J . Phys. Chem. 1991, 95,6372-6379
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tations, but basically the main peaks for alcohol are MH20+(MW + 18), M-H+ (M - l), M-OH (M - 17), and then fragments with less and less CH2 units. When the mixture of CA in H3P0, is heated, one can expect the interaction of the long-chain hydroxyl group of CA with a free H3P04molecule.m But from the above spectroscopic studies it is evident that there is no esterification reaction or dehydration of alcohol.
0 303
Conclusions The above studies apart from revealing numerous textures associated with various phases have enabled us to reach the following conclusions. Mixtures with different concentrations between 2 and 50% of CA in H3P04 exhibit similar textures depending upon the temperatures. The drastic change in the values of density, refractive index, conductivity, and anisotropy of polarizability with temperatureunambiguously compond to smectic E phase. The mixtures with concentrations 505%of CA and above exhibit smectic A and smectic E phases only. X-ray results also lend support to the above observations. The spectroscopic investigations reveal that there is neither esterification reaction due to orthophosphoric acid nor dehydration of alcohol and hence the system is just an additive between CA and H3P04exhibiting polymorphism. This type of polymorphism is rare in nonmesogenic binary mixtures.
313
323
333
343
353
--+
363
373
383
343
403
413
T(K1
Figure 5. Variation of electroconductivity u ( r l for 40 wt % of CA in H3P04.
m-l)
with temperature
from smectic B to smectic E phase. This behavior is generally observed in hexagonal phase of lyotropic systems.I9 6. NMR Studies. 'H NMR spectra for the mixture of 40% of CA were recorded at 297 K. The singlet peaks observed at 0.9 and 1.25 ppm in the spectrum correspond to CH3 and 14 CH2 protons, respectively. The triplet peak at 3.6 ppm is due to the C H 2 0 H group. The broad peak at 8 ppm corresponds to CH,O+( 0 H ) H . 31PNMR spectrum recorded at 297 K gives quite weak signals. The main peak is broad around 0 ppm since the H3P04 is in emulsion. These results fully agree with those from mass spectral analysis. Mass spectrum (CI in methane) gives more fragmen(19) Franeois, J. Kolloid, Z. Z. Polym. 1971, 216, 606.
Acknowledgment. We are thankful to Dr. Hugo Gotlieb, Department of Chemistry, Bar-llan University, Israel, for NMR and mass spectral analysis and to Dr. Jagadetsh, SJCE, Mysore, India, for his help in getting the DSC thermograms. We are thankful to the reviewers and Dr. R. Somashekhar for their valuable suggestions. M.M. thanks the University Grants Commission, New Delhi, India, for financial assistance. Registry No. H3P0,, 7664-38-2; cetyl alcohol, 36653-82-4. (20) Dehn,
W.M.;Jackson, K. E. J . Am. Chsm. Soc. 1933, 55, 4285.
Pkosecond Time-Resolved Resonance Raman Spectroscopy of Bacteriorhodopdn's J, K, and KL Intermediates Stephen J. b i g , Philip J. Reid, and Richard A. Mathim* Chemistry Department, University of California, Berkeley, California 94720 (Received: April 26, 1991)
Resonance Raman spectroscopy has been used to obtain structural and kinetic information on the primary photointermediates of bacteriorhodopsin with 3-ps time resolution. A synchronously pumped dye laser was amplified at 50 Hz to produce a probe pulse at 589 nm while a second, spectrally distinct, pump pulse at 550 nm was generated by amplification of a IO-nm portion of a continuum produced from the probe pulse. This apparatus was used to record Stokes Raman spectra of the photoproduct from 0 ps to 13 ns as well as anti-Stokes spectra from 0 to 10 ps. At 0 p, the Stokes spectrum, assigned to J, has strong hydrogen out-of-plane (HOOP) intensity at lo00 and 956 cm-I, the fingerprint region consists of a broad band of lines from 1155 to 1200 cm-l, and the ethylenic line is found at 1518 cm-'. By 3 ps the relative HOOP intensity drops to its lowest value and the fingerprint collapses to a single strong mode at 1189 cm-', while the ethylenic remains at 1518 cm-I. The lifetime of the initially strong anti-Stokes scattering is -2.5 ps, indicating that the J K transition is due, in large part, to vibrational cooling of the chromophore. We conclude that the chromophore in J is highly twistad and thermally excited but that it cools and conformationally relaxes to a more planar 1 3 4 s chromophore within 3 ps to form K. Between 3 and 40 ps there is a resurgence in Stokes HOOP intensity which remains large and nearly constant thereafter and the ethylenic frequency shifts from 1518 to 1521 cm-I within 200 ps. These changes are assigned to the conversion of K to a more twisted and bluer-absorbing KL species between 20 and 100 ps which must be caused by an isomerization-induced protein conformational change.
-.
Introduction The halophilic bacterium Halobacterium halobjum UM sunlight as an energy Source when normal respiratory avenues are not availab1e.l The energy of the absorbed photon drives a proton
* Author to whom correspondence should be addressed.
pump which generates an electrochrmical potential gradient a m a s the cell membrane. This gradient is then utilized for ATP synthesis. The elucidation of this phototransduction mechanism is important not only because it serves as the source of energy which ( I ) Stowkenius,W.;bgomolni, R. A. Annu. Rev. Blochem. 1962,5l,587.
0022-365419112095-6312$02.50/0 Q 1991 American Chemical Society
Picosecond Raman Spectra of Bacteriorhodopsin 2 0 0 9
I,
The Journal of Physical Chemistry, Vol. 95, No. 16, 1991 6373
-.qfs
BR
“640
/
Figure 1. The bacteriorhodopsin photocycle. Kinetic constants were taken from absorption or Raman spectroscopic
powers all subsequent changes in the chromophore and protein, but also since it is a model for artificial solar transduction and energy storage schemes. The cornerstone of this system is bacteriorhodopsin (BR), a 26 000-Da intrinsic membrane protein comprised of 248 amino acids arranged in a closely knit set of seven a-helicese2 The photoactive moiety, all-tram-retinal (vitamin A), is attached to the protein through a protonated Schiff base linkage with lysine 216. Upon absorption of a photon, the chromophore undergoes a rapid trans-to-cis isomerization about the C I 4 1 4 double bond and 15 kcal/mol(-30%) of the incident photon energy is to red.^^^ BR then proceeds through a series of structurally distinct intermediatesbefore returning to its initial state after several milliseconds (Figure 1). Recent investigations have determined the kinetics and structures of the later photocycle intermediates relevant to proton pumping (L through 0) and considerable progress has been made toward understanding this mechanism at the molecular l e ~ e l . ~ - ~ Determining the primary phototransduction and energy storage mechanism in BR will require a detailed characterization of the picosecond intermediates. Consequently, the paucity of structural data in this time domain has impeded attempts to develop a detailed molecular model. Optical absorption studies have shown that the excited state is rapidly depopulated (
