Couette flow fluorescence detected linear dichroism for analytes in lipid bilayers.

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J-1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut-off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the "leakage" of photons by the long pass filters used is also discussed.

New Approaches to Stretched Film Sample Alignment and Data Collection for Vibrational Linear Dichroism.

Rapid measurements of vibrational linear dichroism (VLD) infrared spectra are shown to be possible by using stretched polymer films and an extension of existing instrumentation designed for vibrational circular dichroism spectroscopy. Earlier techniques can be extended using additional inexpensive polymer substrates to record good-quality VLD spectra of a significantly wider range of compounds with comparatively short sample-preparation times. The polymer substrates used, polyethylene and polytetrafluoroethylene, are commonly available and inexpensive, and samples are more easily prepared than that for many earlier stretched-film and crystal studies. Data are presented for neutral hydrophobic organic molecules on hydrophobic films including acridine, anthracene, fluorene, and recently synthesized S-(4-((4-cyanophenyl)ethynyl)phenyl)ethanethioate. We extend the approach to polar or ionic species, including 2,2'-bipyridine, 1,10-phenanthroline, and sodium dodecyl sulfate, by oxidizing polyethylene films to change their wetting properties. The combination of new instrumentation and modified sample preparation methods is useful in basic spectroscopy for untangling and assigning complicated infrared spectra. Nevertheless, it is not a panacea as surface-adsorbed molecules are often not monodispersed, and higher analyte concentrations can lead to aggregation and resonance phenomena that have previously been observed for infrared spectra on surfaces. These effects can be assessed by varying the sample concentration. The focus of this paper is experimental, and detailed analysis of most of the spectra lies outside its scope, including some well-studied compounds such as acridine and anthracene that allow comparisons with earlier research.

Roles of the ether oxygen in hydration of tetrahydrofuran studied by IR, NMR, and DFT calculation methods.

We studied the concentration dependence of nu(C-H)'s in IR and (1)J(C,H) in NMR for binary water-tetrahydrofuran (THF) mixtures and found different trends for the two types of CH(2) groups in the five-membered ring. The changes of the nu(C-O) spectra showed that complexes of THF associated with water are formed, in which the number of water molecules increases with the water concentration. We suggested that hydration proceeds through the formation of 1:1, and 1:2 complexes of [THF:water] up to X(H(2)O) approximately 0.9, where X(H)((2))(O) is the mole fraction of the water in the mixtures. We carried out ab initio MO and DFT calculations to optimize the geometries of a THF dimer as a model of THF molecules in pure liquid, and 1:1 and 1:2 complexes of [THF:water] to simulate observed concentration dependence of nu(C-H)'s in IR and (1)J(C,H) in NMR. The changes of the calculated nu(C-H) spectra and (1)J(C,H) values for the optimized complexes are in agreement with those observed with varying X(H)((2))(O), supporting our proposal. From the vibrational and NBO analyses of the optimized complexes, the observed blue shift of nu(C-H)'s and the increase of (1)J(C,H) for the CH(2) groups neighboring to the ether oxygen were explained in terms of the changes in the stereoelectronic effect, resulting from HO-H...O< hydrogen bonding. The optimized 1:2-complex contains two weak C-H...OH(2) hydrogen bonds, and blue shift of nu(C-H)'s and increase of (1)J(C,H) were demonstrated from the same analyses of the complexes. This result of simulation also supports that the blue shift of nu(C-H)'s and increase of (1)J(C,H) observed for both the type of CH(2) groups at 0.6 X(H)((2))(O) < 0.9 are attributed to these interactions. On the basis of all these results, we propose that the formation of the 1:2-complex involving weak C-H...OH(2) hydrogen bonds is responsible dominantly for the hydrophobic hydration of THF.