COUPLED EXTERNAL REFLECTANCE FT-IR MINIATURIZED SURFACE-FILM APPARATUS FOR BIOPHYSICAL STUDIES

An FT-IR spectrophotometer has been interfaced to a miniaturized surfa ce film apparatus for external reflection studies of insoluble monolay ers in situ at the air/water interface. Signal-to-noise ratios of 200: 1 were routinely achieved for the CH2 stretching vibrations of phospho lipids. We have monitored, using the acyl chain symmetric CH2 stretchi ng frequency near 2850 cm-1 as a structural probe, lipid conformationa l order changes that occur during the surface pressure-induced two-dim ensional phase transition in monolayers of 1,2-dipalmitoylphosphatidyl serine. In addition, the small volume of the miniaturized film apparat us (30 mL) permitted replacement of H2O with D2O in the subphase. This capability, in turn, permits the acquisition of spectral data in the amide I region of proteins. We report the first external reflection FT -IR spectrum of an insoluble protein monolayer. The protein studied is pulmonary surfactant SP-C.