scholarly journals A quantitative test for superoxide radicals produced in biological systems

1982 ◽  
Vol 203 (3) ◽  
pp. 551-558 ◽  
Author(s):  
H Kuthan ◽  
V Ullrich ◽  
R W Estabrook
Keyword(s):  
Cytochrome C ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Homogeneous System ◽  
Horse Heart Cytochrome ◽  
Ferrocytochrome C ◽  
Ferricytochrome C ◽  
Primary Amino ◽  
Horse Heart Cytochrome C

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH-cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.

Cytochrome c reduction by cysteine plus copper: a pseudosubstrate system for cytochrome c oxidase

1980 ◽  
Vol 58 (6) ◽  
pp. 499-503 ◽  
Author(s):  
Bruce C. Hill ◽  
Peter Nicholls
Keyword(s):  
Electron Transfer ◽  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Electrode ◽  
Horse Heart Cytochrome ◽  
Ferricytochrome C ◽  
Cytochrome C Reduction ◽  
Low Ionic Strength ◽  
Horse Heart Cytochrome C

Cysteine alone reduces horse heart cytochrome c very slowly [Formula: see text] with a rate constant virtually identical in high and low ionic strength buffers. Copper catalyzes this reaction increasing the rate by a factor of 105 in 50 mM phosphate and by a factor of 106 in 10 mM Tris buffers. When ferricytochrome c and cysteine are mixed in an oxygen electrode a "burst" of oxygen uptake is seen, the decline in which parallels the reduction of cytochrome c. When cytochrome c oxidase is added to such a mixture two routes of electron transfer to oxygen exist: enzymatic and ferricytochrome c dependent nonenzymatic. Both processes are sensitive to cyanide, but azide inhibits only the authentic cytochrome c oxidase catalyzed process and BCS the ferricytochrome c stimulated reaction.

Kinetics and mechanism of the reaction between oxidized cytochrome c and ascorbic acid

1991 ◽  
Vol 56 (2) ◽  
pp. 478-490 ◽  
Author(s):  
Joaquin F. Perez-Benito ◽  
Conchita Arias
Keyword(s):  
Ascorbic Acid ◽  
Cytochrome C ◽  
Redox Reactions ◽  
Arrhenius Equation ◽  
Horse Heart Cytochrome ◽  
First Order ◽  
Acidic Form ◽  
Ph Range ◽  
Horse Heart Cytochrome C ◽  
Mechanism Of The Reaction

The reaction between horse-heart cytochrome c and ascorbic acid has been investigated in the pH range 5.5 – 7.1 and at 10.0 – 25.0 °C. The rate shows a first-order dependence on the concentration of cytochrome c, it increases in a non-linear way as the concentration of ascorbic acid increases, it increases markedly with increasing pH and, provided that the ionic strength of the medium is high enough, it fulfills the Arrhenius equation. The apparent activation energy increases as the pH of the solution increases. The results have been explained by means of a mechanism that includes the existence of an equilibrium between two forms (acidic and basic) of oxidized cytochrome c: cyt-H+ -Fe3+ + OH- cyt -Fe3+ + H2O, whose equilibrium constant is (6.7 ± 1.4). 108 at 25.0 °C, the acidic form being more reducible than the basic one. It is suggested that there is a linkage of hydrogenascorbate ion to both forms of cytochrome c previous to the redox reactions. Two possibilities for the oxidant-reductant linkage (binding and adsorption) are discussed in detail.

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