Probing Adsorption, Orientation and Conformational Changes of Cytochrome c on Fused Silica Surfaces with the Soret Band

Abstract

The Soret absorption band has been utilized as a probe for the adsorption of cytochrome c to the surface of a fused silica prism in direct contact with bulk protein solutions of various concentrations and pH. Employing linear polarized light and a single-pass total internal reflection absorption technique, we examined in detail the adsorption isotherm, molecular orientation, packing density, and conformational change of the protein bound 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 C b = 110 μM, indicating that the protein molecules are essentially closely packed on the surface at saturation. The packing density is sensitive to the solution pH as well as the surface hydrophobicity, a result that the protein−surface interaction is 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 is retained over a wide pH range (4−9) and is fairly independent of the surface coverage on both the hydrophilic and hydrophobic substrates. Reorientation of the protein occurs (41° → 20°) at pH ≈ 3, when the cyt c unfolds and the hydrophobic force becomes dominant in the adsorption process.

Determination of Anisotropic Optical Constants and Surface Coverage of Molecular Films Using Polarized Visible ATR Spectroscopy. Application to Adsorbed Cytochrome c Films

“…40 In addition to the different analytical approaches used in these measurements, we also attribute the different experimental results to the higher dissolved protein concentration used in the cited work (35 µM) vs that used here (10 µM). In a recently published study by Cheng et al, 13 the surface coverage of cyt c on a glass substrate was determined using ATR spectroscopy to be a full monolayer (23 pmol/cm 2 ). In this case, the cyt c film was adsorbed from a much higher dissolved protein concentration (110 µM) and at lower ionic strength (7 mM) vs that used here (10 mM).…”

“…11,12 Cyt c is often used as a model protein to address these questions and immobilized cyt c films have been studied using a variety of surface sensitive spectroscopic techniques. [13][14][15][16][17] Glass and ITO are both negatively charged surfaces with heterogeneous surface chemistries. ITO is a semiconductor which can directly oxidize and reduce adsorbed cyt c, depending on the potential applied to the surface.…”

Probing Redox Reactions of Immobilized Cytochrome c Using Evanescent Wave Cavity Ring‐Down Spectroscopy in a Thin‐Layer Electrochemical Cell

“…Assuming that a monolayer of cytochrome c would completely cover the surface, and considering that it is roughly spherical with a diameter of 3.4 nm, [43] the maximum monolayer surface concentration would be about 2 10 À11 mol cm…”

Recent advances in ceramic implants as drug delivery systems for biomedical applications

“…Moreover, albumins are also capable of binding a wide variety of drugs than can be delivered to sites of pharmacological action (Carter et al 1994). Several proteins have been demonstrated to retain their functionality without being denatured inside the frameworks of silica-based mesoporous materials (Deere et al 2002;Cheng et al 2003;Lei et al 2004;Vinu et al 2004;Hartmann 2005;Katiyar et al 2005;Lee et al 2006;Slowing et al 2007).…”