A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.

External reflection Fourier transform infrared (FT-IR) experiments are reported for insoluble monomolecular films of an equimolar mixture of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylserine (DPPS) at the A/W interface as a function of surface pressure and Ca2+ ion presence. The separate components showed a surface pressure-induced conformational ordering of the acyl chains. The conformational ordering occurred more cooperatively for the DPPS. Acyl chain perdeuteration of the DPPC permitted the observation of the response of the individual components in the binary mixture to changes in surface tension and to the presence of Ca2+. Plots of surface pressure versus CH2 or CD2 stretching frequencies were analyzed with a two-state model. At each surface pressure within the two-state region, the fraction of disordered form was the same for each lipid component, suggesting that they are well mixed on the surface. Calcium ion (5 mM in the subphase) produces almost no effect on the pressure-induced acyl chain ordering of the DPPC in a single component film, whereas the same levels of Ca2+ induce acyl chain ordering at all surface pressures in both components of the binary mixture. Thus, unlike the bulk phase mixture of DPPC/DPPS, the binary lipids in this mixed monolayer film appear to retain their miscibility in the presence of Ca2+. Finally, Ca(2+)-induced dehydration of the phosphate group was observed through characteristic frequency shifts in the asymmetric PO2- stretching mode.

We report on the gel-state microaggregation in binary mixtures of diacylphosphatidylcholines over temperatures ranging from -19 degrees C to near the gel-to-liquid crystal transition. Microaggregates with lateral dimensions in the range 1-100 chains were detected and measured with an isotope infrared method that relates the splitting or the shape of the methylene scissors band to aggregate size. Measurements were made on fully hydrated dispersions of diC18DPC/diC20HPC, diC18DPC/diC22HPC, and diC18DPC/diC24HPC at molar ratios of 4:1. Low levels of aggregation were determined with reference to the spectrum of the random mixture diC18DPC/diC18HPC. For diC18DPC/diC20HPC at -19 degrees C, which previous calorimetric measurements have indicated is a nearly ideal, we found about 4% of the minority component chains to be involved in aggregates. For diC18DPC/diC22HPC, the value increased to about 11%. DiC18DPC/diC24HPC was found to be highly fractionated, in agreement with the earlier studies. The unit subcell, which defines the type of acyl-chain packing, was determined for the components of the mixtures. The temperature behavior of the phases and the temperatures at which the minority component domains undergo dissolution were determined.

Fourier transform-infrared (IR) spectroscopic and electron microscopic studies are reported for 1,2-dimyristoylphosphatidylserine (DMPS) and for DMPS/1,2-dimyristoylphosphatidylcholine mixtures in the presence and absence of Ca2+ ion. The frequency of the methyl symmetric deformation mode near 1,378 cm-1, previously assumed insensitive to changes in lipid morphology, has been found to respond to cochleate phase formation by undergoing an approximately 8 cm-1 increase. The new IR spectroscopic marker at 1,386 cm-1 has been used to identify and verify structures suggested from the phase diagram of J. R. Silvius and J. Gagné (1984. Biochemistry. 23:3241-3247) for this system. In addition, the ability of Mg2+ ion to induce cochleate formation has been demonstrated. Higher Mg2+ than Ca2+ levels are required for this process. Finally, IR spectroscopy has been used to monitor dehydration of the lipid surface through changes in the asymmetric PO2- stretching mode. Dehydration precedes cochleate phase formation (i.e., occurs at a lower Ca2+/phosphatidylserine level).

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