The oxidation of ferrocytochrome c in nonbinding buffer

1977 ◽  
Vol 55 (8) ◽  
pp. 796-803 ◽  
Author(s):  
B. F. Peterman ◽  
R. A. Morton
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Equilibrium Constant ◽  
Oxidation Reaction ◽  
Potassium Phosphate ◽  
Ionic Strength Dependence ◽  
Horse Heart Cytochrome ◽  
Apparent Equilibrium Constant ◽  
Diffusion Limited ◽  
Apparent Equilibrium

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1–200 mM using Tris–cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox(~ 9 × 109 M−1 s−1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at μ = 0 was the order of 6 × 103), and was not consistent with diffusion-limited kinetics.

Immunochemical approach to the question of inactive subunits in cases of lactate dehydrogenase B subunit deficiency.

1986 ◽  
Vol 32 (1) ◽  
pp. 116-119 ◽  
Author(s):  
M Maekawa ◽  
K Sudo ◽  
T Kanno
Keyword(s):  
Lactate Dehydrogenase ◽  
Equilibrium Constant ◽  
Competitive Reaction ◽  
Apparent Equilibrium Constant ◽  
B Subunit ◽  
B Subunits ◽  
Free Antigen ◽  
Apparent Equilibrium ◽  
Normal Controls

Abstract If there were enzymatically inactive B subunits in a solution containing lactate dehydrogenase (LD) B4 prepared from the hemolysates of heterozygous individuals with LD-B subunit deficiency, the inactive subunits would compete with normal subunits for antibodies to the B4 subunit. Using the activity of normal LD-B4 as an index, we could ascertain the proportion of free antigen (LD-B4) and antibody-antigen complexes (LD-B4-anti-LD-B4) and calculate the apparent equilibrium constant, Keq. Doing so, we found the value for Keq in LD-B subunit deficiency to be smaller than that for normal controls. Evidently the expected competitive reaction takes place. We conclude that heterozygous individuals with LD-B subunit deficiency do indeed produce variant (enzymatically inactive) B subunits of LD.

Ion binding to lysine-modified derivatives of cytochrome c

1977 ◽  
Vol 55 (2) ◽  
pp. 146-151 ◽  
Author(s):  
R. A. Morton ◽  
K. Breskvar
Keyword(s):  
Cytochrome C ◽  
Equilibrium Constant ◽  
Negative Charge ◽  
Positive Charge ◽  
Ion Binding ◽  
Ferrocytochrome C ◽  
Cacodylate Buffer ◽  
Anion Site ◽  
Derivatives Of ◽  
Apparent Equilibrium

The effect of Cl− and K+ ions on the apparent equilibrium constant of the reaction between horse ferricytochrome c and potassium ferrocyanide was studied. Unmodified cytochrome was compared with two lysine-modified derivatives. One, guanidinated, had all lysyl groups converted to homoarginine (but retained the same positive charge); the other was trinitrophenylated at one lysine (measured spectrophotometrically). Both modified derivatives had a somewhat larger equilibrium constant in the reaction of the reduced protein with ferricyanide, but, unlike trifluoroacetylated cytochrome c (which has a negative charge), the redox properties were not dramatically different. The native protein and the lysine-modified cytochromes showed differential K+ binding in Tris–cacodylate buffer at constant ionic strength (0.003–0.005 M). More K+ was bound to ferrocytochrome c. This redox-linked binding, however, was unaffected by modification of lysine. All three derivatives also showed redox-linked differential Cl− ion binding (more Cl− ion was bound to ferricytochrome); however, in this case, the binding was reduced in the lysine-modified molecules. This was interpreted as loss of a single anion site. This anion site critically depends on one or a few lysines which are more reactive with trinitrobenzene sulfonate.

A study on the reaction mechanism of adenosine 5′-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein

1977 ◽  
Vol 55 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Kazuo Adachi ◽  
Isamu Suzuki
Keyword(s):  
Reaction Mechanism ◽  
Cytochrome C ◽  
Direct Reduction ◽  
Potassium Phosphate ◽  
Difference Spectrum ◽  
Order Rate Constant ◽  
Horse Heart Cytochrome ◽  
Thiobacillus Thioparus ◽  
Aps Reductase ◽  
Concomitant Reduction

The reaction mechanism of adenosine 5′-phosphosulfate (APS) reductase (EC 1.8.99.2) from Thiobacillus thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a first order rate constant of 97.1 s−1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone FADH∙) and the concomitant reduction of nonheme iron to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25–35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2−. The activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.

Solubility of Na2O2.8 H2O in the system H2O2-NaOH-KOH-H2O

1982 ◽  
Vol 47 (3) ◽  
pp. 736-743 ◽  
Author(s):  
Jan Balej ◽  
Otomar Špalek
Keyword(s):  
Equilibrium Constant ◽  
Potassium Hydroxide ◽  
Empirical Equation ◽  
Multicomponent System ◽  
Total Content ◽  
Potassium Ions ◽  
Apparent Equilibrium Constant ◽  
Dissolution Reaction ◽  
Apparent Equilibrium

The solubility of Na2O2.8 H2O in the multicomponent system H2O2-NaOH-KOH-H2O has been investigated at temperatures of 12, 20, and 30°C. Comparison of the data with solubilities of the same compound in the simler system H2O2-NaOH-H2) shows that the presence of potassium hydroxide increases the solubility at the same temperature and total content of hydroxide ions. The effect of potassium ions on the solubility of Na2O2.8 H2O is expressed by an empirical equation relating the apparent equilibrium constant of the dissolution reaction and the molalities of the perhydroxyl and potassium ions at a given temperature.

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