Redox Potential Dependence of Peptide Structure Studied Using Surface Enhanced Raman Spectroscopy

Abstract

We would like to thank EastCHEM and the School of Chemistry of UoE for funding. We are also very grateful to Professor E. Campbell and her workgroup for the opportunity to work on Raman and AFM instruments and for fruitful advice. Supporting information:Additional information available free of charge via the internet at http://pubs.acs.org/ Keywords: SERS; nanoshell; peptide; redox; Goodpasture's; proteolysis AbstractWe describe a novel surface enhanced Raman spectroscopy (SERS) sensing approach utilizing AuroshellTM gold Nanoshells and demonstrate its application to analysis of critical redox-potential dependent changes in antigen structure that are implicated in the initiation of a human autoimmune disease. In Goodpasture's disease, an autoimmune reaction is thought to arise from incomplete proteolysis of the autoantigen, α3(IV)NC1(67-85) by proteases including Cathepsin D. We have used SERS to study conformational changes in the antigen that correlate with its oxidation state and toshow that the antigen must be in the reduced state in order to undergo proteolysis. Our approach determined that an intracellular redox-potential of ~-200 mV was sufficient for reduction, prerequisite for productive processing, of the intratramolecular disulfide bond within the antigenic fragment α3(IV)NC1 67-85 . Moreover we demonstrate that interaction of the antigenic fragment with Cathepsisn D, is vastly facilitated by reduction of this disulfide bond and that the peptide bonds subsequently cleaved by Cathepsisn D can be identified by consultation of a SERS library of short synthetic peptides.

Influence of Protein Size on Surface-Enhanced Raman Scattering (SERS) Spectra in Binary Protein Mixtures

“…Surface-enhanced Raman scattering (SERS) is a versatile and sensitive spectroscopic technique that is widely employed for detection of biomolecules. 1–5 In a SERS experiment, quality and reproducibility of the spectra basically rely on chemical properties of substrate and analyte and, dependently, degree of interactions of these two. In the case where colloidal noble metal nanoparticles are employed as substrate, size, shape, and type of nanoparticles define their plasmonic properties, 6–8 whereas surface charge directly affects their interactions with the analyte.…”

Series of Quinone-Containing Nanosensors for Biologically Relevant Redox Potential Determination by Surface-Enhanced Raman Spectroscopy

“…Within our group, we have developed redox potential nanosensors based on the principle of surface-enhanced Raman spectroscopy (SERS), where a noble-metal nanoshell is decorated with a surface coating of redox-responsive small molecules to form a nanosensor (see Figure ). These nanosensors can be introduced into cells without inducing oxidative stress or cell death and then probed passively using a near-infrared laser to obtain Raman spectra that depend on intracellular redox potential. − While Raman spectroscopy is typically very weak, SERS offers an enhancement of many orders of magnitude and has found considerable application in intracellular sensing already . Our nanosensors include a thiol linkage, which is used to form self-assembled monolayers with a gold surface. , …”

Cellular redox potential and the biomolecular electrochemical series: A systems hypothesis

“…The protein is allowed to reach equilibrium in each redox buffer before the ratio of reduced to oxidised protein disulfide is measured. Several techniques have been used to measure the reduced/oxidized ratio of protein disulfides, these include exploiting differences in the fluorescence emission properties of reduced and oxidised forms of the protein [40,42] ; gel electropheresis, Western blot, followed by densitometric analysis [43] ; SERS [44] ; and NMR spectroscopy [45] . However, mass analysing the molecular details of cysteine redox modifications [14,[46][47][48] .…”