Surface-protein studies, FTIR

Adsorption and diffusion phenomena at or within polymer systems were investigated by surface sensitive ATR-FTIR spectroscopy. For a systematic description, a study was made of (1) the competitive adsorption and desorption behaviour of proteins on polymer surfaces, (2) swelling of hydrophilic polymers by water molecules, which can be accompanied by conformational changes, and (3) induced orientational changes of hydrophobically modified polypeptides by apolar solvents. 10 refs. 

Lins et al. 127 have studied five peptide sequences derived from the ApoB-100 protein, which is the protein moiety in low-density lipoproteins (LDC) that transport cholesterol. ApoB-100 is insoluble and binds to the surface of the LDC particle, and these selected sequences for this study have been implicated as being important in the lipid binding. ATR-FTIR studies showed the one core and three C-terminal originating sequences were mostly sheet-like in the presence of unilamellar vesicles but the N-terminal one was different, probably representing a complex mixture of conformers with some helical component. Furthermore, these workers were able to carry out ATR-LD measurements and determine the orientation of the peptide as being oblique to the membrane. These studies are in contrast to ultraviolet ECD results which were adversely affected by scattering artifacts. 

Another label-free optical detection method—FTIR-ATR—has been applied for detection of thrombin by means of DNA aptamers [73], The antithrombin DNA aptamer previously developed by Tasset et al. [17] was immobilized covalently onto Si surface using UV irradiation method. As a quantitative measure, the area of N-H and CH2 bands was used. This method allowed to detect thrombin with a sensitivity around 10 nmol/L. The specificity of binding of protein to aptamer was also investigated using DNA with no binding site for thrombin. It has been noted that for effective binding study by FTIR-ATR method, the concentration of protein should be kept lower than 100 nmol/L. 

To date, there have been no published studies using ATR-FTIR to measure the spectra of just the protein in (bulk) solution. We have found that such solution spectra can be obtained by subtracting the contribution of denatured material irreversibly bound to the IRE surface from that of bulk and adsorbed protein. The strong interactions between polypeptides and IRE materials that immobilize proteins on IRE surfaces have a deleterious effect on the structure of these molecules. The characteristics of the adsorption process and its affects on protein structure will be discussed elsewhere (10). 

GENDREAU ET AL. FTIR Spectroscopy for Protein-Surface Studies 373... 

The transmission FTIR studies of aqueous protein solutions indicate how structural and conformational differences in a protein can be related to spectral changes, and that spectral features can be used to identify proteins in mixtures. However, these studies involve static systems, and our goal was to study flowing systems and the adsorption of proteins onto various surfaces. 

While these conclusions are directed towards the fibrinogen-albumin mixture study, this type of FTIR analysis of proteins has general applicability. These results indicate that FTIR can produce usable spectra of flowing aqueous protein solutions, and these spectra can in turn provide useful molecular-level information concerning protein-surface interactions. 

The FTIR/ATR studies of FN adsorption indicate that the polymer surface plays an important role in determining both the amount and the conformation of adsorbed FN, Comparison of the PEO-PEUU surface with the PDMS-PEUU and PTMO-PEUU surfaces show that on the more hydrophilic PEO-PEUU polymer, less protein adsorbs, and the interactions between the protein and polymer take place more slowly and are less intense as shown by the late appearance of the COOH vibration and the smaller 1669 cm l amide I peak observed at early adsorption times. The protein which adsorbs first appears to interact most strongly with the surface. On all polymers, changes in the amide I and the amide III region of adsorbed FN suggests that the amount of B sheet structure in FN increases upon adsorption. 

The surface deposits on UF PES membranes fouled by skimmed milk have been studied using ATR-FTIR to detect the functional groups of the fouling species [37]. Some milk components (lactose and salts) were eliminated by water rinsing, whereas proteins were only partially removed by chemical cleaning at basic pH. For dynamic conditions. 

As mentioned in the previous section, the first FTIR studies were performed to evaluate the suitable deposition of the low-density lipoprotein on the gold support in order to preserve the lipoprotein structural characteristics. This is an important experimental issue, because the protein structure and surface charging are the most important structural features necessary to be maintained to ensure feasible "ex-vivo" analytical information with respect to oxidative changes induced on cellular membrane by ROS. 

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