cytochrome-c-t and 11-mercaptoundecanoic-acid

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (RRMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.

Topics: Adsorption; Animals; Cytochromes c; Electrochemistry; Electrodes; Fatty Acids; Gold; Microscopy, Atomic Force; Models, Molecular; Protein Conformation; Sulfhydryl Compounds; Time Factors

A cytochrome c/11-MUA heterolayer was fabricated to analyze its electrochemical characteristics in harsh conditions for a stable bioelectronic device. Since a cytochrome c/11-MUA heterolayer has been applied to construct the bioelectronics device such as non-volatile biomemory device, an understanding of electrochemical property of the heterolayer in harsh conditions such as variation of temperature and pH, and repetition of usage is necessary to manufacture a stable platform of bioelectronic device. Cytochrome c, a metalloprotein to have a heme group, was self-assembled on the Au surface via the chemical linker 11-mercaptoundecanoic acid (11-MUA). Immobilization of the heterolayer was confirmed by surface-enhanced Raman spectroscopy (SERS) and scanning tunneling microscopy (STM). The fatigue test was done by investigating the redox properties based on cyclic voltammetry (CV) of the heterolayer. The retention time test and pH dependence, thermal test of the fabricated heterolayer were conducted by CV, which showed that the fabricated film retained redox properties for more than 33 days, and from pH 5.0 to pH 9.0, from 15 °C to 55 °C. Taken together, our results show that a cytochrome c/11-MUA heterolayer is very stable, which could be used as a platform of bioelectronic device.

Topics: Adsorption; Coated Materials, Biocompatible; Conductometry; Cytochromes c; Electronics; Enzyme Activation; Enzyme Stability; Fatty Acids; Materials Testing; Nanoparticles; Protein Binding; Sulfhydryl Compounds

A novel hyperbranched polyethyleneimine (PEI) modified gold surface has been designed, fabricated, and investigated with respect to its ability to resist non-specific adsorption of proteins. The facile synthesis strategy, based on self-assembly, involves immobilization of polyethyleneimine to gold surfaces modified with 11-mercaptoundecanoic acid (MuDA) monolayers using traditional carbodiimide chemistry. The hyperbranched polymer brushes were characterized by X-ray photoelectron spectroscopy (XPS). Reflection absorption infrared spectroscopy (RAIRS) and ellipsometry measurements showed the thickness of the PEI brushes increases with adsorption solution ionic strength. Polymer brush surface concentrations can be improved from 2560 to 3880chains/μm(2) by changing the ionic strength of the adsorption solution (PBS) by varying NaCl concentration from 0 to 650mM. Protein adsorption (pH 7.4) was evaluated under flow injection analysis (FIA) conditions using a quartz crystal microbalance (QCM). The PEI brushes suppress protein adsorption, for example, cytochrome C, bovine serum albumin (BSA), and ribonuclease A, to less than 0.08μg/cm(2) and the protein resistance increases with increasing ionic strength of the carrier solution, performance comparable to that achieved with comparable PEG-coated surfaces. The PEI brushes were exceptionally stable, with adsorption characteristics maintained after 6months storage in aqueous conditions (pH 7.4, 25°C, PBS). The potential of hyperbranched PEI structures as protein-resistant surfaces is discussed.

Topics: Adsorption; Biofouling; Cytochromes c; Fatty Acids; Gold; Osmolar Concentration; Photoelectron Spectroscopy; Polyethyleneimine; Proteins; Ribonuclease, Pancreatic; Serum Albumin, Bovine; Sulfhydryl Compounds; Surface Properties

Post-translational nitration of tyrosine is considered to be an important step in controlling the multiple functions of cytochrome c (Cyt-c). However, the underlying structural basis and mechanism are not yet understood. In this work, human Cyt-c variants in which all but one tyrosine has been substituted by phenylalanine have been studied by resonance Raman and electrochemical methods to probe the consequences of tyrosine nitration on the heme pocket structure and the redox potential. The mutagenic modifications of the protein cause only subtle conformational changes of the protein and small negative shifts of the redox potentials which can be rationalized in terms of long-range electrostatic effects on the heme. The data indicate that the contributions of the individual tyrosines for maintaining the relatively high redox potential of Cyt-c are additive. Nitration of individual tyrosines leads to a destabilization of the axial Fe-Met80 bond which causes the substitution of the native Met ligand by a water molecule or a lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of the heme pocket of Cyt-c due to tyrosine nitration and to local electric fields are independent of each other and occur via different molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the peroxidase function.

Topics: Cytochromes c; Fatty Acids; Heme; Humans; Iron; Kinetics; Lysine; Methionine; Mutation; Nitro Compounds; Oxidation-Reduction; Phenylalanine; Protein Stability; Protein Structure, Quaternary; Recombinant Proteins; Silver Compounds; Static Electricity; Sulfhydryl Compounds; Tyrosine; Water

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

Topics: Biocatalysis; Cytochromes c; Electrochemistry; Electrodes; Enzymes, Immobilized; Fatty Acids; Fatty Alcohols; Gold; Hydrophobic and Hydrophilic Interactions; Oxidation-Reduction; Oxygen; Protein Unfolding; Sulfhydryl Compounds

The biomolecular/organic hetero-structure films (cytochrome c/11-mercapto-undecanoic acid) on gold substrates were controlled and fabricated with molecular level for developing valuable molecular electronic devices. Cytochrome c is a metalloprotein having redox property, which can be directly applicable to biomemory device as a active element. For efficient immobilization of the protein on the gold substrate, 11-mercapto-undecanoic acid (11-MUA) was used as a linker material between protein and inorganic substrate. The proposed nano scaled biomolecular/organic hetero-structure layer (cytochrome c/11-MUA) on gold surface was investigated by using surface plasmon resonance technique. The molecular morphology of the fabricated protein layer was confirmed by scanning tunneling microscopy. Electrochemical properties of fabricated biomolecular/organic hetero layer were verified using cyclic voltammetry. Their redox properties was sustained over 1000 cycles of cyclic voltametry. It proved that the fabricated film was a suitable platform for the bioelectronic device application.

Topics: Biosensing Techniques; Coated Materials, Biocompatible; Crystallization; Cytochromes c; Electronics; Equipment Design; Equipment Failure Analysis; Fatty Acids; Nanostructures; Nanotechnology; Particle Size; Protein Binding; Sulfhydryl Compounds; Surface Plasmon Resonance

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.

Topics: Adsorption; Biosensing Techniques; Cytochromes c; Electrochemistry; Electrodes; Fatty Acids; Hydrogen-Ion Concentration; Oxidation-Reduction; Silver; Spectrum Analysis, Raman; Sulfhydryl Compounds; Surface Properties

Silver electrodes were covered with mixed self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) and 11-mercaptoundecanol (MU) and subsequently coated with alternating layers of cytochrome c (Cyt) and poly(anilinesulfonic acid) (PASA). The immobilized protein is electroactive and retains its native structure. Compared to the case of systems on gold electrodes, the stability of the assembly was found to be decreased. The redox process of Cyt is accompanied by reversible oxidation-reduction of PASA as revealed by the comparative surface-enhanced resonance Raman (SERR) analysis of assemblies including Cyt and the redox-inactive apo-cytochrome c. Time-resolved SERR experiments show a fast electron exchange between the protein and the polyelectrolyte that may play a supporting role in the electric communication of thicker multilayer assemblies employed as sensors.

Topics: Aniline Compounds; Animals; Biosensing Techniques; Coated Materials, Biocompatible; Cytochromes c; Electrodes; Electrolytes; Electron Transport; Fatty Acids; Fatty Alcohols; Gold; Oxidation-Reduction; Silver; Spectrum Analysis, Raman; Sulfhydryl Compounds; Surface Properties