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.