Novel application of sulfur K-edge X-ray absorption and X-ray photoelectron spectroscopies to investigate the structure and reactivity of extracellular proteins

In this interdisciplinary work, two spectroscopic techniques normally utilized in surface science and solid-state physics are applied to biological systems. X-ray Absorption Spectroscopy (XAS) was used to study protein/surface interactions and protein oxidation, and X-ray Photoelectron Spectroscopy (XPS) was used to study cell/surface interactions and dynamic molecule/surface interactions as exhibited in the process of electroosmotic flow (EOF) through microfluidic channels. Finally a theoretical quantum mechanical interpretation of the sulfur K-Edge X-ray Absorption Near Edge Spectroscopy (S K-Edge XANES) spectrum on an array of molecules is presented to provide a fundamental foundation, and prove applicability and signal sensitivity to proteins with low sulfur concentrations.; S K-edge XANES, never before used to study biomaterial interactions, is shown to be a sensitive analytical technique for the investigation of protein folding and protein vulnerability to oxidative attack. Two extra cellular matrix (ECM) proteins, fibronectin (FN) and laminin (LN) were adsorbed onto well-characterized hydrophilic and hydrophobic derivatized surfaces.{09}The adsorption of ECM messenger proteins onto these surfaces induced disruption of S-S (or disulfide) bonds in contact with the surface in one protein (FN) but not in the other (LN).; The first use of in situ sulfur K-edge XANES spectroscopy to simultaneously detect and distinguish between cysteine (RSH), methionine (RSR), and their oxidative products in proteins and biomolecules is reported. Our analysis of two relatively rigid proteins (lysozyme and collagen) illustrated the key relationship between the location of RSR residues within the overall protein structure: lysozyme showed strong resistance to oxidation, while collagen showed very high vulnerability to oxidation.; Compilation of new XANES data into a “reference library” of S-containing hydrocarbon molecules, and comparison with full multiple scattering calculations utilizing the recently developed FEFF8 code, and molecular orbital calculations on model molecules, allowed a full interpretation of the origins of chemical shifts, shoulders and splitting due to increasing valence state, the effect of π-bonding with neighbors, and disulfide bonding.; The properties of electroosmotic flow (EOF) through microfluidic channels were correlated with the surface chemistry of the polymer substrates used to prepare the devices. XPS was also used to characterize the untreated and oxygen plasma-treated surfaces of poly(methyl methacrylate) (PMMA) used for fluid flow experiments. (Abstract shortened by UMI.){09}...