Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides.

Department of Physics and Astronomy, University of Louisville, Louisville, Kentucky 40292, Department of Chemistry, Butler University, 4600 Sunset Avenue, Indianapolis, Indiana 46208.
The Journal of Physical Chemistry C (Impact Factor: 4.84). 05/2009; 113(19):8306-8312. DOI: 10.1021/jp810845e
Source: PubMed

ABSTRACT In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c. Polarized light in the spectral region from 450 to 775 nm was all-coupled into an alumina thin film, which functioned as a single-mode planar optical waveguide. The alumina thin-film waveguide used for this work had a thickness of 180 nm and was deposited on a glass substrate by the atomic layer deposition process. The protein submonolayer was formed on the alumina waveguide surface through electrostatic adsorption from an aqueous buffer solution at neutral pH. The optical properties of the surface-adsorbed cyt c were investigated for bulk protein concentrations ranging from 5 nM to 8200 nM in the aqueous buffer solution. For a protein surface density of 2.3 pmol/cm(2), the molar absorptivity measured at the charge transfer band was 335 M(-1) cm(-1), and for a surface density of 15 pmol/cm(2) was 720 M(-1) cm(-1), which is much closer to the value of cyt c dissolved in an aqueous neutral buffer (830 M(-1) cm(-1)). The modification of the protein molar absorptivity and its dependence on the surface density can most likely be attributed to conformational changes of the surface-adsorbed species.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report here the fabrication, characterization, and application of a single-mode integrated optical waveguide (IOW) spectrometer capable of acquiring optical absorbance spectra of surface-immobilized molecules in the visible and ultraviolet spectral region down to 315 nm. The UV-extension of the single-mode IOW technique to shorter wavelengths was made possible by our development of a low-loss single-mode dielectric waveguide in the UV region based on an alumina film grown by atomic layer deposition (ALD) over a high quality fused silica substrate, and by our design/fabrication of a broadband waveguide coupler formed by an integrated diffraction grating combined with a highly anamorphic optical beam of large numerical aperture. As an application of the developed technology, we report here the surface adsorption process of bacteriochlorophyll a on different interfaces using its Soret absorption band centred at 370 nm. The effects of different chemical compositions at the solid-liquid interface on the adsorption and spectral properties of bacteriochlorophyll a were determined from the polarized UV-Vis IOW spectra acquired with the developed instrumentation. The spectral extension of the single-mode IOW technique into the ultraviolet region is an important advance as it enables extremely sensitive studies in key characteristics of surface molecular processes (e.g., protein unfolding and solvation of aromatic amino-acid groups under surface binding) whose spectral features are mainly located at wavelengths below the visible spectrum.
    The Analyst 01/2014; · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Aggregation of protein into insoluble intracellular complexes and inclusion bodies underlies the pathogenesis of human neurodegenerative diseases. Importance of cytochrome c (cyt c) arises from its involvement in apoptosis, sequence homology and for studying molecular evolution. A systemic investigation of polyethylene glycol (PEG) and trifluoroethanol (TFE) on the conformational stability of cyt c as a model hemeprotein was made using multi-methodological approach. Cyt c exists as molten globule (MG) at 60 % PEG-400 and 40 % TFE as confirmed by far-UV CD, attenuated total reflection Fourier transform infrared spectroscopy, Trp environment, 8-anilino-1-naphthalene-sulfonic acid (ANS) binding and blue shift in the soret band. Q-band splitting in MG states specifies conformational changes in the hydrophobic heme-binding pocket. Aggregates were detected at 90 % PEG-400 and 50 % TFE as confirmed by increase thioflavin T and ANS fluorescence and shift in Congo red absorbance. Detection of prefibrils and protofibrils at 90 % PEG-400 and 50 % TFE was possible after 72-h incubation. Single cell gel electrophoresis of prefibrils and protofibrils showed DNA damage confirming their toxicity and potential health hazards. Scanning electron microscopy and XRD analysis confirmed prefibrillar oligomers and protofibrils of cyt c.
    Amino Acids 04/2014; · 3.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An optical impedance spectroscopy (OIS) technique based on a single-mode electro-active integrated optical waveguide (EA-IOW) was developed to investigate electron-transfer processes of redox adsorbates. A highly sensitive single-mode EA-IOW device was used to optically follow the time-dependent faradaic current originated from a sub-monolayer of cytochrome c undergoing redox exchanges driven by a harmonic modulation of the electric potential at several DC bias potentials and at several frequencies. To properly retrieve the faradaic current density from the AC-modulated optical signal we introduce here a mathematical formalism that i) accounts for intrinsic changes that invariably occur in the optical baseline of the EA-IOW device during potential modulation and ii) provides accurate results for the electro-chemical parameters. We are able to optically reconstruct the faradaic current density profile against the DC bias potential in the working electrode, identify the formal potential, and determine the energy-width of the electron-transfer process. In addition, by combining the optically-reconstructed faradaic signal with simple electrical measurements of impedance across the whole electrochemical cell and the capacitance of the electric double-layer, we are able to determine the time-constant connected to the redox reaction of the adsorbed protein assembly. For cytochrome c directly immobilized onto the ITO surface we measured a reaction rate constant of 26.5 s-1. Finally, we calculate the charge-transfer resistance and pseudo-capacitance associated with the electron-transfer process and show that the frequency dependence of the redox reaction of the protein sub-monolayer follows as expected the electrical equivalent of an RC-series admittance diagram. Above all, we show here that OIS with single-mode EA-IOW's provide strong analytical signals that can be readily monitored even for small surface-densities of species involved in the redox process (e.g., femto-moles/cm2, 0.1% of a full protein monolayer). This experimental approach, when combined with the analytical formalism described here, brings additional sensitivity, accuracy, and simplicity to electro-chemical analysis, and is expected to become a useful tool in investigations of redox processes.
    Analytical Chemistry 01/2014; · 5.83 Impact Factor

Full-text (2 Sources)

Available from
May 22, 2014