PM-IRRAS Spectra Research Articles

Interfacial complex of α-lactalbumin with oleic acid: effect of protein concentration and PM-IRRAS study

We used the Langmuir technique to investigate the binding of the bovine α-lactalbumin (BLA III) to the oleic acid (OA) monolayer in gastric conditions (pH 2.0 and 36.6 °C). Both substances are components of the BAMLET complex, which is beneficial to human health. The monolayer studies have been completed with BAM imaging and PM-IRRAS spectra. This paper explores the interfacial behavior of the lipid monolayer in the presence of the protein. It proves that the higher the concentration of protein in the subphase, the higher the increase in the relative surface pressure of the OA monolayer is, until the additional amount of protein affects the maximmum film response. We also discussed the MIP (maximum insertion pressure) value and analyzed the infrared spectra to prove that BLA III interacts with OA at the interface. We focused on the kinetics of the interaction between these components. We noticed that after protein adsorption at the interface (which rate depends on BLA III concentration), the protein penetrates the lipid monolayer, followed by film rearrangement (the protein forces on fatty acid molecules to change their orientation) until finally a stable lipid-protein complex is formed at the air-water interface. This multi-stage process is relevant to the course of a relaxation curve, which demonstrates the monolayer response on protein injection. This paper explores the physicochemical phenomena which fully recognized may open the door to numerous applications of these lipid-protein system.

Mechanism of lysozyme adsorption onto gold surface determined by quartz crystal microbalance and surface plasmon resonance

In this study, the physicochemical characterization of lysozyme adsorbed on gold was investigated. Through the use of MP-SPR it was possible to establish that the orientation of molecules changes from side-on to between or end-on with increasing surface coverage. The data confirms that the process of adsorption is driven primarily by electrostatic interactions but also by hydrophobic forces. MP-SPR data was compared with the Random Sequential Adsorption model for a molecule with an ellipsoidal shape. Contact angle measurements showed that higher surface coverage also translates in more hydrophilic properties of obtained lysozyme layer. Comparison of CD and PM-IRRAS spectra in solution and adsorbed state respectively showed changes in the secondary structures of lysozyme. These changes are dependent on pH, but fundamentally they go in the direction of the increase of β-turn/random content with a simultaneous decrease in β-sheet fraction, which suggests that aggregation is not occurring. The combination of MP-SPR and QCM-D measurements allowed the estimation of the number of water molecules associated with the lysozymes films. It has been observed that hydration decreases from 70% in pH = 4 to 30% in pH = 11. This data indicates that hydration is driven mainly by the degree of protonation of lysozyme molecules.

Self-Assembled Monolayer Films Derived from Tridentate Cyclohexyl Adsorbates with Alkyl Tailgroups of Increasing Chain Length

Tridentate cyclohexyl-based alkanethiolate SAMs generated from a series of adsorbates of the form R3C6H6(CH2SH)3, where R = -(CH2)nH and n = 3, 8, and 13 (3CnCyTSH), were examined. Characterization of the SAMs by X-ray photoelectron spectroscopy (XPS) revealed that all sulfur atoms of the tridentate adsorbates were bound to the surface of gold, and that the tailgroups were in general less densely packed than the SAM derived from octadecanethiol (C18SH). For each of the SAMs, the relative molecular coverage on the surface was estimated from the XPS-derived C1s/Au4f ratios. The trend in conformational order for these SAMs as determined by the surface interactions with contacting liquids and the relative crystallinity of the alkyl chains as revealed by the PM-IRRAS spectra were found to decrease as follows: C18SH >> 3C13CyTSH > 3C8CyTSH > 3C3CyTSH. A preliminary study of the thermal stability of the SAMs as evaluated by XPS indicates that the SAM generated from the cyclohexyl-based adsorbate with the longest alkyl chain, 3C13CyTSH, is markedly more stable than the SAM generated from C18SH. Overall, the results suggest that the stability of the SAMs are influenced by both the length of the alkyl chains and the chelate effect associated with the tridentate adsorbates.

Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is expressed in mammalian cells in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7. The different intracellular localization of these two isoforms suggests distinct function in membrane dynamics. The aim of this work was to analyze the behavior of AnxA6 isoforms at the air/water interface alone and in the presence of membrane mimicking lipid monolayers. Using Langmuir technique showed that AnxA6-2 was less adsorbed to the neat air–water interface than AnxA6-1 at acidic pH and minor differences in their PM-IRRAS spectra were observed. Both isoforms exhibited similar behavior towards cholesterol monolayer. However, the interactions of AnxA6-2 with cholesterol ester monolayer were most favorable compared to AnxA6-1. Our experimental data are discussed in relation with the different intracellular localization of the two isoforms and with our constructed model of AnxA6-2 with the known crystal structure of AnxA6-1 showing the persistence of the 516–529 α-helix in AnxA6-2 despite the absence of the 524-VAAEIL-529 sequence.

Determination of the Contribution of the Myristoyl Group and Hydrophobic Amino Acids of Recoverin on its Dynamics of Binding to Lipid Monolayers

It has been postulated that myristoylation of peripheral proteins would facilitate their binding to membranes. However, the exact involvement of this lipid modification in membrane binding is still a matter of debate. Proteins containing a Ca 2+-myristoyl switch where the extrusion of their myristoyl group is dependent on calcium binding is best illustrated by the Ca 2+-binding recoverin, which is present in retinal rod cells. The parameters responsible for the modulation of the membrane binding of recoverin are still largely unknown. This study was thus performed to determine the involvement of different parameters on recoverin membrane binding. We have used surface pressure measurements and PM-IRRAS spectroscopy to monitor the adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers in the presence and absence of calcium. The adsorption curves have shown that the myristoyl group and hydrophobic residues of myristoylated recoverin strongly accelerate membrane binding in the presence of calcium. In the case of nonmyristoylated recoverin in the presence of calcium, hydrophobic residues alone are responsible for its much faster monolayer binding than myristoylated and nonmyristoylated recoverin in the absence of calcium. The infrared spectra revealed that myristoylated and nonmyristoylated recoverin behave very different upon adsorption onto phospholipid monolayers. Indeed, PM-IRRAS spectra indicated that the myristoyl group allows a proper orientation and organization as well as faster and stronger binding of myristoylated recoverin to lipid monolayers compared to nonmyristoylated recoverin. Simulations of the spectra have allowed us to postulate that nonmyristoylated recoverin changes conformation and becomes hydrated at large extents of adsorption as well as to estimate the orientation of myristoylated recoverin with respect to the monolayer plane. In addition, adsorption measurements and electrophoresis of trypsin-treated myristoylated recoverin in the presence of zinc or calcium demonstrated that recoverin has a different conformation but a similar extent of monolayer binding in the presence of such ions.

SAMs functionalised by α-quaterthiophene as model for polythiophene grafting

A model of polythiophene grafting on a SAM functionalised by α-quaterthiophene is presented. The elaboration and characterisation of the dodecanethiol monolayer terminated by α-quaterthiophene are reported. Blocking level of substrates, structure of the alkyl chains and organisation of the quaterthiophene units as well as their chemical behaviour are analysed. HREELS spectra showed that the extreme surface of the monolayers is entirely covered by quaterthiophene units after typical adsorption times of 12 h. PM-IRRAS spectra allow to follow the orientation of molecules within the monolayer versus adsorption time. It is shown that the long axis of quaterthienyl groups stand slightly tilted with respect to the perpendicular. In addition, interfacial reactions of the SAM layer with a model molecule, the end-capped terthiophene, were electrochemically induced via the formation of radical cations. The number of grafted molecules that depends on the 4T film structure is discussed in regard to the 4T moiety reactivity.

Calibration Procedure to Derive IRRAS Spectra from PM-IRRAS Spectra

A calibration procedure is presented to relate Fourier transform infrared reflection-absorption spectroscopy (IRRAS) spectra obtained by polarization modulation (PM) and conventional techniques. This procedure concerns only PM-IRRAS measurements obtained with a lock-in amplifier. At the first stage, it consists in measuring two calibrated spectra, by adding a linear polarizer after the sample. The corresponding corrected PM-IRRAS spectra display band intensities close to those observed on IRRAS spectra (at about 6%), but the value of the baseline is lower. The calibration procedure can be performed at the second stage, by normalizing the corrected PM-IRRAS spectra of the sample and the bare substrate. In this case, the band intensities and the baseline of the normalized corrected PM-IRRAS and IRRAS spectra are similar.

Polarization-Modulated FT-IR Spectroscopy of a Spread Monolayer at the Air/Water Interface

This study devoted to the FT-IR spectroscopy of monolayers spread at the air/water interface is, to our knowledge, the first report presenting complete mid-infrared monolayer spectra perfectly extracted from the strong water vapor bands. This has been possible with the use of the polarization-modulated IRRAS method, which is not sensitive to the isotropic absorptions of the sample environment. On the basis of theoretical modeling and experiments, the best angle of incidence has been found near 76° for detection of intraplane as well as out-of-plane oriented monolayer absorptions. With the use of such experimental conditions, on the normalized difference (covered vs. uncovered water) PM-IRRAS spectra, monolayer vibrational bands come out upward or downward, depending on the orientation of their transition moment with respect to the interface. Application to the study of deuterated arachidic acid and arachidate monolayers allows observation of the vibrational modes of the polar head groups interacting with the liquid water molecules and provides some evidence of their symmetrical anchoring. The vibrational modes of the liquid water subphase contribute to these difference spectra as broad dips that certainly contain information on a possible restructuring of the water molecules at the interface.