OPTICAL SPECTROELECTROCHEMISTRY OF PROTEIN FILMS

Project: Research project

Description

DESCRIPTION: Intramolecular electron transfer (ET) reactions in metalloproteins have been widely studied using diffusionless (direct) electrochemical methods, in which the protein is strongly adsorbed on the electrode surface. Kinetic and mechanistic aspects of ET reactions are determined from these studies, which implies that the structure of the adsorbed protein film is well understood. However, such knowledge is usually lacking, which is due primarily to the experimental difficulty of structurally characterizing a protein (sub)monolayer at a solid-liquid interface. New analytical techniques that can yield structural information complementary to the kinetic data obtained from electrochemical methods are clearly needed. The major objective of this project is to develop a powerful new technology, broadband electroactive planar waveguide spectroscopy, which will combine the information content of multiwavelength spectroelectrochemistry with the inherent sensitivity of attenuated total reflection (ATR) spectroscopy at the surface of a planar integrated optical waveguide (IOW). Implementing this technology will enable relationships between protein film structure and ET kinetics in surface-confined metalloprotein (sub)monolayers to be elucidated at an unprecedented level of detail. The Specific Aims are designed to develop and test a prototype spectrophotometer, and initiate studies of structure and intramolecular ET in protein films: 1. A planar electroactive IOW (EA-IOW) and an achromatic coupler that will cancel the refractive index dispersion of the EA-IOW in the visible region will be designed, fabricated, and assembled into a single-component device. The device will be evaluated and refined to achieve a functional combination of spectral bandwidth, potential window, coupling efficiency, propagation loss and conductivity. 2. A broadband EA- IOW spectrophotometer will be constructed; its performance will be evaluated by measuring polarized ATR spectra of thin films of redox-active chromophores as a function of applied potential. The technology will then be extended to studies of structure-ET relationships in an electrostatically adsorbed protein film (cytochrome C on indium-tin oxide). 3. Broadband EA-IOW technology in combination with other surface analytical methods, will be applied to systematically investigate relationships between porphyrin orientation, protein conformation, electrode-porphyrin separation distance, and ET kinetics in heme protein films. Initial studies will focus on cytochrome c and cytochrome b5 immobilized via electrostatic adsorption and site-directed covalent bonding methods on EA-IOW devices.
StatusFinished
Effective start/end date5/1/999/30/01

Funding

  • National Institutes of Health
  • National Institutes of Health: $106,050.00

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Spectroelectrochemistry
Electrons
Optical waveguides
Proteins
Metalloproteins
Kinetics
Spectrophotometers
Porphyrins
Monolayers
Spectroscopy
Hemeproteins
Cytochromes b5
Electrodes
Planar waveguides
Cytochromes
Chromophores
Cytochromes c
Crystal orientation
Conformations
Electrostatics

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)