Units Of Superoxide Dismutase Research Articles

Kinetics, mechanism, and identification of photodegradation products of phenazine-1-carboxylic acid

ABSTRACTPhenazine-1-carboxylic acid (PCA) is a broad-spectrum antibiotic against many plant pathogens, produced by Pseudomonas and other species. The biosynthesis and regulation of PCA has been well documented, but there is no report about its photochemical properties. Herein, the photodegradation of PCA was carried out in an aqueous solution under the irradiation of visible light to investigate the kinetics, mechanism, and identification of photodegradation products of PCA. Results revealed that photodegradation of PCA accorded well with first-order reaction kinetics. The measured half-life of PCA was 2.2 days at pH 5.0 and increased to 37.6 days at pH 6.8 when exposed to visible light. When oxygen was removed from its solution, the half-life of PCA was doubled. Different units of superoxide dismutase (SOD) enzyme (i.e. 0, 300, and 3000 units) and varying concentrations of sodium azide (i.e. 0 mg, 5 mg, 10 mg, and 20 mg) were used to decipher the mechanism for PCA photodegradation. Hydroxyl PCA and hydroxy phenazine were tentatively identified as the degradation products of PCA photodegradation process by high-performance liquid chromatography (HPLC). The obtained degradation products were further characterized and confirmed by HPLC-mass spectrometry and LC-MS/MS-based analytical approaches. In conclusion, the degradation of PCA was found to be light dependent, which could be accelerated by hydrogen ion and oxidant in the solution. The results suggest that PCA was more stable when stored in a neutral or alkaline environment or in the dark. Therefore, it is important to modify the PCA structure or use a suitable dosage for its broad-spectrum applications.

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Superoxide anion generation in effective and ineffective soybean root nodules

Summary Superoxide anion generation was measured in soybean root nodules through the autoxidation of epinephrine, resulting in the formation of adrenochrome. Superoxide anion production was initially high in effective nodules but it rapidly declined following an increase in nitrogen fixation capacity. Superoxide anion levels sharply increased after a drop in the nitrogen fixation rate and reached their maximum rates between 35 and 42 days post-inoculation. Superoxide anion rate of formation was 2 to 4 times higher in the nodules than their respective adjacent root tissue. The rate of epinephrine autoxidation was partially dependent on EDTA and NAD(P)H. Superoxide anion production was inhibited between 80 to 90 percnt; by 100 mmol/L of HEPES, MES, MOPS, Tiron, thiourea and two units of superoxide dismutase whereas BSA, DMSO, mannitol, methionine, phenylalanine and sodium benzoate (quenchers of hydroxyl radicals) caused only a partial suppression of superoxide anion formation. On the other hand, both sodium azide and mercapto-ethanol, stimulated O2*− production.

Generation of chemiluminescence in experimental autoimmune uveitis.

Using experimental model of uveitis, the inflammation-associated chemiluminescence was measured by luminol amplification method. The induction of uveitis in Lewis rats was carried out by immunizing bovine S-antigen in complete Freund's adjuvant on hind foot-pad. The chemiluminescence activity from six sets of retina and choroid measured at the peak of inflammation was found to be in the range of 300,000-400,000 counts, amounting to nearly 20-fold increase from non-immunizated control animals under the same conditions. The phagocyte-derived superoxide anion and hydroxyl radical appear to be the two major radicals in the chemiluminescent species. This was revealed by in vitro suppression of chemiluminescence by 5,400 units of superoxide dismutase and 10 mM of D-mannitol. The decreases in counts were 21% and 35% for superoxide dismutase and D-mannitol respectively. The presence of hydrogen peroxide was not established since catalase with dose up to 20,000 units did not cause any significant suppression in counts. In the time sequence studies covering the entire course of uveitis development, the level of chemiluminescent species at day 9 postimmunization increased to 1.5-fold that of day 0, continued to increase to a maximum of 18-fold on day 12 and decreased slowly to about 3-fold in day 19. The pattern of increase appears to coincide with our previous findings in the number of polymorphonuclear leukocytes, the extent of membrane lipid peroxidation and the degree of retinal degeneration. Thus, these radicals play an important role in initiation as well as perpetuation of the membrane oxidative processes that lead to retinal degeneration.

Prevention of Reperfusion Injury of the Ischemic Spinal Cord: Use of Recombinant Superoxide Dismutase

We investigated the effect of recombinant superoxide dismutase, an oxygen free radical scavenger, on the prevention of reperfusion injury of the ischemic spinal cord. Somatosensory evoked potentials (SEPs) were obtained in 23 dogs. Spinal cord ischemia was produced by cross-clamping the descending thoracic aorta just distal to the origin of the left subclavian artery through a left thoracotomy. Mean proximal aortic blood pressure was maintained between 90 and 100 mm Hg by partial exsanguination. Serial SEPs were obtained at 60-second intervals until the SEP disappeared. Aortic cross-clamping was continued for 10 additional minutes after the disappearance of the SEP. In Group 1 (N = 8), no medication was given when the aortic cross-clamp was removed. In Group 2 (N = 8), a bolus of 25,000 units of superoxide dismutase was injected into the proximal aorta prior to removal of the aortic cross-clamp, and was followed by 5,000 units per minute for 10 minutes after release of the cross-clamp. In Group 3 (N = 7), 50,000 units of superoxide dismutase was administered as a bolus prior to removal of the aortic crossclamp, followed by an additional 10,000 units per minute for 10 minutes as in Group 2. The postoperative neurological status was assessed by Tarlov's criteria. There was no significant difference in aortic cross-clamp time among the three groups. Paraplegia developed in 4 animals in Group 1; the remaining 4 dogs had paraparesis. In Group 2, paraparesis developed in 2 of 8 dogs; the other 6 had no neurological injury. All the animals in Group 3 had complete recovery. Group 1 animals (control) had a significantly ( p < .001, Fisher's exact test) increased incidence of neurological injury compared with the superoxide dismutase–treated animals (Groups 2 and 3). There was no significant difference in the incidence of paraparesis between Groups 2 and 3. We conclude that (1) oxygen free radicals generated during reperfusion may compound the ischemic injury, (2) the ischemic spinal cord injury is limited by the use of recombinant superoxide dismutase, and (3) further studies are indicated to elucidate the dose-response phenomenon of recombinant superoxide dismutase in this animal model.

Production of the Superoxide Radical by Horseradish Peroxidase

The reaction mixture contained, in a total volume of 1.00m1, enzyme, p-coumaric acid (2.5pmol), dihydroxyfumaric acid (30pmol), KH2P04 (8.3pmol) and sufficient KOH to adjust the pH to 6. Incubations were carried out for 0.5h at 25°C and caffeic acid production was assayed as described by Halliwell(l975). 100% corresponded to a rate of 60nmol of caffeic acid formed in 0.5h. Oxidation of dihydroxyfumarate was followed by the decrease in E300 (althoughp-coumaric acid also absorbs light at 300nm, very little is used up during a 30min incubation). Samples (10~1) were removed from the reaction mixture at intervals, added to lOml of water and the absorbance recorded at once. Units of superoxide dismutase were as described by McCord & Fridovich (1969).