J. Phys. Chem. 1980, 84, 2691-2692
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Electron Spln-Echo Studies of Nitroxlde Spin Probes in Lipid Bilayers. Direct Measurement of Transverse Relaxation Times as a Sensltlve Probe of Molecular Motion Keith Madden,' Larry Kevan,**' Philip D. Morse,% Department of Chemlstty and Department of Bbbgbal Sciences, Wayne State Unlvefstty, Detroit, Michlgen 48202
and Robert N. Schwartrs Department of Chemlstry, UnlversHy of Illlnols at Chicago Circle, Chicago, Illlnols 60680 (Received: July 25, 7080)
Electron spin-echo spectroscopy has been used to make the first direct measurement of the transverse electron spin-spin relaxation time T2of doxylcholestane nitroxide spin probes in egg yolk lecithin bilayers. The temperature dependence of T2from 75 to 100 "C shows a maximum at 95 "C and reflects contributions due to rotational reorientation and translational motion of the spin probe. The local viscosity in the bilayers is shown to be greater than in n-hexadecane. Spin-echoes have also been observed in lecithin vesicles.
Introduction The study of molecular motion in organized molecular assemblies like micelles, bilayers, and vesicles is of considerable importance for understanding their chemical and biochemical functions, The motion of nitroxide spin probes in such assemblies has been extensively studied by continuous wave electron spin resonance (ESRh4 The dynamical information comes from the transverse spinspin relaxation time IT2)which is inversely related to the homogeneous ESR line widths. However, even in ordinary solutions in the fast, tumbling region the nitroxide ESR lines are inhomogeneously broadened due to unresolved intramolecular hyperfine interactions? Thus Tz is not obtainable directly from the experimental line widths and must be derived indirectly from an often complex spectral simulation frequently based on estimated hyperfine couplings." This indirect analysis becomes even more complex when applied to nitroxide probes in molecular assemblies such as lipid bilayers.8 Electron spin-echo (ESE) spectroscopy provides a method to directly measure T2in sol~tion.~ A 90°-1800pulse sequence separated by time r generates an echo at time r after the second pulse. The echo decays exponentially according to exp(-2r J T2).The equivalence of T2measured by ESE spectroscopy and by CW ESR has recently been verified in solutionolo Here we report the first direct measurements of T2in nitroxide-labeled lipid bilayers by ESE spectroscopy. The temperature dependence of T2 gives information about the motional processes in these bilayer systems. Experimental Section Unsonicated dispersions of egg yolk lecithin (Sigma) labeled with 0.5 mol % 3-doxyl-5a-cholestane (Molecular Probes, Inc.) were prepared in a nitrogen atmosphere by the method of Israelachvili et a1.8 Degassed solutions of the 1mM doxylcholestane probe in n-hexane and n-hexadecane were also examined. Two pulse ESE decay curves were recorded with a home-built X-band ESE spectrometerl' fitted with a Varian E-4540 variable-temperature accessory. Results Two pulse echoes were obtained from the center MI= 0022-3654/80/2084-2691$01.00/0
0 line of the ESR three-line nitroxide ESR spectrum, and they decayed as single exponential5 according to exp(-2r/T2). T2is plotted as a function of temperature in Figure 1. Note that ESR signals could only be observed at relatively high temperatures in the bilayer systems.
Discussion The temperature dependence of T2in the hexane, hexadecane, and bilayer samples goes through a maximum with increasing temperature. This is similar to the higher temperature behavior of the anthracene-& anion in 2methyltetrahydrofuran glass also measured by ESE spectroscopy.12 This temperature dependence can be qualitatively explained as follows. In the lower temperature range of echo observation, increasing temperature leads to faster reorientation of the nitroxide probe which averages out some of the hyperfine anisotropy and increases T2 At still higher temperatures T2is maximized and then decreases as radical-radical collisions become more frequent and introduce the additional relaxation mechanism of Heisenberg spin e x ~ h a n g e . ~ This l ~ J ~latter mechanism depends on the translational motion of the nitroxide probe. The temperature at which the maximum in T2 is observed increases from 42 "C in n-hexane to 62 OC in nhexadecane to 95 "C in the lipid bilayers. This is in order of increasing viscosity and implies that the effective viscosity of the local environment in the bilayer is higher than in n-hexadecane. This is of interest since the lecithin components have hydrocarbon chain lengths similar to n-hexadecane. The increased effective viscosity in the bilayer is attributed to the organization of the molecular assembly. This direct measurement of T2makes possible a more sensitive investigation of changes in the rotational and translational motions due to foreign molecules incorporated into the bilayers than by simulation of ESR spectra. The effect of sonication to form vesicles on the motional freedom of the nitroxide probes is also amenable to detailed study. Current experiments on lecithin vesicles labeled with doxylcholestaneshow a two-component echo decay behavior possibly associated with faster nitroxide reorientation and with slower vesicle reorientation. Such detail is unavailable from CW ESR experiments. We 0 1980 American Chemical Society
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J. Phys. Chem. lS80, 84, 2692-2694
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I mM n-decane I rnM n-hexadecane
Acknowledgment. The work was partially supported by NSF grant INT77-21688.
Radiation Leboratay, Notre Dame LkrkerSity, Notre Dame, IN 46556. (a) Department of Chemistry, Unlverslty of Houston, Houston, TX 77004. (b) Department of Sciences, Wayne State ullverslty. On leave at the Department of Chemlstry, University of Callfornia, Los Angeles, CA 90024. B d h , L. J., Ed. “Sph LabelhgTheory and Applicatkns”; Academic Press: New York, 1978. Stlllman, A. E.; Schwartz, R. N. J. Man. Reson. 1978. 22. 269. Hwang, J. S.; Mason, R. P.; Hwang, L. P.; Freed, J. H. J. &. 1975, 79, 489. Bales, 8. L. J. Magn. Reson. 1980, 38, 193. IsraelachvlH, J.; SJosten,J.; ErYtsson, L. E. (3.; Ehrslrom, M.; oraslund, A.; Ehrenberg, A. Biochem. Blophys. Acfa 1975, 382,125. Stlllman, A. E.; Schwartz, R. N. In “Time Domain Electron Spin Resonance”; Kevan, L.; S c h w a , R. N., Ed.; Wlley: New Yolk, 1979; Chapter 5. Schwartr, R. N.; Jones, L. L.; Bowman, M. K. J. phys. Chem. 1979, 83,3429. Ichkawa, T.; Kevan, L.; Narayana, P. A. J. Phys. Chem. 1978, 83, 3378. Brown, I. M. In “Tlme Domain Electron Spln Resonance”; Kevan, L.; Schwartz, R. N., Ed.; Wlley: New York, 1979; Chapter 6.
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Fwre 1. Transverse relaxation time ( T2)as a functbn of temperature In (0)1 mM doxylcholestanelndecane, (A) 1 mM doxylcholestanel n-hexadecane, and ( 0 )doxylcholestanelegg leckhln (1:100 mol ratio) mukibllayers In aqueous buffer.
believe that we have demonstrated the potential of direct measurement of Tzby ESE methods to give valuable new insights into the subtleties of motional processes in molecular assemblies and biological subassemblies.
Surface-Enhanced Raman Scattering from Molecules Adsorbed on Mercury R. Naaman, S. J. Buelow, 0. Cheshnovsky, and D. R. Herschbach” Depellment of Chemlstty, Haward University, CambrMge, Messachusetts 02138 (Received: August 5, 1080)
Raman scattering from pyridine, benzene, or cyclohexane molecules adsorbed on a liquid mercury surface is observed to be strongly enhanced relative to the same molecules in the gas phase or solution. The nominal enhancement factors (estimated for monolayer absorption, as conventional) are of the order of lo4to Previous observations of such strong surface-enhancedRaman scattering have all involved roughened solid metal surfaces; the prevalent view is that surface roughness on the order of a few hundred angstroms is an essential condition. Our results show this does not hold for liquid mercury surfaces.
le.
Intense Raman scattering from molecules adsorbed on metal surfaces has now been observed for many systems.’ This phenomenon, known as surface-enhanced Raman scattering (SERS), involves scattering cross sections as much as six orders of magnitude larger than those for the same molecules in solution or gas phase. Other characteristic features include the following: (1)enhancement is prominent only for certain vibrational modes; (2) broad continua appear underneath the Raman spectra; (3) the enhancement is increased by surface roughness on the order of a few hundred angstroms;2 (4) often the enhancement depends strongly on the angle of incidence of the exciting light.3 Theoretical models for SERS have not yet reached a consensus; a t present there are two chief approaches. The first emphasizes intensity borrowing from specific chemical bonds formed between the molecule and the surface? The second approach considers dielectric or electromagnetic properties of the surface; this includes effects such as image dipolesF6high local fields produced by surface irregularity! and resonant excitation of surface plasm~ns.~J For some of these electromagnetic mechanisms, surface roughness is an essential condition for strong SERS. Previous observations of strong SERS have all involved roughened solid surfaces. Here we report strong enhancement of Raman scattering from molecules adsorbed on a liquid mercury drop, This result challenges the prevalent idea that surface roughness, on the scale of a few 0022-3654/80/2084-2692$0 1.0010
hundred angstroms, is a requisite for SERS. For these experiments, a hanging drop electrode (Metrohm EA290) which produced a mercury drop with a known surface area was suspended in a Pyrex cell. The mercury used was triply distilled (Eastern Smelting and Refining Corp.). The cell could be filled with solution or connected at a vacuum manifold to introduce gas. The experiments were conducted at room temperature (22 “C). No voltage was applied to the electrode. An argon ion laser beam (Spectra Physics 164) was aimed at the drop through the bottom of the cell. A lens of focal length 30 cm collimated the 514.5-nm beam to a diameter of 2 mm a t the drop. The light scattered at 90° to the laser beam was collected by a Raman spectrometer (Cary 82). For experiments in which gas-phase molecules were adsorbed onto the mercury surface, the system was evacuated to a pressure of torr. Thus impurities such as HzO and COz may have been present on the surface. Several minutes were required for the SERS signal to build up to full intensity; this is attributed to gradual adsorption of the gas molecules onto the mercury surface. The SERS signal was found to increase linearly with the laser intensity at low power levels. Further increase in the laser intensity (above 100 mW) caused nonlinear changes in the signal, probably due to heating of the drop resulting from absorption by mercury at the excitation wavelength? Large enhancements of the Raman scattering were observed for pyridine, benzene, and cyclohexane adsorbed 0 1980 American Chemical Society
