Protein attenuated total reflection studies

One can distinguish between methods in which absorption of the evanescent surface wave in different wavelength regions is measured (these are often called attenuated total reflection methods), and methods which use the evanescent wave to excite other, spectroscopic phenomena, like fluorescence and Raman scattering or light scattering. As the methods of conventional fluorescence spectroscopy have been shown to be exceptionally successful in studies of proteins and other biopolymers, their evanescent surface-sensitive counterparts will be reviewed first. 

There is substantial history regarding the application of conventional vibrational spectroscopy methods to study the intact surface of skin, the extracted stratum corneum and the ceramide-cholesterol-fatty acid mixtures that constitute the primary lipid components of the barrier. The complexity of the barrier and the multiple phases formed by the interactions of the barrier components have begun to reveal the role of each of these substances in barrier structure and stability. The use of bulk phase IR to monitor lipid phase behavior and protein secondary structures in the epidermis, as well as in stratum corneum models, is also well established 24-28 In addition, in vivo and ex vivo attenuated total reflectance (ATR) techniques have examined the outer layers of skin to probe hydration levels, drug delivery and percutaneous absorption at a macroscopic level.29-32 Both mid-IR and near-IR spectroscopy have been used to differentiate pathological skin samples.33,34 The above studies, and many others too numerous to mention, lend confidence to the fact that the extension to IR imaging will produce useful results. 

Protein-lipid interactions and particularly peptide-lipid interactions have been studied in supported bilayers by attenuated total reflection (ATR) FTIR spectroscopy. A slightly dated, but still valid comprehensive review on this method applied to supported bilayers has been published (20). Because IR light probes the vibrational properties of different classes of covalent bonds, this method is useful to examine lipids, peptides, and interactions between the two in the same sample. The most common parameter for assessing lipid structure and order is to study the stretching vibrations of the lipid acyl chains, for example as a function of peptide concentration or temperature. Such studies have lead to the conclusion that fusion peptides from viruses increase the lipid chain order of fluid phase bilayers and that... 

Attenuated Total Reflectance FTIR (ATR-FTIRf) is a method that has been applied by a number of workers for the study of protein conformation. ATR has been used for monitoring adsorption of proteins or blood components to sur ces (1,2), and for the structural analysis of proteins dried onto an IRE (thin film) (3,4). It has also been used for exploring the effects of solution conditions on the structure of proteins irreversibly adsorbed to an IRE (5,6,7), and has been shown to be usefel for studying the secondary structure and ligand binding properties of membrane proteins (8,9). 

Oberg and Fink (1994) The Application of Attenuated Total Reflection FTIR to the Study of Protein Structure In Solution. Ms. in preparation. 

The experimental techniques used in these studies have been detailed in the original papers (13, 14) and will only be briefly discussed here. Studies of pressure and pH variation were carried out on dissolved proteins while studies of solvent and pH variation were done on adsorbed protein films. The adsorbed film studies utilized a liquid attenuated total reflection (ATR) cell with the proteins adsorbed on the ATR crystal (Ge). In this arrangement the pH of the saline or the solvent itself could easily be varied. As in the previous section, virtually all of the spectra were obtained in aqueous media with the infrared spectrum of water removed from the final spectrum by computer substraction. Where non-aqueous solvents were used (14), the spectrum of the solvent was also removed by spectral subtraction. 

Fourier Transform Infrared Spectroscopic and Attenuated Total Reflectance Studies of Protein Adsorption in Flowing Systems... 

The complexity of the FT-IR spectra collected in the Attenuated Total Reflectance (ATR) mode which can include contributions from (a) the adsorbed protein layer or (b) the soluble proteins in the liquid layer adjacent to the ATR surface but within the "depth of penetration" of the evanescent field of the IR beam (this so-called "bulk" effect is especially significant when near-physiological protein concentrations are studied) ... 

In early work (8) we used infrared spectroscopy coupled with attenuated total reflection optics. This work was done before the availability of infrared equipment based on Fourier transform methods. Due to their relative speed these methods now permit in situ, real time measurements with a resolution of 1 sec or less (9), and continue to yield valuable data, particularly in the hands of the Battelle group in a series of studies dating from 1979 (10). In our early infrared work we had to be content to rinse and dry the surface before obtaining the infrared reflection spectrum Nevertheless the values of surface concentration were remarkably close to those determined more recently. Infrared studies of proteins suffer generally from the fact that the main features of protein spectra are similar for all proteins and therefore it is difficult to distinguish one from another. 

The technique of attenuated total reflection (ATR) has been used for some time and recently for in situ studies by Fringeli, Gendreau and others in the investigation of protein... 

Applying attenuated total reflection Fourier transform infrared and total internal reflection fluorescence spectroscopy, the same group studied the secondary structure and aggregation properties of different proteins that are adsorbed onto planar PAA brushes (Hollmann, Steitz, Czeslik, 2008 Reichhart Czeslik, 2008). They found... 

---