Infrared spectroscopy, protein adsorption

Morrissey 53) used transmission infrared spectroscopy to study protein adsorption onto silica particles in a heavy water (DzO) buffer. By observing the shift in the amide I absorption band, he could deduce the fraction of protein carbonyl groups involved in bonding to the silica surface. He found that bovine IgG had a bound fraction of 0.20 at low bulk solution concentrations, but only about 0.02 at high solution concentrations. However, neither prothrombin nor bovine serum albumin exhibited a change in bound fraction with concentration. Parallel experiments with flat silica plates using ellipsometry showed that the IgG-adsorbed layers had an optical thickness of 140 A and a surface concentration of 1.7 mg/m2 at low bulk solution concentration — in concentrated solutions the surface amount was 3.4 mg/m2 with a thickness of 320 A (Fig. 17). 

ATR spectroscopy in the infrared has been used extensively in protein adsorption studies. Transmission IR spectra of a protein contain a wealth of conformational information. ATR-IR spectroscopy has been used to study protein adsorption from whole, flowing blood ex vivo 164). Fourier transform (FT) infrared spectra (ATR-FTIR) can be collected each 5-10 seconds165), thus making kinetic study of protein adsorption by IR possible 166). Interaction of protein with soft contact lens materials has been studied by ATR-FTIR 167). The ATR-IR method suffers from problems similar to TIRF there is no direct quantitation of the amount of protein adsorbed, although a scheme similar to the one used for intrinsic TIRF has been proposed 168) the depth of penetration is usually much larger than in any other evanescent method, i.e. up to 1000 nm water absorbs strongly in the infrared and can overwhelm the protein signal, even with spectral subtraction applied. 

In THE PAST DECADE, IMPROVEMENTS IN infrared spectroscopic instrumentation have contributed to significant advances in the traditional analytical applications of the technique. Progress in the application of Fourier transform infrared spectroscopy to physiochemical studies of colloidal assemblies and interfaces has been more uneven, however. While much Fourier transform infrared spectroscopic work has been generated about the structure of lipid bilayers and vesicles, considerably less is available on the subjects of micelles, liquid crystals, or other structures adopted by synthetic surfactants in water. In the area of interfacial chemistry, much of the infrared spectroscopic work, both on the adsorption of polymers or proteins and on the adsorption of surfactants forming so called "self-assembled" mono- and multilayers, has transpired only in the last five years or so. 

Spectroscopic methods, such as fluorescence recovery and quenching, Fourier-transform infrared spectroscopy (FT-IR), and light reflection technique have been used for studies of adsorbed proteins (for example Burghardt Axelrod 1981, Thompson et al. 1981, van Wagenen et al. 1982), and surfactant adsorption layers (for example Ldsche et al. 1983, L6sche Mohwald 1989, Daillant et al. 1991, Henon Meunier 1992, Mohwald 1993). Considerable progress has been made in recent years with respect to the sensitivity of detectors and the efficiency of computers, so that the power of these methods has increased remarkably. 

Due to the fundamental importance of the adsorbed protein film, many methods have been used to characterize its nature. These methods include ellipsometry (3,A), Fourier transform infrared spectroscopy (FTIR) (5,6), multiple attenuated internal reflection spectroscopy (MAIR) (7,8) immunological labeling techniques (9), radioisotope labeled binding studies (j ), calorimetric adsorption studies (jj ), circular dichroism spectroscopy (CDS) (12), electrophoresis (j ), electron spectroscopy for chemical analysis (ESCA) (1 ), scanning electron microscopy (SEM) (15,16,9), and transmission electron microscopy (TEM) (17-19). 

Infrared spectroscopy has been used in many ways in clinical laboratories. The infrared analysis of serum and other fluids from healthy individuals and from patients with various diseases has been an aid in the diagnosis of those diseases. The adsorption of plasma proteins on the surface of polymers which are to be exposed to the bloodstream has been and is actively being investigated by means of internal reflection techniques. Normal and diseased skin surfaces have been examined by the ATR method... 

The stmctural and conformational analysis of proteins adsorbed to solid surfaces is difficult because most common analytical methods are not compatible with the presence of the interacting solids. With recent developments in instrumentation and techniques, our understanding of protein adsorption behavior has improved considerably [4, 14]. The most commonly used techniques include attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), radiolabeling techniques, immunofluorescence enzyme-linked immunosorbent assay (ELISA), ellipsometry, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), and amide HX with nuclear magnetic resonance (NMR). Atomic force microscopy (AFM) and scanning... 

How is electron transfer between electrodes and redox proteins facilitated by cast surfactant films X-ray photoelectron spectroscopy (XPS), surface infrared spectroscopy, and voltammetric studies showed that competitive adsorption on an electrode surface between surfactant, protein, and macromolecular impurities in the protein solution is invariably won by the surfaetant when present in large enough amounts [19]. Adsorption of proteins and macromolecular... 

Distribution between trains and loops in molecules adsorbed at solid/Iiquid interfaces is also possible and has been shown to occur for flexible polymers. There are some indications that protein molecules at solid/liquid interfaces do not always undergo the drastic conformational changes that occur at fluid/fluid interfaces. At a solid/liquid interface, an adsorbing molecule cannot penetrate the solid phase. Furthermore, adsorption may be confined to sites and thus be localized. Using infrared difference spectroscopy, Morrissey and Stromberg (1974) found a bound fraction (number of carbonyl surface... 

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