Several noninvasive optical and electrochemical techniques were adapted to examine partitioning of protein from seawater onto polished titanium with the use of the plant enzyme ribulose-1,5,-bisphosphate carboxylase-oxygenase (Rubisco) as a model. Protein films, varying in surface concentrations from 0.011 to 3.606 μg cm<sup>−2</sup>, were prepared by exposing polished Ti surfaces to seawater amended with 0.04-90.80 μg mL<sup>−1</sup> of <sup>3</sup>H-Rubisco. Mean film thickness, <i>d,</i> measured by ellipsometry, increased linearly over most of the range of irreversibly bound protein (Γ<sub>irr</sub> = 0.011-2.491 μg cm<sup>−2</sup>). Spatial coverages of the films were more heterogeneous at low surface coverages, indicative of heterogeneous adsorption resulting in barren Ti oxide surface sites and insular protein clusters. The thickness of the underlying Ti oxide layer, also measured by ellipsometry, was highly variable and indicated that oxidation of the surface was suppressed at high protein coverages during two-hour exposures to seawater. Vibrational spectra of surface films, from submonolayer (0.03 μg cm<sup>−2</sup>) to multilayer (3.61 μg cm<sup>−2</sup>), were obtained with the use of Fourier transform infrared reflection-absorption spectrometry (FT-IRAS). Peak areas of amide I and II bands varied linearly with Γ<sub>irr</sub>, permitting noninvasive measurement of protein mass at the surface. Relative intensities of the amide II/amide I bands, band composition of the amide III, and peak frequencies varied with surface concentration, indicating unfolding of adsorbed proteins. Vibrational spectroscopic and ellipsometric evidence suggests that protein structure is most altered at low surface concentrations. Electrochemical impedance spectroscopy (EIS) performed from 100 μHz to 100 kHz on replicate test surfaces revealed that the electrochemical behavior of the titanium/protein interface was consistent with that of a parallel RC circuit. The charge transfer resistance, <i>R</i><sub>ct</sub>, of the interface varied as a two-state function of protein concentration. The <i>R</i><sub>ct</sub> increased more rapidly within the monolayer domain (0.12 to 2.8 MΩ cm<sup>2</sup>) than in the multilayer domain (2.8 to 4.9 MΩ cm<sup>2</sup>), indicating that impedance to electron flow across the interface is most influenced by protein monolayer formation and is less affected by additional layers. Estimations of rates of oxidation or dissolution of the substratum were inversely proportional to protein surface concentrations. Together these techniques provide internally consistent measurements of surface film thickness, adsorbate mass, gross chemical composition, interface organization, electrical impedance, capacitance, and oxide layer thickness. These data are useful for determining the physical state of the interface, its dynamics, and the potential oxidation rates of the substratum underlying the surface film.
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