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  • Title: Direct electrochemical and spectroscopic assessment of heme integrity in multiphoton photo-cross-linked cytochrome C structures.
    Author: Lyon JL, Hill RT, Shear JB, Stevenson KJ.
    Journal: Anal Chem; 2007 Mar 15; 79(6):2303-11. PubMed ID: 17288462.
    Abstract:
    Multiphoton excitation (MPE) lithography offers an effective, biocompatible technique by which three-dimensional architectures comprised of proteins, enzymes, and other relevant materials may be fabricated for use in biological studies involving cellular signal transduction and neuronal networking. We present a series of studies designed to investigate the integrity of cytochrome c (cyt c) photo-cross-linked via MPE. Specifically, we have used electrochemical methods and surface-enhanced Raman spectroscopy (SERS) to determine whether photo-cross-linked cyt c retains its well-characterized Fe(II/III) heme redox activity. Cyt c is observed to retain its native FeII/III electron-transfer properties, as the apparent electron-transfer rate constant, k0ET, for cyt c photo-cross-linked onto an indium-doped tin oxide (ITO) substrate was 8.4 +/- 0.2 s-1, on the same order of magnitude as literature values though somewhat slower than other immobilized cyt c studies, most likely due to unoptimized entrapment in the photo-cross-linked matrix. SERS data reveals peaks corresponding to vibrational modes of an intact porphyrin ring with the Fe center intact. Cyt c has also been shown to demonstrate peroxidase-like activity, and we have evaluated the turnover rate of H2O2 at photo-cross-linked matrices relative to that at adsorbed monolayers of cyt c on glass substrates. The photo-cross-linked cyt c samples demonstrate apparent Michaelis-Menten parameters of Vm = 0.34 fmol/s and kcat/Km on the order of 104 s-1 M-1, in agreement with previously published results for aqueous cyt c. Fluorescence data obtained for mediated H2O2 turnover also indicated enzymatic activity specifically at photo-cross-linked cyt c structures.
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