STUDIES ON STATES OF CYTOCHROME-C MOLECULES IN AQUOUS SOLUTIONS USING SYNCHRONOUS FLUORESCENCE SPECTROSCOPY

The states of cytochrome C molecules in aquous solution were studied with synchronous fluorescence spectroscopy, It was found that the synchronous fluorescent spectra of cytochrome C were contributed by tyrosine and tryptophan residues separately at Delta lambda = 20 nm and Delta lambda = 80 nm, The peak position in synchronous fluorescent spectra of tyrosine residues in cytochrome C molecule does not change with its concentration, but that of tryptophan residue changes with its concentration, Only one peak at 340.0 nm was observed in the dilute solution of cytochrome C, With increasing the concentration of cytochrome C, a new peak at 304. 0 nm appeared. The peak at 340.0 nm disappeared and only one peak at 304.0 nm was observed at a higher concentration of cytochrome C, It may originate from the change of aggregation states of cytochrome C molecules and it was considered that the peak at 340.0 nm was attributed to the monomer and peak at 304.0 nm was due to the dimmer or oligomers. When urea was added into cytochrome C solution in which both monomer and dimmer or oligomers exist, cytochrome C molecules do not denature in the range of the specific concentrations of urea. The concentration of monomer of cytochrome C molecules increased and that of aggregation slates decreased by adding urea, Therefore, the synchronous fluorescence spectroscopy can be used to identify monomer and aggregation states of cytochrome C molecules.

VOLTAMMETRY OF CYTOCHROME-C ENTRAPPED IN HYDROGEL MEMBRANE ON GRAPHITE ELECTRODE

The voltammetric behavior of cytochrome c entrapped in hydrogel membranes at paraffin wax-impregnated spectroscopic graphite electrodes (WISGE) was studied in this paper. A pair of well-defined peaks appeared at +70 mV (vs. Ag/AgCl). Beside these two peaks, another pair of peaks emerged at around +225 mV. Further investigations suggested that at least three states of cytochrome c existed in the membranes due to the special structure of the hydrogel. The native conformation of cytochrome c molecules was stabilized by the hydrophilic environment that was formed by the hydroxyl structure of the membranes and facilitated the cytochrome c electron transfer reaction at +70 mV. The molecules directly adsorbed on the surface of the graphite electrode were responsible for the redox peaks at around +225 mV. Whether the adsorption peaks were detectable or not was related to the thickness of membranes and the pre-retaining time before the formation of membranes.

PROMOTER EFFECT OF HALOGEN ANIONS ON THE DIRECT ELECTROCHEMICAL REACTION OF CYTOCHROME-C AT GOLD ELECTRODES

The promoter effect of halogen anions for heterogeneous electron transfer between cytochrome c and a gold electrode was studied. It was found that the order of the promoter ability of halogen anions is I- > Br- > Cl- > F-. In addition, factors which can affect the promoter effect were discussed.

BINDING OF CYTOCHROME-C WITH 4-PYRIDYL DERIVATIVES MODIFIED ON THE GOLD ELECTRODE

In this work, the adsorption or binding of cytochrome c with 4-pyridyl derivatives modified on the gold electrode was studied. It was found that the concentrations of electrolyte had much influence on the adsorption of cytochrome c. At lower concentration

ELECTROCHEMICAL REACTION OF CYTOCHROME-C AT GOLD ELECTRODES MODIFIED WITH THIOPHENE CONTAINING ONE FUNCTIONAL-GROUP

The electrochemical reactions of cytochrome c were studied at a thiophene-modified gold electrode. It was demonstrated that thiophene is an effective promoter, although there is only one functional group in the molecule. Based on this result, the mechanis

DIRECT ELECTROCHEMICAL REACTIONS OF CYTOCHROME-C AT IODIDE-MODIFIED ELECTRODES

Quasi-reversible and direct electron transfer was observed between an iodide-modified Au electrode and cytochrome c, as well as between cytochrome c in an iodide-containing solution and a bare Au electrode. The results suggest that an electrostatic intera

REDOX THERMODYNAMICS OF CYTOCHROME-C AT THE BARE GLASSY-CARBON ELECTRODE

Investigation of the redox thermodynamics of horse heart cytochrome c at bare glassy carbon electrodes has been performed using cyclic voltammetry with a nonisothermal electrochemical cell. The thermodynamic parameters of the electron-transfer reaction of cytochrome c have been estimated in different component buffer solutions. The change DELTAS(re)-degrees in reaction center entropy and the formal potential E-degrees' (at 25-degrees-C, vs. standard hydrogen electrode (SHE)) for cytochrome c are found to be -64.1 J K-1 mol-1 and 0.251 V in phosphate buffer, -64.8 J K-1 mol-1 and 0.257 V in Tris + HCl buffer, -65.6 J K-1 mol-1 and 0.261 V in Tris+CH3COOH buffer (pH 7.0, ionic strength 100 mM). The temperature dependence of the formal potential obtained in phosphate buffer with or without NaCl in the range 5-55-degrees-C shows biphase characteristics in an alkaline solution with an intersection point at ca. 44-degrees-C or 42-degrees-C, which should be due to a structural change in the protein moiety of cytochrome c. However, in acidic and neutral solutions only a monotonic relationship between E-degrees' and temperature is observed. The effect of the buffer component on E-degrees' for cytochrome c is also discussed.

EFFECT OF PH ON THE ELECTRON-TRANSFER OF CYTOCHROME-C ON A GOLD ELECTRODE MODIFIED WITH BIS(4-PYRIDYL)DISULPHIDE

The adsorption of bis(4-pyridyl)disulphide (PySSPy) and 4.4'-bipyridyl (PyPy) on a gold electrode was studied using cyclic voltammetry. The adsorption isotherms and equilibrium constants (1 X 10(6) mol-1 l for PyPy and 6 x 10(6) Mol-1 l for PySSPy) were determined. The effect of pH on the electrochemical behaviour of cytochrome c was studied on the PySSPy-modified gold electrode. The results show that cytochrome c can only transfer electrons on a deprotonated electrode surface. When the pH is decreased, the standard heterogeneous rate constant of cytochrome c on the modified gold electrode decreases and the electrochemical behaviour changes from a quasi-reversible to an irreversible process.

REEVALUATION OF 2,2-BIPYRIDINE AND PYRAZINE AS PROMOTERS FOR DIRECT ELECTRON-TRANSFER BETWEEN CYTOCHROME-C AND METAL-ELECTRODES

Results from previous electrochemical studies have indicated that 2,2'-bipyridine and pyrazine do not function as promoters for heterogeneous electron transfer between cytochrome c and metal electrodes. Their lack of activity was attributed to the improper positioning of the two functional groups in 2,2'-bipyridine and the inefficient length of pyrazine. In the present study it was determined that both 2,2'-bipyridine and pyrazine act as promoters when self-absorbed over a sufficiently long dipping time or at roughened electrodes. The promoter characteristics of these two molecules were studied and compared with those of 4,4'-bipyridine. The difference in their promoter behavior appears to result primarily from their different strengths of adsorption and not because electrodes modified with 2,2'-bipyridine or pyrazine are unsuitable for accelerating direct electron transfer reactions in cytochrome c. These results have implications regarding the mechanism(s) of promoter effects in electrochemical reactions of cytochrome c.

ELECTROCATALYSIS OF POLYPYRROLE-METHYLENE BLUE FILM MODIFIED ELECTRODE FOR DIRECT REDOX REACTION OF CYTOCHROME-C

The electron transfer process of hemeproteins on the electrode surface is considered a promising subject in the area of bioelectrochemistry. Electrochemists believe that electron transfer between electroactive proteins and electrode surface might be expected to simulate the electron transfer between proteins. This research provides information about the electron transfer mechanism in biological system. Cytochrome c is a typical electron transferring protein,

The direct electrochemistry behavior of Cyt c on the modified glassy carbon electrode by SBA-15 with a high-redox potential

It is discovered that SBA-15 (santa barbara amorphous) can provide the favorable microenvironments and optimal direct electron-transfer tunnels (DETT) of immobilizing cytochrome c (Cyt c) by the preferred orientation on it. A high-redox potential (254 mV vs. Ag/AgCl) was obtained on glassy carbon (GC) electrode modified by immobilizing Cyt c on rod-like SBA-15. With ultraviolet-visible (UV-vis), circular dichroism (CD), FTIR and cyclic voltammetry, it was demonstrated that immobilization made Cyt c exhibits stable and ideal electrochemical characteristics while the biological activity of immobilized Cyt c is retained as usual.

基质辅助激光解吸电离飞行时间质谱表征细胞色素C

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF—MS) technique was used for analysis of moleculear weight of cytochrome C.The effects of three kinds of matrix,such as 2,5-dihydroxybenzoic acid(DHB),a-cyano-4-hydroxycinnamic acid(a-CHC) and sinapinic acid(SA),were used to compared and a suitable a-CHC was found.Experimental data showed that this method was properable to analysis of the congeneric biochemical samples.

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon 'nanotubes (SWNTs) modified glass carbon electrode (SWNTs/ GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK(a) of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.