scholarly journals Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals

1976 ◽  
Vol 157 (1) ◽  
pp. 237-246 ◽  
Author(s):  
M C Berman ◽  
C M Adnams ◽  
K M Ivanetich ◽  
J E Kench
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Phosphate Buffer ◽  
Electron Flow ◽  
Cytochrome B5 ◽  
Significant Proportion ◽  
Intrinsic Property ◽  
Order Rate Constant ◽  
Rapid Phase ◽  
Autoxidation Reaction

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 × 10(−3) +/− 0.19 × 10(−3) S-1 (mean +/− S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 × 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 × 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.

Kinetics of superoxide scavenging by glutathione: an evaluation of its role in the removal of mitochondrial superoxide

2003 ◽  
Vol 31 (6) ◽  
pp. 1337-1339 ◽  
Author(s):  
C.M. Jones ◽  
A. Lawrence ◽  
P. Wardman ◽  
M.J. Burkitt
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Superoxide Radical ◽  
Spin Trap ◽  
Significant Proportion ◽  
Mitochondrial Respiratory Chain ◽  
Oxidize Glutathione ◽  
Mitochondrial Superoxide ◽  
The Matrix ◽  
Kinetics Of

Superoxide radicals are produced in trace amounts by the mitochondrial respiratory chain. Most are removed rapidly by superoxide dismutase in the matrix. Superoxide is also known to react with glutathione. Reported values of the rate constant for this reaction range from 102 to in excess of 105 M−1·s−1. The magnitude of this rate constant has important physiological implications because, if it is at the upper end of the reported range, a significant proportion of mitochondrial superoxide will evade removal by superoxide dismutase, and will oxidize glutathione to the potentially harmful glutathionyl radical. Using EPR spectroscopy to monitor competition between glutathione and the spin trap 5,5-dimethyl-1-pyrroline N-oxide for reaction with superoxide, we have estimated that the rate constant for the reaction between superoxide and glutathione is only ~200 M−1·s−1. Hence superoxide dismutase will always out-compete glutathione for reaction with the superoxide radical, thereby preventing formation of the glutathionyl radical.

scholarly journals Reaction of Peroxynitrite with Mn-Superoxide Dismutase

2001 ◽  
Vol 276 (15) ◽  
pp. 11631-11638 ◽  
Author(s):  
Celia Quijano ◽  
Daniel Hernandez-Saavedra ◽  
Laura Castro ◽  
Joe M. McCord ◽  
Bruce A. Freeman ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Active Site ◽  
Manganese Superoxide Dismutase ◽  
Order Rate Constant ◽  
Metal Centers ◽  
Manganese Ion ◽  
Hydroxyphenylacetic Acid

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitratedin vitroand potentiallyin vivoby peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant andEscherichia coliMn-SOD with a second order rate constant of 1.0 ± 0.2 × 105and 1.4 ± 0.2 × 105m−1s−1at pH 7.47 and 37 °C, respectively. TheE. coliapoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <104m−1s−1for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e.manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 ± 0.1 × 104m−1s−1and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.

scholarly journals The effects of halothane on hepatic microsomal electron transfer

1975 ◽  
Vol 148 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M C Berman ◽  
K M Ivanetich ◽  
J E Kench
Keyword(s):  
Electron Transfer ◽  
Desaturase Activity ◽  
Anaesthetic Agent ◽  
Reductase Activity ◽  
Electron Flow ◽  
Cytochrome B5 ◽  
Order Rate Constant ◽  
Redox Equilibrium ◽  
Microsomal Cytochrome B5 ◽  
Nadh Cytochrome

1. The effects of halothane (CF3CHBrCl), a volatile anaesthetic agent, on electron transfer in isolated rat liver microsomal preparations were examined. 2. At halothane concentrations achieved in tissues during clinical anaesthesia (1-2mM), halothane shifts the redox equilibrium of microsomal cytochrome b5 in the presence of NADPH towards the oxidized form. Halothane accelerates stoicheiometric consumption of NADPH and O2, increases the rate of reoxidation of NADH-reduced microsomal ferrocytochrom b5, but does not affect NADPH- or NADH-cytochrome c reductase activity. The enhanced microsomal electron flow seen in the presence of halothane is not diminished by CO nor is it increased by pretreatment of the animals with phenobarbital. 3. The effects of halothane are maximum in microsomal preparations isolated from animals fed on a high-carbohydrate diet to induce stearate desaturase activity. Changes in microsomal electron transfer caused by halothane are in all cases abolished by low concentrations (1-2mM) of cyanide. Microsomal stearate desaturase activity is unaffected by halothane. 4. The first-order rate constant for oxidation of membrane-bound ferrocytochrome b5 in the absence of added substrate (k1 equals 1.5 times 10(-3)A-1) is similar to that for autoxidation of purified ferrocytochrome b5(k1 equals 7 times 10(-3)S-1) the rate of autoxidation of soluble ferrocytochrome b5 is unaffected by halothane. 5. It is concluded that the effects of halothane on microsomal electron transfer are not related to cytochrome P-450 linked metabolism but rather arise from the interaction of halothane with the cyanide-sensitive factor of the stearate desaturase pathway.

Spontaneous hydrolysis of heroin in buffered solution

1978 ◽  
Vol 56 (4) ◽  
pp. 665-667 ◽  
Author(s):  
Paul T. Smith ◽  
M. Hirst ◽  
C. W. Gowdey
Keyword(s):  
Liquid Chromatography ◽  
Rate Constant ◽  
Phosphate Buffer ◽  
Internal Standard ◽  
Order Rate Constant ◽  
Gas Liquid Chromatography ◽  
First Order ◽  
Order Rate ◽  
Spontaneous Hydrolysis ◽  
Hydrolysis Of

Electron-capture gas–liquid chromatography was used to study the spontaneous hydrolysis of heroin in phosphate buffer (pH 6.4 and pH 7.4) at 23 °C. Aliquots of solution were taken over a 24-h period. After extraction at pH 8.9 into propan-2-ol (10%) – ethyl acetate, deacetylated products were made into heptafluorobutyrate derivatives which were analyzed quantitatively using nalorphine as the internal standard. Heroin decomposes to O6-monoacetylmorphine (O6-MAM) under these conditions. Further decomposition to morphine was not observed. Spontaneous hydrolysis was faster at pH 7.4 (first-order rate constant, 9.6 × 10−5 min−1) than at pH 6.4 (first-order rate constant, 3.0 × 10−5 min−1). In 24 h, the decomposition to O6-MAM was 13 and 4%, respectively.

scholarly journals A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi

1977 ◽  
Vol 161 (1) ◽  
pp. 3-11 ◽  
Author(s):  
F Lavelle ◽  
M E McAdam ◽  
E M Fielden ◽  
P B Roberts ◽  
K Puget ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Pulse Radiolysis ◽  
Ferric Iron ◽  
Catalytic Mechanism ◽  
Order Rate Constant ◽  
Sodium Ascorbate ◽  
Paramagnetic Resonance ◽  
Photobacterium Leiognathi ◽  
Ph Range

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.

Improvement of a direct spectrophotometric assay for routine determination of superoxide dismutase activity

1991 ◽  
Vol 37 (11) ◽  
pp. 1993-1999 ◽  
Author(s):  
B J Bolann ◽  
R J Ulvik
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Spectrophotometric Method ◽  
Order Rate Constant ◽  
Spectrophotometric Assay ◽  
Alkaline Ph ◽  
First Order ◽  
Pseudo First Order ◽  
Superoxide Dismutase Mimics

Abstract The growing interest in measuring superoxide dismutase (EC 1.15.1.1) in many diseases calls for useful routine assays. For this purpose, the direct spectrophotometric method of Marklund (J Biol Chem 1976;251:7504-7) was improved to offer an alternative to the imprecise, indirect assays currently used. The decay of O2.- (from KO2) at pH 9.5 was monitored as the decrease in delta A (delta A = A250nm-A360nm). Superoxide dismutase was determined from the pseudo-first-order rate constant of O2.- dismutation. The precision of the assay was improved by increasing the concentration of O2.- and expanding the interval for measurements of O2.- concentrations to 4-16 mumol/L. Other assay characteristics, including temperature, were also optimized. In hemolysate the assay had a within-day CV of 5.5-13% and a between-day CV of 4%. Mn-superoxide dismutase and some superoxide dismutase mimics are inhibited at alkaline pH. Therefore, the method is primarily recommended for Cu,Zn-superoxide dismutase.

scholarly journals Iron oxidation and transferrin formation by phosvitin

1975 ◽  
Vol 151 (3) ◽  
pp. 519-525 ◽  
Author(s):  
S Osaki ◽  
R C Sexton ◽  
E Pascual ◽  
E Frieden
Keyword(s):  
Catalytic Activity ◽  
Rate Constant ◽  
Phosphate Buffer ◽  
Iron Oxidation ◽  
Order Rate Constant ◽  
Half Time ◽  
First Order ◽  
Order Rate ◽  
O2 Concentration

The catalytic activity of phosvitin in Fe(II) oxidation and the addition of iron to transferrin were studied under various conditions. It was concluded that the Fe(II) oxidized by phosvitin would bind to apotransferrin, although an appreciable fraction of Fe(III) remained bound to phosvitin. Fe(III) also migrated from phosvitin to apotransferrin. This reaction was first-order with respect to Fe(III)-phosvitin concentration with a half-time (t1/2) of 10 min, and a first-order rate constant, k=0.069min−1, in 700 μM-phosphate buffer, pH 7.2, at 30 degrees C. The catalysis of the oxidation of Fe(III) by phosvitin was proportional to O2 concentration, and is quite different from the relative O2 independence of Fe(II) oxidation as catalysed by ferroxidase. A scheme for the mobilization and transfer of iron in the chicken, including the role of ferroxidase, phosyitin and transferrin, is presented.

Development of Sustained Release Multi-Component Microparticulate System of Diclofenac Sodium and Tizanidine Hydrochloride

1970 ◽  
Vol 1 (1) ◽  
pp. 97-105
Author(s):  
Kamlesh Dashora ◽  
Shailendra Saraf ◽  
Swarnalata Saraf
Keyword(s):  
Particle Size ◽  
Sustained Release ◽  
Cellulose Acetate ◽  
Rate Constant ◽  
Diclofenac Sodium ◽  
Order Rate Constant ◽  
Anomalous Transport ◽  
First Order ◽  
Sem Study ◽  
Transport Type

Sustained released tablets of diclofenac sodium (DIC) and tizanidine hydrochloride (TIZ) were prepared by using different proportions of cellulose acetate (CA) as the retardant material. Nine formulations of tablets having different proportion of microparticles developed by varied proportions of polymer: drug ratio ‘’i.e.’’; 1:9 -1:3 for DIC and 1:1 – 3:1 for TIZ. Each tablet contained equivalent to 100 mg of DIC and 6mg of TIZ. The prepared microparticles were white, free flowing and spherical in shape (SEM study), with  the particle size varying from 78.8±1.94 to 103.33±1.28 µm and 175.92± 9.82 to 194.94±14.28µm for DIC  and TIZ, respectively.  The first order rate constant K1 of formulations were found to be in the range of  K1 = 0.117-0.272 and 0.083- 0.189 %hr-1for DIC and TIZ, respectively. The value of exponent coefficient (n) was found to be in the range of 0.6328-0.9412  and 0.8589-1.1954 for DIC and TIZ respectively indicates anomalous  to  non anomalous transport type of diffusions among different formulations

scholarly journals Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence

1991 ◽  
Vol 275 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Y K Li ◽  
J Boggaram ◽  
L D Byers
Keyword(s):  
Isotope Effect ◽  
Rate Constant ◽  
Ph Dependence ◽  
Thiol Group ◽  
Order Rate Constant ◽  
Acidic Group ◽  
Irreversible Inhibitors ◽  
Effects Of Ph ◽  
Solvent Isotope ◽  
Bell Shaped Curve

Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.

scholarly journals Specificity of activated human protein C

1985 ◽  
Vol 230 (2) ◽  
pp. 497-502 ◽  
Author(s):  
S R Stone ◽  
J Hofsteenge
Keyword(s):  
Amino Acid ◽  
Rate Constant ◽  
Amino Acid Residue ◽  
Protein C ◽  
Activated Protein C ◽  
Order Rate Constant ◽  
Human Protein ◽  
Functional Protein ◽  
Hydrophobic Residues ◽  
Apolar Residue

Peptide p-nitroanilide substrates and peptidylchloromethane inhibitors were used to examine the specificity of activated human Protein C. Substrates with arginine in the P1 position had the highest activity. The best substrates and inhibitors, as judged by the second-order rate constant for their interaction with the enzyme, had an apolar residue in the P2 position. In contrast with thrombin [Kettner & Shaw (1981) Methods Enzymol. 80, 826-842], activated Protein C was able to accommodate large hydrophobic residues such as phenylalanine and leucine in the P2 position. In the P3 position, the enzyme preferred an apolar D-amino acid residue. The results of the present study have also indicated a suitable substrate and inhibitor to be used in the assay of functional protein C and of thrombomodulin.

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