The theory and practice of external infrared reflection absorption spectrometry (IRRAS) as applied to monomolecular films at the air-water interface are reviewed. The observed IR frequencies for films of amphiphilic species provide information about the conformational states of the hydrocarbon chains and the hydrogen bonding and ionization states of the polar head groups, under conditions of controlled surface pressure. Determination of molecular orientation is also feasible and requires detailed consideration of the reflection-absorption properties of the three- phase (air-monolayer-water) system. Current theoretical approaches are described. Applications of IRRAS to the study of single- and double-chain amphiphiles and proteins are reviewed, and initial excursions into biochemistry (interfacial enzyme catalysis) and physiology (pulmonary surfactant function) are reported.

Monolayers of behenic acid methyl ester at the air/H2O and air/D2O interfaces provide a convenient test for quantitative analysis of infrared reflection−absorption spectroscopy (IRRAS) intensities. Spectra were acquired for both s- and p-polarized radiation at angles of incidence of 35°, 40°, 45°, and 50°. The observed ∼10 cm-1 splitting (at a surface pressure of 14 mN/m) for both the methylene scissoring and rocking modes provides direct evidence for the occurrence of a perpendicular orthorhombic subcell structure and for the existence of all-trans acyl chains. Analysis of the IRRAS intensities of the methylene and carbonyl stretching vibrations reveals that, of the limited set of chain tilt angles possible with respect to the surface normal, a chain tilt angle of 0° provides by far the best fit to the data. For each vibration, data for both polarizations at all four angles were fit with a single set of three parameters:  chain tilt angle, effective extinction coefficient (k max), and the overall degree of polarization (determined by the efficiency of the polarizer and the error in its optical alignment). This last parameter was found to be important in accounting for observed IRRAS intensities, especially for p-polarized radiation close to the Brewster angle. Finally, the feasibility of using the observed unequal intensities in the components of the split methylene scissoring bands to determine the angle that the orthorhombic subcell makes in the x,y (water surface) plane is demonstrated.

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a non-crystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca(2+). Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

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