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The peptide hormone human calcitonin (hCT) has a marked tendency to aggregate in aqueous solutions, resulting in viscous and turbid dispersions consisting of long fibrils approximately 80 A in diameter. Both transmission (T-FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) experiments were applied on hCT adsorption and aggregation kinetics. By means of the surface sensitive ATR-FTIR spectroscopy at a hydrophobic/hydrophilic interface, early adsorption and aggregation steps of hCT could be followed in situ under real time conditions. Since the aggregation of hCT is associated with conformational changes, the secondary structure sensitive amide I'-band (D2O) could be used as a diagnostic marker. ATR-FTIR spectra recorded during the aggregation kinetics of hCT showed an increase of the amide I'-band intensity by a factor of 3.4, interpreted as pronounced adsorption of hCT molecules from bulk solution to the germanium plate. Furthermore, variations in the line shape of the amide I'-band were interpreted. At the beginning, hCT adopted a random coil conformation followed by distinct formations of alpha-helical and intermolecular parallel beta-sheet structures. Finally, the random coil content declined to 63%, whereas alpha and beta contents rose to 8% and 29%, respectively. From our kinetics results the alpha-structures were formed faster than the beta-structures. This was interpreted as an initial induction of amphiphilic helices during the adsorption process of hCT monomers. ATR-FTIR spectroscopy provides a sensitive analytical tool suggested to monitor interfacial adsorption and aggregation phenomena also of other peptides and proteins.

In situ attenuated total reflection (ATR) Fourier transform (FT) spectroscopy is presented as an adequate tool for studying molecular structure and function of biomembranes. In this article emphasis was directed to the production of suitable model bilayer membranes for optimum mimicking of natural biomembranes, and to special FTIR ATR techniques to achieve enhanced selectivity as well as time resolved information on complex membrane assemblies. In this context, the preparation of supported bilayers according to the LB/vesicle method is presented and the use of such model membranes to build more complex assemblies, e.g. with creatine kinase, a surface bound enzyme, and alkaline phosphatase, a membrane anchored enzyme. A comprehensive summary of equations used for quantitative ATR spectroscopy is given and applied to determine the surface concentration and orientation of membrane bound molecules. The use of supported bilayers for drug membrane interaction studies is demonstrated by the local anesthetic dibucaine. Besides of structural information's, such studies result also thermodynamic date, such as adsorption isotherm and partition coefficient. A special ATR setup for more precise background compensation is presented enabling the conversion of a single beam spectrometer into a pseudo double beam spectrometer. This optical component may be placed in the sample compartment of the spectrometer, and is referred to as single-beam-samplereference (SBSR) attachment. Finally, a short theoretical introduction into time resolved modulation spectroscopy is given. Temperature modulated excitation of reversible conformational changes in the polypeptide poly-L-lysine and the enzyme RNase are shown as examples.

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