- By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists
have built sensors based upon CytoC immobilized onto metal electrodes. Previous studies have shown that the
electron transfer rate through the protein is a function of the position of this heme group with respect to the
electrode surface. In this study a detailed examination of CytoC orientation when electrostatically immobilized onto
both amine (NH₃⁺) and carboxyl (COO⁻) functionalized gold is presented. Protein coverage, on both surfaces, was
monitored by the change in the atomic % N, as determined by x-ray photoelectron spectroscopy. Spectral features
within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively
and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well
ordered film. Time of flight secondary ion mass spectrometry data demonstrated a clear separation between the
two samples based on the intensity differences of secondary ions stemming from amino acids located
asymmetrically within CytoC (cysteine: C₂H₆NS⁺; glutamic acid: C₄H₆NO⁺ and C₄H₈NO₂⁺; leucine: C₅H₁₂N⁺). For a
more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of
secondary-ions stemming from the amino acid residues at either end of the protein. The 50 % increase in this ratio,
observed between the protein covered NH₃⁺ and COO⁻ substrates, indicates opposite orientations of the CytoC on
the two different surfaces.
- Baio, J. E., Weidner, T., Ramey, D., Pruzinsky, L., & Castner, D. G. (2013). Probing the orientation of electrostatically immobilized cytochrome C by time of flight secondary ion mass spectrometry and sum frequency generation spectroscopy. Biointerphases, 8(1), 1-8. doi:10.1186/1559-4106-8-18
|Funding Statement (additional comments about funding)
- This work was supported by NIH Grant EB-002027 (NESAC-BIO). T.W. thanks the German Research Foundation (DFG grants We 4478/1-1 and We 4478/2-1). J.E.B. thanks the National Science Foundation for a research fellowship(NSF grant #1202620).
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