Cytochrome adsorbed

Fig. 14.37. Speculative cartoon of cytochrome adsorbed c on a COOH-termi-nated self-assembled monolayer on gold. A crys-talline region for HS(CH2)15COOH SAM is depicted. Alkanethiol molecules are represented as end-capped cylinders with a COOH terminus (black) and a thiolate (gray) attachment to the gold electrode. Polypeptide line diagrams of cytochrome c molecule are shown in an electrostatically favored orientation. For clarity, the heme group has been blackened. (Reprinted from Bowden, "Wiring Mother Nature, Interface 6(4) 40-45, Fig. 2, 1997. Reproduced by permission of the Electrochemical Society, Inc.)...

The highest interfacial electron transfer rate constant yet reported (about 14,000 s ) is for a c-type cytochrome from Aquifex aolicus This protein has a 62-amino acid linker domain by which it is usually anchored to the periplasmic side of the inner membrane this linker has a cysteine as the terminal residue before the signal region, and the sulfur atom provides an anchor point. The cytochrome adsorbs strongly onto a Au electrode that is already modified with a hexane-thiol SAM (note this requires that the molecules in the SAM move or vacate to allow this). The results are striking. 

Second, most membrane materials adsorb proteins. Worse, the adsorption is membrane-material specific and is dependent on concentration, pH, ionic strength, temperature, and so on. Adsorption has two consequences it changes the membrane pore size because solutes are adsorbed near and in membrane pores and it removes protein from the permeate by adsorption in addition to that removed by sieving. Porter (op. cit., p. 160) gives an illustrative table for adsorption of Cytochrome C on materials used for UF membranes, with values ranging from 1 to 25 percent. Because of the adsorption effects, membranes are characterized only when clean. Fouling has a dramatic effect on membrane retention, as is explained in its own section below. 

Figure 11.15 Cation-exchange mia O-LC analysis of a mixture of model proteins (a) the original sample consisting of myoglobin (M), cytochrome C (C) and lysozyme (L) (b) and (c) proteins adsorbed on to and then released from the polyaaylic acid coated fibre with exti ac-tion times of 5 and 240 s, respectively. Reprinted from Journal of Microcolumn Separations, 8, J.-L. Liao et al., Solid phase mia O exti action of biopolymers, exemplified with adsorption of basic proteins onto a fiber coated with polyaaylic acid, pp. 1-4, 1996, with permission from Jolm Wiley Sons, New York.

The protein recovery was found to be 95% of the amount injected, whereas, on the untreated carrier they were almost totally irreversibly adsorbed. Meanwhile, some reduction in the pore volume of the carrier could be deduced from the results of the chromatographic test. The calculated pore volume available for phtalic acid was 0.67 cm2/g (V) whereas for cytochrome C — 0.5 cm2/g. A detailed description of the experiment allows the evaluation of the effective thickness (teff) of the polymeric stationary phase. The tcff calculated as V/Ssp is 2.3 nm. The value... 

Armstrong FA, Bond AM, Buchi FN, Hanmett A, Hill HAO, Lannon AM, Lettington OC, Zoski CG. 1993. Electrocatalytic reduction of hydrogen-peroxide at a stationary pyrol3ftic-graphite electrode surface in the presence of cytochrome-c peroxidase— A description based on a microelectrode array model for adsorbed enzyme molecules. Analyst 118 973-978. 

The prevalence of the heme in O2 metabolism and the discovery in the 1960s that metallophthalocyanines adsorbed on graphite catalyze four-electron reduction of O2 have prompted intense interest in metaUoporphyrins as molecular electrocatalysts for the ORR. The technological motivation behind this work is the desire for a Pt-ffee cathodic catalyst for low temperature fuel cells. To date, three types of metaUoporphyrins have attracted most attention (i) simple porphyrins that are accessible within one or two steps and are typically available commercially (ii) cofacial porphyrins in which two porphyrin macrocycles are confined in an approximately stacked (face-to-face) geometry and (iii) biomimetic catalysts, which are highly elaborate porphyrins designed to reproduce the stereoelectronic properties of the 02-reducing site of cytochrome oxidase. 

Figure 3.92 shows SERS spectra of adsorbed SSBipy and PySH at 0 V in the absence of the solution species, together with the Raman spectra of PySH in solution and crystalline SSBipy. The activities of the modified electrodes were first confirmed in solution containing cytochrome c. 

At this point it was clear how SSBipy was adsorbed on the electrode and the timescale over which this occurred, as well as the role of the concentration of the initial solution. However, the actual mode of action of the adsorbed species still remained somewhat obscure. An important insight into this was provided by the work of Hill et al. in 1987 who studied the effect of partial substitution of the layer of adsorbed promoter on the electrochemistry of cytochrome c. 

K.L. Egodage, B.S. de Silva, and G.S. Wilson, Probing the conformation and orientation of adsorbed protein using monoclonal antibodies cytochrome c3 films on a mercury electrode. J. Am. Chem. Soc. 119, 5295-5301 (1997). 

C. Hinnen, R. Parsons, and K. Niki, Electrochemical and spectroreflectance studies of the adsorbed horse heart cytochrome c and cytochrome c3 from D. vulgaris, miyazaki strain, at gold electrode. J. Electroanal. Chem. 147, 329-337 (1983). 

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). 

Figure 3 Cyclic voltammograms recorded at different scan rates in aqueous solution (pH 7) of cytochrome c, under the following experimental conditions (a) protein adsorbed on the Sn02 electrode surface (b) Au electrode pretreated with bipyridyl protein in solution...

Recently, an inorganic promoter as [Ru(CN)5(Spy)]4 (Spy = 4-thio-pyridine) adsorbed on a gold electrode surface also proved to be very effective in the direct electrochemistry of cytochrome c the tetraanion not... 

After the production of the solution of proteins, the purification of the protein requires six different steps. Silica gel powder is added to the solution and filtered off, to adsorb large quantities of cytochrome C3. Several steps of column chromatography follow. 

Additives such as polyethylene glycol, cationic antibiotics, polymers, small uncharged molecules, and negatively charged proteins have been used extensively in order to avoid the denaturing of enzymes or to improve the sensitivity and operational stability of biosensors. DNA has been proposed as an additive to improve the response and stability of biosensors based on CP. The biomolecules studied, such as tyrosinase [93], peroxidase [94], cytochrome C [95], have been shown to improve its performance by using adsorbed DNA within CP as an additive. 

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