U. P. Fringeli

Infrared Membrane Spectroscopy

Application of vibrational spectroscopy to the problem of structure determination of molecules of biological interest goes back to the early uses of raman and infrared spectroscopy in the study of organic molecules. For reviews of earlier work the reader is referred to compilations by Kohlrausch (1943) and by Jones and Sandorfy (1956), whereas more recently a comprehensive discussion has been presented by Bellamy (1975). These compilations accentuate the correlation of vibrational spectra with molecular structure from an essentially empirical point of view and culminate in the establishment of empirical correlation charts. For typical examples the reader is referred to Weast (1974) and Bellamy (1975). There have been many treatments of the theoretical basis of molecular vibrational spectroscopy. Among them the classical work by Herzberg (1945) and by Wilson et al. (1955) should be mentioned. Applications of infrared spectroscopy (IR) to structure problems of biological interest have been summarized by Susi (1969), Fraser and MacRae (1973), and Wallach and Winzler (1974). It was remarked quite eraly that relevant structural information about biological systems often requires study in aqueous solution, which forms the natural environment for most biologically important systems. Besides critical control of experimental conditions and samples the conventional methods of raman spectroscopy may be applied to aqueous solutions in a quite straightforward manner, cf. the contribution by Lord and Mendelson, Chapter 8.

Quantitative modulated excitation Fourier transform infrared spectroscopy

The detection of weak absorption changes induced by an external excitation is often hindered by intense background absorptions as well as by noise. Modulation spectroscopy is an adequate tool to be applied in such a case, provided the system may be periodically stimulated, leading to a periodic reversible or pseudoreversible response. In modulated excitation (ME) Fourier transform infrared spectroscopy the phase sensitive detection (PSD) used for the demodulation of the periodic system response is generally performed during data acquisition, i.e., applied to the intensity of the interferogram. This leads to a number of problems in quantitative analysis and the requirement of optional equipment. In this article, a method is presented to perform an off-line vector PSD of conventional time-resolved spectra after data acquisition. A detailed mathematical analysis of PSD applied to the spectral intensity, the interferogram intensity, and to time-resolved spectra is presented. It is shown, that vector PSD applied to a set of time-resolved spectra is straightforward and avoids any additional mathematical corrections. Furthermore, it will be shown how ME spectroscopy can be used for experimental separation of overlapping bands and a detailed description for the determination of absolute modulation amplitudes and phase lags is given.The detection of weak absorption changes induced by an external excitation is often hindered by intense background absorptions as well as by noise. Modulation spectroscopy is an adequate tool to be applied in such a case, provided the system may be periodically stimulated, leading to a periodic reversible or pseudoreversible response. In modulated excitation (ME) Fourier transform infrared spectroscopy the phase sensitive detection (PSD) used for the demodulation of the periodic system response is generally performed during data acquisition, i.e., applied to the intensity of the interferogram. This leads to a number of problems in quantitative analysis and the requirement of optional equipment. In this article, a method is presented to perform an off-line vector PSD of conventional time-resolved spectra after data acquisition. A detailed mathematical analysis of PSD applied to the spectral intensity, the interferogram intensity, and to time-resolved spectra is presented. It is shown, that vector PSD appli...

Instability of langmuir-blodgett layers of barium stearate, cadmium arachidate and tripalmitin, studied by means of electron microscopy and infrared spectroscopy

Results of an investigation of the stability of n-layers of barium stearate, cadmium arachidate and tripalmitin by means of electron microscopy and attenuated total reflection infrared spectroscopy are reported. Odd and even numbered barium stearate n-layers with n=1,2,3.4,5 are found to rearrange spontaneously from a regular film into ultrastructures of irregular, flat islands of varying thickness. The kinetics of the phase transformation of the first layer depends on the substrate, that of n-layers appears to be dependent on n, the temperature, and the surrounding medium. The kinetic behaviour of odd and even numbered layers is distinctly different. Similar studies on cadmium arachidate layers reveal much slower kinetics of the rearrangement process. In the case of tripalmitin n-layers it is shown that electron microscopy and infrared spectroscopy yield valuable complementary information about ultrastructure and molecular structure of the layers in correlation with the rearrangement process, which also occurs with this system. Consequences of the results of this paper for work published in various fields are briefly discussed.

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.

Distribution and diffusion of alamethicin in a lecithin/water model membrane system

SummaryAttenuated total reflection infrared spectroscopy has been used to determine the equilibrium distribution of the peptide antibiotic alamethicinRF30 between dipalmitoyl phosphatidylcholine bilayers and the aqueous environment. The distribution coefficientK=ceqW/ceqM turned out to be concentration dependent, pointing to alamethicin association in the membrane with increasing concentration in the aqueous phase (ceqW). This concentration was varied within 28 and 310nm, i.e., in a range typical for black film experiments. Furthermore, diffusion coefficients of alamethicin in the hydrophobic phase of the membrane (DM) and across the membrane/water interface (DI) have been estimated from the time course of the equilibration process. It was found that the diffusion rate of the uncharged analogueRF50 is about 10 times higher than that of theRF30 component, exhibiting one negative charge at theC-terminus. The time constants for transmembrane diffusion of alamethicinRF30 varied between 2.2 hr at low concentration and 3.2 hr at higher concentration. The corresponding low concentration value of theRF50 component was found to be 0.25 hr.

A new method of phase sensitive detection in modulation spectroscopy applied to temperature induced folding and unfolding of RNase A

Abstract Modulation spectroscopy can be an adequate tool to separate a weak system response from a huge background absorption, provided the process under consideration enables a periodic external stimulation. Phase sensitive detection (PSD) is then used to demodulate the periodic system response. We introduce a new method of PSD that can be used in modulated excitation (ME)-FTIR spectroscopy without the need of a lock-in amplifier or spectrometer build-in hardware. An advantage of this method is that only standard procedures included in all FTIR instruments, such as the measurement of time-resolved spectra, are required. The principal mathematical formalism of the used PSD is described. As an example we show phase-resolved spectra of the amid I ′ region obtained from temperature modulated FTIR spectroscopic experiments of RNase A. The advantage of the ME compared to a relaxation process is shown by the power of separation of overlapping absorption bands.

Hydration sites of egg phosphatidylcholine determined by means of modulated excitation infrared spectroscopy

Conventional and modulated excitation infrared spectra of egg phosphatidylcholine are measured in the spectral range of 4000-900 cm-1 by modulation of relative humidity with an amplitude of +/- less than 5% at a mean value of 75% (T=30 degrees C). From the modulated-excitation spectrum, hydration sites are found to be the greater than PO2 group, the greater than C=O group and the choline group.

Radiation damage in tripalmitin layers studied by means of infrared spectroscopy and electron microscopy

Structural deteriorations in biomembranes, as inevitably induced while structural information is gathered by electron optical methods, were evaluated by infrared spectroscopy. Tripalmitin model membranes were irradiated with 100 keV-electrons in an electron microscope. The intensity decay of group vibrations over the dose reveals the sequence of damage in the polar and nonpolar part of the molecule. The C-C backbone, being the most important structural feature, shows a significant latency effect up to 0.6 e-/A2 and is completely disordered by 3 e-/A2, corresponding to about three inelastic processes per molecule.

The Structure of Lipids and Proteins Studied by Attenuated Total-Reflection (ATR) Infrared Spectroscopy

The structure of tripalmitin multilayers prepared by the Langmuir-Blodgett technique has been investigated within room temperature and 62°C using infrared internal reflection spectroscopy (ATR). It was found that the bilayer structure exhibits crystalline features with pseudo hexagonal structure. The methylene groups of the hydrocarbon chains are in the all-trans-zig-zag conformation, oriented perpendicularly to the germanium internal reflection plate. Increasing temperature produces at first a continuous conformational change in the glycerol part of the molecule. This process is extended over nearly 10° and followed by sudden melting (disordering) of the hydrocarbon chains within 1°. In comparison microcrystalline tripalmitin shows an identical infrared spectrum and analogous behaviour at increased temperatures. This indicates that the bilayer structure (head-head, tail-tail) also exicts in the crystal.

ATR and Reflectance IR Spectroscopy, Applications

Optical reflection spectroscopy in the mid- and near-IR (infrared) range occupies an important complementary position for interface studies. Many reflection techniques permit in situ applications, and if applied in the mid-IR, result in quantitative and structural information on a molecular level. The basic equipment consists of a commercial IR spectrometer and a suitable reflection accessory that usually fits into the sample compartment of the spectrometer. There is a wide range of different spectroscopic reflection techniques. One should distinguish between internal (total) reflections and external reflections. Attenuated total reflection technique (ATR) belongs to the first group, whereas specular reflection (SR) belongs to the second one. These techniques together with a brief presentation of the theory behind, are presented in this article.