SPECTROSCOPIC EXAMINATION OF PROTEIN ADSORPTION FROM SEAWATER ONTO TITANIUM

Several noninvasive optical and electrochemical techniques were adapte d to examine partitioning of protein from seawater onto polished titan ium with the use of the plant enzyme ribulose-1,5,-bisphosphate carbox ylase-oxygenase (Rubisco) as a model. Protein films, varying in surfac e concentrations from 0.011 to 3.606 mug cm-2, were prepared by exposi ng polished Ti surfaces to seawater amended with 0.04-90.80 mug mL-1 o f H-3-Rubisco. Mean film thickness, d, measured by ellipsometry, incre ased linearly over most of the range of irreversibly bound protein (GA MMA(irr) = 0.011-2.491 mug cm-2). Spatial coverages of the films were more heterogeneous at low surface coverages, indicative of heterogeneo us adsorption resulting in barren Ti oxide surface sites and insular p rotein clusters. The thickness of the underlying Ti oxide layer. also measured by ellipsometry, was highly variable and indicated that oxida tion of the surface was suppressed at high protein coverages during tw o-hour exposures to seawater. Vibrational spectra of surface films, fr om submonolayer (0.03 mug cm-2) to multilayer (3.61 mug cm-2), were ob tained with the use of Fourier transform infrared reflection-absorptio n spectrometry (FT-IRAS). Peak areas of amide I and II bands varied li nearly with GAMMA(irr) permitting noninvasive measurement of protein m ass 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. Vibr ational spectroscopic and ellipsometric evidence suggests that protein structure is most altered at low surface concentrations. Electrochemi cal impedance spectroscopy (EIS) performed from 100 muHz 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, R(ct), of the interface v aried as a two-state function of protein concentration. The R(ct) incr eased more rapidly within the monolayer domain (0.12 to 2.8 MOMEGA cm2 ) than in the multilayer domain (2.8 to 4.9 MOMEGA cm2), indicating th at impedance to electron flow across the interface is most influenced by protein monolayer formation and is less affected by additional laye rs. Estimations of rates of oxidation or dissolution of the substratum were inversely proportional to protein surface concentrations. Togeth er these techniques provide internally consistent measurements of surf ace film thickness, adsorbate mass, gross chemical composition, interf ace organization, electrical impedance, capacitance, and oxide layer t hickness. 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.