Direct electrochemical and spectroscopic assessment of heme integrity in multiphoton photo-cross-linked cytochrome C structures.
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.
Lyon
Jennifer L
JL
Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station MC A5300, Austin, Texas 78712, USA.
Hill
Ryan T
RT
Shear
Jason B
JB
Stevenson
Keith J
KJ
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D004563:Electrochemistry; D006418:Heme / Q000737:chemistry*; D009195:Peroxidase / Q000378:metabolism; D010777:Photochemistry; D017785:Photons; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods*
D003432:Cross-Linking Reagents; D006418:Heme; D045304:Cytochromes c; D009195:Peroxidase