Cytochrome immobilization

Heering HA, Wiertz FGM, Dekker C, de Vries S. 2004. Direct immobilization of native yeast Iso-1 cytochrome c on bare gold Fast electron relay to redox enzymes and zeptomole protein-film voltammetry. J Am Chem Soc 126 11103-11112. 

Heering HA, Williams KA, de Vries S, Dekker C. 2006. Specific vectorial immobilization of oligonucleotide-modified yeast cytochrome c on carbon nanotubes. Chem Phys Chem 7 1705-1709. 

K. Tammeveski, T. Tenno, A.A. Mashirin, E.W. Hillhouse, P. Manning, and CJ. McNeil, Superoxide electrode based on covalently immobilized cytochrome c modeling studies. Free Radical Biol. Med. 25, 973-978 (1998). 

J.C. Cooper, G. Thompson, and C.J. McNeil, Direct electron transfer between immobilized cytochrome c and gold electrodes. Mol. Cryst. Liq. Cryst. 235, 127-132(1993). 

B. Ge and F. Lisdat, Superoxide sensor based on cytochrome c immobilized on mixed-thiol SAM with a new calibration method. Anal. Chim. Acta. 454, 53-64 (2002). 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Z.Q. Feng, S. Imabayashi, T. Kakiuchi, and K. Niki, Electroreflectance spectroscopic study of the electron transfer rate of cytochrome c electrostatically immobilized on the w-carboxyl alkanethiol monolayer modified gold electrode. J. Electroanal. Chem. 394, 149-154 (1995). 

The positive effect of convection of the substrate solution on mass transfer can be observed even better with macromolecular substrates that undergo processes such as protein digestion. For example, Fig. 9 compares reversed-phase chromatograms of cytochrome c digests obtained by cleavage with trypsin immobilized in both packed and molded column reactors, and clearly demonstrates the much higher activity of the monolithic device under otherwise similar circumstances [90]. 

Fig. 9. Reversed-phase separations of cytochrome c digests obtained with trypsin-modified beads (left) and trypsin-modified monolithic reactor (right) in a tandem with a chromatographic column (Reprinted with permission from [90]. Copyright 1996 Wiley-VCH). Conditions digestion (left curve) trypsin-modified beads reactor, 50 mm x 8 mm i.d., 0.2 mg of cytochrome c, digestion buffer, flow rate 0.2 ml/min, 25 °C, residence time, 15 min (right curve) trypsin immobilized onto molded monolith other conditions the same as with trypsin-modified beads. Reversed-phase chromatography column, Nova-Pak C18,150 mm x 3.9 mm i.d., mobile phase gradient 0-70% acetonitrile in 0.1% aqueous trifluoroacetic acid in 15 min, flow rate, 1 ml/min, injection volume 20 pi, UV detection at 254 nm...

Figure 3 shows the cyclic voltammograms of cytochrome c recorded in aqueous solution either when the protein is adsorbed (or immobilized) on the electrode (Sn02) surface (a), or when it diffuses to a gold electrode pretreated by adsorption of 4,4 -bipyridyl (b). 

Direct electron transfer has also been achieved with many metalloproteins such as cytochrome C, horseradish peroxidase, microperoxidase (MP-11), myoglobin, hemoglobin, catalase, azurin, and so on, immobilized on different CNT-modified electrodes [45, 61, 144—153]. 

Q Yang, X-Y Liu, M Hara, P Lundahl, J Miyake. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin-biotin binding. Anal Chem 280 94-102, 2000. 

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