| Abstract
| - The Soret absorption band has been utilized as a probe for the adsorption of cytochrome c to the surface ofa fused silica prism in direct contact with bulk protein solutions of various concentrations and pH. Employinglinear polarized light and a single-pass total internal reflection absorption technique, we examined in detailthe adsorption isotherm, molecular orientation, packing density, and conformational change of the proteinbound to the bare (hydrophilic) and silanized (hydrophobic) glass surfaces. An adsorbate density of Γ = 1.4× 1013 molecules/cm2 was determined for the hydrophilic substrate at pH 7.2 and Cb = 110 μM, indicatingthat the protein molecules are essentially closely packed on the surface at saturation. The packing density issensitive to the solution pH as well as the surface hydrophobicity, a result that the protein−surface interactionis governed by both electrostatic and hydrophobic forces. The same forces also govern the molecular orientation,yielding an angle of θμ = 41° between the heme plane and the surface normal at neutral pH. The angle isretained over a wide pH range (4−9) and is fairly independent of the surface coverage on both the hydrophilicand hydrophobic substrates. Reorientation of the protein occurs (41° → 20°) at pH ≈ 3, when the cyt cunfolds and the hydrophobic force becomes dominant in the adsorption process.
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