Direct Electrochemical and Spectroscopic Assessment of Heme Integrity in Multiphoton Photo-Cross-Linked Cytochrome c Structures

详细信息    查看全文

• 作者：Jennifer L. Lyon ; Ryan T. Hill ; Jason B. Shear ; Keith J. Stevenson
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：March 15, 2007
• 年：2007
• 卷：79
• 期：6
• 页码：2303 - 2311
• 全文大小：288K
• 年卷期：v.79,no.6(March 15, 2007)
• ISSN：1520-6882

文摘

Multiphoton excitation (MPE) lithography offers an effective, biocompatible technique by which three-dimensionalarchitectures comprised of proteins, enzymes, and otherrelevant materials may be fabricated for use in biologicalstudies involving cellular signal transduction and neuronal networking. We present a series of studies designedto 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-linkedcyt c retains its well-characterized Fe(II/III) heme redoxactivity. Cyt c is observed to retain its native Fe^II/IIIelectron-transfer properties, as the apparent electron-transfer rate constant, k⁰_ET, for cyt c photo-cross-linkedonto an indium-doped tin oxide (ITO) substrate was 8.4± 0.2 s^-1, on the same order of magnitude as literaturevalues though somewhat slower than other immobilizedcyt c studies, most likely due to unoptimized entrapmentin the photo-cross-linked matrix. SERS data reveals peakscorresponding to vibrational modes of an intact porphyrinring with the Fe center intact. Cyt c has also been shownto demonstrate peroxidase-like activity, and we haveevaluated the turnover rate of H₂O₂ at photo-cross-linkedmatrices relative to that at adsorbed monolayers of cyt con glass substrates. The photo-cross-linked cyt c samplesdemonstrate apparent Michaelis-Menten parameters ofV_m = 0.34 fmol/s and k_cat/K_m on the order of 10⁴ s^-1M^-1, in agreement with previously published results foraqueous cyt c. Fluorescence data obtained for mediatedH₂O₂ turnover also indicated enzymatic activity specifically at photo-cross-linked cyt c structures.