chem. 1989, 93, 4282-4286. (23) Lo Nostro, P.; Stubicar, N.; Chen, S.-H. Langmuir 1994, 10, 1040-1043. (24) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47.
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vesicular aggregates.12,25 If one eliminates in a corresponding kinetic analysis contributions from enzyme binding to aggregated substrates, which most efficiently occurs in the presence of anionic reaction products or bound anions from added salts, the initial rates of hydrolysis of medium- to long-chain phosphatidylcholines (diC6PC f diC14PC) are of the same order of magnitude, independent of whether present as monomers, micelles, or vesicles.25 The small differences in the rate constants for the decomposition of the phospholipase-PC complex on the aggregate (micelles or vesicles), expressed as kcat*, on going from diC6PC micelles to diC14PC (vesicles) may, however, reflect the trend in the conformational equilibrium, as indicated by [Θ]λ,max for the same lipids; see Table 1. In other words, the two highest kcat* values correlate with the two highest [Θ]λ,max values measured within the series of phosphatidylcholines. This finding supports the idea that the substrate conformation plays a certain role in the kinetics of the phospholipase catalyzed hydrolysis of aggregated phosphatidylcholines,26 and it may help one to better understand certain molecular aspects of the interfacial phospholipase activation.27 (25) Berg, O. G.; Rogers, J.; Yu, B.-Z.; Yao, J.; Romsted, L. S.; Jain, M. K. Biochemistry 1997, 36, 14512-14530. (26) (a) Wells, M. A. Biochemistry 1972, 11, 1030-1041. (b) Dennis, E. A.; Danke, P. L.; Deems, R. A.; Kensil, R. A.; Pluckthun, A. Mol. Cell. Biochem. 1981, 36, 36-45. (c) Thuren, T.; Virtanen, J. A.; Kinnunen, P. K. J. Biochemistry 1987, 26, 5816-5819. (27) It is worthwhile to note that kcat* also correlates with the micelle size: the larger the micelles, the higher kcat* is.
Walde et al.
Concluding Remarks The aggregation behavior of short-chain phosphatidylcholines has been studied extensively in the past, mainly with the aim of understanding the kinetic behavior of phospholipases. In the present work, we have shown that circular dichroism is a convenient tool to investigate the conformational properties of short-chain phosphatidylcholine monomers and micelles: micelle formation of diC6PC in water, for example, could be followed by recording the CD spectrum, the cmc (at 25 °C) being 15 mM (Figure 4B), a value which agrees with literature data,4,12,13 which were based on a completely different methodology. Furthermore, the CD spectra indicate that major conformational differences in water exist on going from monomeric diC3PC (or diC4PC) to monomeric diC5PC (see Figure 5). These differences are most likely due to differences in the interactions between the two acyl chains. DiC5PC seems to be more “rigid”, with closer contact of the hydrophobic chains, if compared with diC3PC (or diC4PC). Acknowledgment. We would like to thank Helmut Hauser for his discussion and comments. The work has been supported by the Swiss National Science Foundations (CHiral 2, grant No. 21-36734.92). LA9810814
